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Cytometry Nov 2000DNA fluorescence dyes have been used to study DNA dynamics, chromatin structure, and cell cycle analysis. However, most microscopic fluorescence studies of DNA use only...
BACKGROUND
DNA fluorescence dyes have been used to study DNA dynamics, chromatin structure, and cell cycle analysis. However, most microscopic fluorescence studies of DNA use only steady-state measurements and do not take advantage of the additional information content of the time-resolved fluorescence. In this paper, we combine fluorescence imaging of DNA with time-resolved measurements to examine the proximity of donors and acceptors bound to chromatin.
METHODS
We used frequency-domain fluorescence lifetime imaging microscopy to study the spatial distribution of DNA-bound donors and acceptors in fixed 3T3 nuclei. Over 50 cell nuclei were imaged in the presence of an AT-specific donor, Hoechst 33258 (Ho), and a GC-specific acceptor, 7-aminoactinomycin D (7-AAD).
RESULTS
The intensity images of Ho alone showed a spatially irregular distribution due to the various concentrations of DNA or AT-rich DNA throughout the nuclei. The lifetime imaging of the Ho-stained nuclei was typically flat. Addition of 7-AAD decreased the fluorescence intensity and lifetime of the Ho-stained DNA. The spatially dependent phase and modulation values of Ho in the presence of 7-AAD showed that the Ho decay becomes nonexponential, as is expected for a resonance energy transfer (RET) with multiple acceptors located over a range of distances. In approximately 40 nuclei, the intensity and lifetime decrease was spatially homogeneous. In approximately 10 nuclei, addition of 7-AAD resulted in a spatially nonhomogeneous decrease in intensity and lifetime. The RET efficiency was higher in G(2)/M than in G(0/1) phase cells.
CONCLUSIONS
Because RET efficiency depends on the average distance between Ho and 7-AAD, data suggest that the heterogeneity of lifetimes and spatial variation of the RET efficiency are caused by the presence of highly condensed regions of DNA in nuclei.
Topics: 3T3 Cells; AT Rich Sequence; Animals; Bisbenzimidazole; Cell Nucleus; Chromatin; DNA; Dactinomycin; Fluorescent Dyes; GC Rich Sequence; Mice; Microscopy, Fluorescence; Spectrometry, Fluorescence; Time and Motion Studies
PubMed: 11042614
DOI: 10.1002/1097-0320(20001101)41:3<178::aid-cyto4>3.0.co;2-n -
The American Journal of Pathology Aug 1971The morphologic effects of actinomycin D (ACTD) on normal and regenerating rat pancreas at three dosage levels were studied. ACTD at 1.0 and 0.5 mug/g total body weight...
The morphologic effects of actinomycin D (ACTD) on normal and regenerating rat pancreas at three dosage levels were studied. ACTD at 1.0 and 0.5 mug/g total body weight produced reversible nucleolar and cytoplasmic changes in the acinar cells of the normal rat pancreas. The nucleolar alteration, zonal segregation, preceded the appearance of cytoplasmic lesions, implying a precursor relationship of the former to the latter. ACTD-induced cytoplasmic lesions were similar to those produced by ethionine. Since ethionine failed to produce nucleolar changes, it is suggested that the primary mechanisms of action of these two compounds are different, although the cytoplasmic lesions may occur by a common metabolic route. Regeneration of exocrine pancreas after injection of ethionine was affected by ACTD to different degrees depending upon the time of ACTD administration. Regeneration was prevented when ACTD, 0.5 mug/g, was given at day 11, the first day after the ethionine regimen; it was markedly decreased when ACTD was given at day 12, but much less affected when ACTD was given at later days. The effects at days 11 and 12 may have been related to prevention of DNA transcription and the formation of mRNAs at these days. The 0.05 mug/g dosage of ACTD had much less effect on normal and regenerating acinar cells, causing reduced cytoplasmic basophilia, decreased ergastoplasm and fewer free ribosomes, which suggests impairment of rRNA synthesis.
Topics: Animals; Cell Nucleus; Chromatin; Cytoplasm; Dactinomycin; Ethionine; Male; Microscopy, Electron; Pancreas; Rats; Rats, Inbred Strains; Regeneration; Ribosomes; Time Factors
PubMed: 5142270
DOI: No ID Found -
Biophysical Journal Sep 1969The age response for lethality of Chinese hamster cells to ultraviolet light shows that they are resistant in G(1), sensitive as they move into and through the S phase...
The age response for lethality of Chinese hamster cells to ultraviolet light shows that they are resistant in G(1), sensitive as they move into and through the S phase and resistant again in G(2) and mitosis. Survival curves determined at different times in the cycle reveal that mitotic cells are the most resistant fraction, much more resistant than S cells, and more resistant than either G(1) or G(2) cells. The extent to which the age response is ilfluenced by nucleic acid and protein synthesis was investigated by using inhibitors of these processes. In the presence of inhibitors of DNA or protein synthesis added to G(1) cells before exposure, cell survival neither declines to the minimum survival of S cells nor rises subsequently to the resistance of G(2) cells. If, before exposure, DNA synthesis is arrested in the middle of S, when survival is at a minimum, the subsequent rise in survival during G(2) is not prevented. However when cycloheximide is added before exposure, during the middle of S, this rise is prevented. When actinomycin D, an inhibitor of RNA synthesis is added prior to exposure the age response is affected only slightly. Postirradiation treatment of G(1) and mid-S cells with inhibitors of DNA or protein synthesis maintains survival at a level characteristic of the age of the cells.
Topics: Aging; Animals; Cell Line; Cricetinae; Culture Techniques; Cycloheximide; Dactinomycin; Mitosis; Radiation Effects; Ultraviolet Rays
PubMed: 5807224
DOI: 10.1016/S0006-3495(69)86444-1 -
Lancet (London, England) May 1990
Topics: Chemical and Drug Induced Liver Injury; Child; Dactinomycin; Drug Administration Schedule; Drug Evaluation; Humans; Wilms Tumor
PubMed: 1971368
DOI: No ID Found -
Therapeutic Drug Monitoring Dec 2010To develop a method for drug dosing and pharmacokinetic (PK) sampling in children with cancer from a single indwelling central venous catheter that minimized drug...
OBJECTIVES
To develop a method for drug dosing and pharmacokinetic (PK) sampling in children with cancer from a single indwelling central venous catheter that minimized drug contamination.
METHODS
A benchtop system was designed to simulate dosing and clearing actinomycin-D (AMD) and vincristine (VCR) from central venous catheters. The authors evaluated the effects of flush volume, composition and pH, timed drug instillation, and number of blood-draw return cycles on residual drug concentrations. A proof-of-principle study was conducted in three pediatric patients with cancer with paired PK samples obtained by both central and peripheral catheters.
RESULTS
Nearly complete removal of drug from the catheter was obtained after five blood-draw return cycles consisting of 5 mL of whole blood. Residual concentration of AMD was 0.18 ± 0.02 ng/mL or 0.16% of the initial infusion concentration. VCR exhibited lower propensity for catheter adsorption than AMD with residual concentrations undetectable after three blood-draw return cycles. In patients in which the clearance procedure was used, higher drug concentrations were generally observed from centrally cleared samples at most time points, but differences relative to peripherally obtained samples were not statistically significant for either AMD or VCR. Two of three patients had higher exposure for AMD based on PK samples obtained from central catheters, whereas exposure for VCR was similar for both sampling catheters in all patients.
CONCLUSIONS
A reliable procedure to efficiently reduce AMD and VCR contamination during PK sampling has been established and is currently being used in a PK study being conducted by the Children's Oncology Group.
Topics: Antineoplastic Agents; Blood Specimen Collection; Catheterization, Central Venous; Catheters, Indwelling; Child; Dactinomycin; Drug Monitoring; Drug Therapy, Combination; Humans; Neoplasms; Pilot Projects; Vincristine
PubMed: 20962707
DOI: 10.1097/FTD.0b013e3181fa3c68 -
The Journal of Biological Chemistry Sep 1978This communication describes a novel effect of actinomycin D (AcD) in inhibiting the nuclear processing or turnover of estrogen receptors in the human breast cancer cell...
This communication describes a novel effect of actinomycin D (AcD) in inhibiting the nuclear processing or turnover of estrogen receptors in the human breast cancer cell line. MCF-7. In the absence of AcD, estradiol treatment results in rapid (5 min) hormone binding and translocation of unfilled cytoplasmic receptors (Rc) and binding of unfilled nuclear receptors (Rn). Thereafter, filled nuclear receptors (RnE) progressively deplete and, by 3 to 5 h, 70% are lost or processed. We now show that 1 to 2 micrometer AcD or chromomycin A3, both of which intercalate at G-C base-pairs on DNA, selectively and completely block RnE processing. In contrast, estrogen binding, translocation of receptor complex, and RnE accumulation in the nucleus are completely insensitive to AcD inhibition. At 1 to 2 micrometer, all other intercalators and inhibitors tested, including other inhibitors of transcription and replication, or inhibitors of translocation or of other functions, fail to prevent binding, translocation, or the nuclear processing step. AcD intranslocation, or the nuclear processing step. AcD inhibition of RnE processing is dependent on dose; at lower doses (100 nM decreasing to 1 nm), progressively greater RnE depletion occurs. AcD completely prevents RnE depletion if given together with or within 30 min after estradiol; at any time between 30 min and 5 h after estradiol, the processing of RnE is stopped instantly by addition of AcD. Because of the complexity of actinomycin action, several mechanisms can be proposed to explain its effect on nuclear ER levels.
Topics: Breast Neoplasms; Cell Nucleus; Cells, Cultured; Dactinomycin; Dose-Response Relationship, Drug; Estradiol; Receptors, Estrogen
PubMed: 681355
DOI: No ID Found -
The Journal of Biological Chemistry May 1996Mechanisms regulating the intracellular level of endogenous U6 small nuclear RNA were studied by transient transfection of ectopic U6 gene constructs into immortalized...
Mechanisms regulating the intracellular level of endogenous U6 small nuclear RNA were studied by transient transfection of ectopic U6 gene constructs into immortalized normal and malignant human cell lines. Transfection and expression of a modified U6 gene containing native promoter, capping, and termination sequences but lacking all highly conserved internal spliceosome sequences produced dose-dependent effects on endogenous U6 gene expression. At low transfection doses, no significant changes in endogenous U6 RNA levels or half-life were noted. However, as the dose of the transfected gene and its expression increased, native U6 RNA levels dramatically decreased in association with an apparent decrease in U6 RNA half-life. Down-regulation of native U6 RNA levels was transient, with recovery noted within 48-96 h in conjunction with declining expression of the ectopic gene. These modulatory effects appeared specific to endogenous U6 transcripts, because no changes were noted in 7sk, U1, U3, or 5S RNA levels or half-lives. Transfection with an unmodified U6 gene did not alter total U6 transcript levels but did produce a similar dose-dependent decrease in U6 RNA half-life. These studies suggest a hitherto unrecognized U6-specific intracellular regulatory mechanism, through which over-accumulation of U6 small nuclear RNA is prevented.
Topics: Cell Line; Dactinomycin; Humans; RNA Processing, Post-Transcriptional; RNA, Small Nuclear; Transfection; Tumor Cells, Cultured
PubMed: 8631843
DOI: 10.1074/jbc.271.18.10477 -
Infection and Immunity Apr 2007The Clostridium perfringens epsilon-toxin causes a severe, often fatal illness (enterotoxemia) characterized by cardiac, pulmonary, kidney, and brain edema. In this...
The Clostridium perfringens epsilon-toxin causes a severe, often fatal illness (enterotoxemia) characterized by cardiac, pulmonary, kidney, and brain edema. In this study, we examined the activities of two neutralizing monoclonal antibodies against the C. perfringens epsilon-toxin. Both antibodies inhibited epsilon-toxin cytotoxicity towards cultured MDCK cells and inhibited the ability of the toxin to form pores in the plasma membranes of cells, as shown by staining cells with the membrane-impermeant dye 7-aminoactinomycin D. Using an antibody competition enzyme-linked immunosorbent assay (ELISA), a peptide array, and analysis of mutant toxins, we mapped the epitope recognized by one of the neutralizing monoclonal antibodies to amino acids 134 to 145. The antibody competition ELISA and analysis of mutant toxins suggest that the second neutralizing monoclonal antibody also recognizes an epitope in close proximity to this region. The region comprised of amino acids 134 to 145 overlaps an amphipathic loop corresponding to the putative membrane insertion domain of the toxin. Identifying the epitopes recognized by these neutralizing antibodies constitutes an important first step in the development of therapeutic agents that could be used to counter the effects of the epsilon-toxin.
Topics: Animals; Antibodies, Bacterial; Antibodies, Monoclonal; Bacterial Toxins; Cell Line; Cell Membrane Permeability; Clostridium perfringens; Dactinomycin; Dogs; Enzyme-Linked Immunosorbent Assay; Epitope Mapping; Mutation; Neutralization Tests; Peptides
PubMed: 17261609
DOI: 10.1128/IAI.01643-06 -
Antimicrobial Agents and Chemotherapy Dec 1972The effect of incubation with interferon prior to the stimulation of interferon production (priming) and of sequential treatment with cycloheximide and actinomycin D...
The effect of incubation with interferon prior to the stimulation of interferon production (priming) and of sequential treatment with cycloheximide and actinomycin D (superinduction) on the interferon yield from polyinosinic-polycytidylic acid (poly I.poly C)-induced diploid human foreskin cell cultures (FS-3 strain) was studied. Suitable priming with interferon produced, on the average, about an eightfold increase over the control yield, with a greater increase noted on some occasions when the control interferon yield was very low. Under the optimal conditions carefully defined in our experiments, superinduction produced about a 100-fold increase over the average control yield, resulting in interferon yields of about 10,000 reference units from cultures containing about 10(6) cells. Combined superinduction and priming did not produce yields markedly higher than obtainable by superinduction alone. Essentially similar results were obtained in cultures of human embryonic kidney cells and in FS-3 cells stimulated with other double-stranded polynucleotide inducers. However, stimulation of cells with certain concentrations of a mixture of diethylaminoethyl-dextran and poly I.poly C altered the interferon response; the yield was considerably higher than in cells stimulated with poly I.poly C alone, but it could not be markedly increased further by superinduction.
Topics: Cell Line; Cells, Cultured; Cycloheximide; Dactinomycin; Diploidy; Humans; Interferons; Kidney; Poly I-C; Skin; Stimulation, Chemical
PubMed: 4670440
DOI: 10.1128/AAC.2.6.476 -
Proceedings of the National Academy of... Aug 1994Apoptosis and necrosis are two types of cell death with different morphologic features. We report here the isolation of a monoclonal antibody, BV2, that specifically...
Apoptosis and necrosis are two types of cell death with different morphologic features. We report here the isolation of a monoclonal antibody, BV2, that specifically recognizes cells undergoing developmental programmed cell death in different tissues of the chicken and zebra-finch embryos. The antigen recognized by BV2 monoclonal antibody is detected in vitro in primary chicken embryonic fibroblasts induced to die by actinomycin D, as well as fibroblasts induced to die by chemical anoxia. The expression of this specific antigen during necrosis appears to require active protein synthesis. These findings provide evidence that cells from different embryonic tissues undergoing programmed cell death during vertebrate development express similar antigens and indicate that apoptosis and necrosis may share similar biochemical features.
Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Apoptosis; Birds; Cells, Cultured; Chick Embryo; Dactinomycin; Fluorescent Antibody Technique; In Vitro Techniques; Necrosis
PubMed: 8078937
DOI: 10.1073/pnas.91.18.8641