Denaturation of Nucleic Acids Induced by Intercalating Agents. Biochemical and Biophysical Properties of Acridine Orange-DNA Complexes

Abstract

At high binding denstities acridine orange (AO) forms complexes with ds DNA which are insoluble in aqueous media. These complexes are characterized by high red- and minimal green-luminescence, 1:1 (dye/P) stoichiometry and resemble complexes of AO with ss nucleic acids. Formation of these complexes can be conveniently monitored by light scatter measurements. Light scattering properties of these complexes are believed to result from the condensation of nucleic acids induced by the cationic, intercalating ligands. The spectral and thermodynamic data provide evidence that AO (and other intercalating agents) induces denaturation of ds nucleic acids; the driving force of the denaturation is high affinity and cooperativity of binding of these ligands to ss nucleic acids. The denaturing effects of AO, adriamycin and ellipticine were confirmed by biochemical studies on accessibility of DNA bases (in complexes with these ligands) to the external probes. The denaturing properties of AO vary depending on the primary structure (sugar-and base-composition) of nucleic acids.     

An approach to diagnosis of T-cell lymphoproliferative disorders by flow cytometry

T-cell lymphoproliferative disorders are among the most challenging diagnoses in hematopathology. Unlike the more common B-cell disorders, in which clonality is often readily discernible by surface immunoglobulin light chain restriction, there is no specific immunophenotypic signature that is diagnostic of a clonal T-cell population. Immunophenotypic criteria that are helpful in the diagnosis of T-cell neoplasms include T-cell subset antigen restriction, anomalous T-cell subset antigen expression, deletion or diminution of one of the pan T-cell antigens, a precursor T-cell phenotype, and expression of additional markers (e.g., CD30, CD20, major myeloid antigens, and TCRgammadelta). Analysis of the inherent forward and orthogonal light scatter properties of the cell can also provide important diagnostic clues. None of these features is 100% specific, however, for aberrant expression of pan-T antigens may be seen in viral infections, B-cell malignancies, or in reactive changes following administration of certain medications. An increased CD4:CD8 ratio is often observed in Hodgkin's lymphoma. Based on the analysis of 87 neoplastic and 80 control cases, we conclude that flow cytometric features that are most suspicious for malignancy include the loss or markedly dim expression of CD45; complete loss of one or more pan-T antigens; diminished expression of more than two pan-T antigens in conjunction with altered light scatter properties; and CD4/CD8 dual-positive or dual-negative expression (except thymic lesions).     

Influence of the length of the phosphate chain in mRNA 5' cap analogues on their interaction with eukaryotic initiation factor 4E

The recognition of the 5'mRNA cap structure m7G(5')ppp(5')N by one of the components of the initiation translation machinery, the eIF4E factor, plays a pivotal role in regulation of the protein synthesis. In the present study we have shown two opposing roles of the cap phosphate chain in the specific eIF4E-cap interaction. The extension of the phosphate chain enhances the binding of the cap to the unphosphorylated eIF4E but destabilises the eIF4E-cap complex in case of the phosphorylated protein.     

Binding studies of eukaryotic initiation factor eIF4E with novel mRNA dinucleotide cap analogues

Studies on the interaction of the murine translation initiation factor 4E with two new-synthesized cap-analogues, modified at C2' of 7-methylguanosine, have been performed by means of the fluorescence titration method. No difference in the binding affinity for eIF4E was observed compared with the "anti reversed" cap analogues, possessing the analogous modifications at C3'. Potential significance of the novel caps as research tools for examination of the nuclear cap binding complex CBC80/20 has been discussed.     

Interaction between yeast eukaryotic initiation factor eIF4E and mRNA 5' cap analogues differs from that for murine eIF4E

Measurements of interaction of 7-methyl-GTP eIF4E from S. cerevisiae were performed by means of two methods: Isothermal Titration Calorimetry (ITC) and fluorescence titration. The equilibrium association constants (Kas) derived from the two methods show significantly different affinity of yeast eIF4E for the mRNA 5' cap than those of the murine and human proteins. The observed differences in the Kas values and the enthalpy changes of the association (deltaH(o)) suggest some dissimilarity in the mode of binding and stabilization of cap in the complexes with eIF4E from various sources.     

Thermodynamics of 7-methylguanosine cation stacking with tryptophan upon mRNA 5' cap binding to translation factor eIF4E

All eukaryotic nuclear transcribed mRNAs possess the cap structure, consisting of 7-methylguanosine linked by the 5'-5' triphosphate bridge to the first nucleoside. The goal of the present study is to dissect the enthalpy and entropy changes of association of the mRNA 5' cap with eIF4E into contributions originating from the interaction of 7-methylguanosine with tryptophan. The model results are discussed in the context of the thermodynamic parameters for the association of eIF4E with synthetic cap analogues.     

"Liquidless" cell staining by dye diffusion from gels and analysis by laser scanning cytometry: potential application at microgravity conditions in space

BACKGROUND: Conventional staining of cells or tissue sections on microscope slides involves immersing the slides into solutions of dyes then rinsing to remove the unbound dye. There are instances, however, when use of stain solutions is undesirable-e.g., at microgravity conditions in space, where the possibility of accidental spill (many dyes are known carcinogens) introduces health hazard. Likewise, transporting bulk of liquid stains and rinses may be burdensome in certain situations such as field expeditions or combat. METHODS: The "liquidless" staining procedure is proposed in which the dyes are contained in thin strips of hydrated polyacrylamide or gelatin gels that have been presoaked in the stain solutions. Fluorochromes that have affinity to DNA (propidium iodide, PI; 4,6-diamidino-2-phenylindole, DAPI, Hoechst 33342) or to protein (sulforhodamine 101) were used to saturate the gels. The gel strips were placed over the prefixed cells or tissue sections deposited on microscope slides and relatively low (20 g/cm2) pressure was applied to ensure the contact. The cells were also stained by using commercially available mounting media into which DAPI or PI were admixed. Intensity of fluorescence of the PI stained cells was measured by laser scanning cytometry (LSC). RESULTS: Satisfactory cell and tissue staining, with minimal background, was achieved after 10-20 min contact between the cells and gels. Optimal concentrations of the dyes in the solutions used to presoak the gels was found to be 2-4-fold higher than the concentrations used routinely in cytometry. The measurements of intensity of cellular fluorescence by LSC revealed that the staining of DNA was stoichiometric as reflected by the characteristic cellular DNA content frequency histograms with distinct G1, S, and G2/M cell populations and 2:1 ratio of G2/M to G1 peak fluorescence. Individual gels can be saturated with more than a single dye-e.g., to obtain differential DNA and protein staining. Cell staining with DAPI or PI in the gelatin-based mounting media led to high fluorescence background while staining with DAPI in "aqueous" medium was satisfactory. CONCLUSIONS: Relatively fast staining of cells or tissue sections on microscope slides can be achieved by nonconvective dye diffusion using hydrated gels permeated with the dyes, applied to cells at low pressure. The quality of the staining provided by this methodology is comparable to conventional cell staining in dye solutions.     

Unique analytical capabilities of laser scanning cytometry (LSC) that complement flow cytometry

Flow cytometry has become an indispensable instrumentation in many disciplines of biology and medicine. There are some limitations of flow cytometry, inherent to the fact that the cells are measured in flow, which limit its usefulness in some applications. The microscope-based laser scanning cytometer (LSC) has many features similar to flow cytometry but few restrictions of the latter and therefore it is useful in many new applications. This review briefly outlines the applications that are unique to LSC, particularly related to its morphometric capabilities and the possibility of cell relocation. Potential future applications of LSC are also discussed.     

Activation of caspases measured in situ by binding of fluorochrome-labeled inhibitors of caspases (FLICA): correlation with DNA fragmentation

Activation of caspases is the key event of apoptosis and new methods are needed to assay this event, particularly in situ, in individual cells. To measure in situ caspases activation in the present study we employed fam-VAD-fmk and fam-VEID-fmk, the fluorochrome (fam)-labeled inhibitors of caspases (FLICA), which through the fluoromethylketone (fmk) moiety bind to active center of the activated enzymes. The peptide moiety of these inhibitors defines their specificity; VAD is generic to most caspases and VEID is caspase-6 specific. The frequencies of cells showing caspases activation were compared with those showing DNA fragmentation (detected by the TUNEL assay) in the same cultures. Apoptosis of HL-60 cells was induced by DNA topoisomerase I inhibitor camptothecin (CPT) or tumor necrosis factor-alpha combined with cycloheximide (TNF-alpha + CHX). The cells that bound FLICA had morphological changes typical of apoptosis. The intensity of their fluorescence was measured by laser scanning cytometry. Maximal rate of activation of the caspases, measured by the increase in frequency of the cells that bound fam-VAD-fmk, occurred between 30 and 90 min after the administration of TNF-alpha + CHX and between 2 and 4 h after the administration of CPT. In the CPT-treated cultures about 30% fewer cells bound fam-VEID-fmk than fam-VAD-fmk which suggests that the activation of caspase-6 was delayed or was not induced in some cells. A strong overall correlation between the cytometric assays of the apoptotic index based on the detection of caspases activation by the FLICA and the TUNEL assay was observed. The data indicate that FLICA offers a rapid and convenient method of assessing caspase's activation in individual cells and can also be used to estimate the frequency of apoptosis.