-
Journal of Virology Jan 1971The in vitro product of mouse leukemia virus deoxyribonucleic acid (DNA) polymerase can be separated into two fractions by sedimentation in sucrose gradients. These two...
The in vitro product of mouse leukemia virus deoxyribonucleic acid (DNA) polymerase can be separated into two fractions by sedimentation in sucrose gradients. These two fractions were analyzed for their content of single-stranded DNA, double-stranded DNA, and DNA-ribonucleic acid (RNA) hybrid by (i) digestion with enzymes of known specificity and (ii) equilibrium centrifugation in Cs(2)SO(4) gradients. The major fraction early in the reaction contained equal amounts of single-stranded DNA and DNA-RNA hybrid and little double-stranded DNA. The major fraction after extensive synthesis contained equal amounts of single-and double-stranded DNA and little hybrid. In the presence of actinomycin D, the predominant product was single-stranded DNA. To account for these various forms of DNA, we postulate the following model: the first DNA synthesis occurs in a replicative complex containing growing DNA molecules attached to an RNA molecule. Each DNA molecule is displaced as single-stranded DNA by the synthesis of the following DNA strand, and the single-stranded DNA is copied to form double-stranded DNA either before or after release of the single strand from the RNA. Actinomycin blocks this conversion of single-to double-stranded DNA.
Topics: Carbon Isotopes; Centrifugation, Density Gradient; Cesium; DNA Replication; Dactinomycin
PubMed: 5543423
DOI: 10.1128/JVI.7.1.106-111.1971 -
The Journal of Antibiotics Dec 1986Streptomyces sp. X-14873 (ATCC 31679) has been found to produce a number of secondary metabolites. Three have been identified as the novel actinomycins, X-14873B, C and...
Streptomyces sp. X-14873 (ATCC 31679) has been found to produce a number of secondary metabolites. Three have been identified as the novel actinomycins, X-14873B, C and D, each of which contains both proline and 3-hydroxy-5-methylproline. Potentially, the most important microbial product from this fermentation is the novel polyether antibiotic X-14873A which differs from lysocellin only in the substituents at carbons C-4 and C-5 in the tetrahydropyranyl ring. The methyl group and proton in lysocellin are replaced by an ethyl and hydroxyl group, respectively in X-14873A. In addition, two other novel polyethers, X-14873H and G, were isolated and shown to differ from 1 in lacking a carboxyl group and in the case of 3, possessing an ether bridge across the terminal tetrahydrofuranyl ring system.
Topics: Animals; Anti-Bacterial Agents; Chemical Phenomena; Chemistry; Crystallization; Dactinomycin; Mice; Molecular Conformation; Streptomyces; Thallium
PubMed: 2434456
DOI: 10.7164/antibiotics.39.1704 -
Clinical and Experimental Immunology Jan 1979Dactinomycin treatment a of group of (NZB X NZW)F1 hybrid female mice was delayed until the age of 6-6 1/2 months, by which time the immune complex disease was well...
Dactinomycin treatment a of group of (NZB X NZW)F1 hybrid female mice was delayed until the age of 6-6 1/2 months, by which time the immune complex disease was well established. Three animals of the original twenty-eight had already died, ten had heavy proteinuria and a few were oedematous. The dactinomycin dose was 3.5 microgram per day, which was suspended when significant weight loss occurred. Twelve of the thirteen experimental mice were alive at 12 months of age, eleven at 15 months, but only eight by 20 months, whereas all twelve control animals had died by the age of 11 months. These results and the supporting data on body weight and renal function indicate that dactinomycin can at least arrest the disease process and may improve it. The mechanism is not known, but it may be the result of a reduced availability of DNA or an alteration in its properties following combination with dactinomycin.
Topics: Animals; Body Weight; Dactinomycin; Female; Lupus Erythematosus, Systemic; Mice; Mice, Inbred Strains; Time Factors
PubMed: 428145
DOI: No ID Found -
European Journal of Biochemistry Feb 1988When Trypanosoma cruzi epimastigotes are exposed to temperatures of 37-41 degrees C there is a drastic decline in total protein synthesis. Analysis of the proteins...
When Trypanosoma cruzi epimastigotes are exposed to temperatures of 37-41 degrees C there is a drastic decline in total protein synthesis. Analysis of the proteins synthesized at 41 degrees C by one-dimensional gel electrophoresis showed three major bands of Mr 83,000, 70,000 and 60,000. A similar pattern of heat-shock proteins was found in two different strains of T. cruzi (Tulahuen and GM strains) and in exponentially growing or in stationary epimastigotes. Actinomycin D prevented the appearance of these polypeptide bands, suggesting that the heat-shock proteins in T. cruzi epimastigotes are induced at the level of transcription. Analysis of the proteins synthesized by metacyclic forms at different temperatures suggests that heat-shock proteins in these cells are already synthesized at 27 degrees C. Elevation of temperature above 37 degrees C blocks the synthesis of most proteins in metacyclic forms except for major bands of Mr 83,000, 70,000, 60,000 and 55,000. More detailed analyses by high-resolution two-dimensional gel electrophoresis of the proteins synthesized at 27 degrees C or 37 degrees C by epimastigotes indicates that the heat-shock protein pattern is more complex than that demonstrated by one dimension, and at least ten new polypeptides are identified in two-dimensional gels. A similar analysis of metacyclic forms shows that most if not all the proteins present at 39 degrees C are also present at 27 degrees C. This result led us to the suggestion that the differentiation of T. cruzi to metacyclic forms involves the induction of heat-shock proteins, which prepares the parasite to infect the mammalian host.
Topics: Animals; Dactinomycin; Electrophoresis, Polyacrylamide Gel; Heat-Shock Proteins; Hot Temperature; Species Specificity; Trypanosoma cruzi
PubMed: 3278903
DOI: 10.1111/j.1432-1033.1988.tb13863.x -
Translational Stroke Research Aug 2016Cell-based therapies including bone-marrow derived mononuclear cells (MNCs) are now widely being studied because of their pleotropic effects and promising results to...
Various Cell Populations Within the Mononuclear Fraction of Bone Marrow Contribute to the Beneficial Effects of Autologous Bone Marrow Cell Therapy in a Rodent Stroke Model.
Cell-based therapies including bone-marrow derived mononuclear cells (MNCs) are now widely being studied because of their pleotropic effects and promising results to improve recovery after stroke in animal models. Unlike other types of cell therapies, MNCs is a mixture of lymphoid, myeloid, erythroid, and stem cell populations. Which cell population(s) accounts for the beneficial effects of MNCs in stroke recovery is unclear. In this paper, we employed a mouse stroke model with middle cerebral artery occlusion (MCAo), and used positively and negatively sorted autologous MNCs by MACs to determine which fractions of the MNCs contribute to their beneficial effects. We evaluated the benefits of neurofunctional recovery produced by individual cell lineages within MNCs in a long-term observation study up to 28 days after stroke. Mortality and modulation of inflammation were also compared among different sub-populations. We further studied the impact of neurotoxicity posed by activated microglia in the presence of different cell lineages within MNCs. We concluded that myeloid cell lineage and stem cell/progenitors appeared to be important components within MNCs that contribute to improved outcomes after stroke.
Topics: Animals; Antigens, CD; Atrophy; Bone Marrow Cells; Bone Marrow Transplantation; Cells, Cultured; Coculture Techniques; Dactinomycin; Disease Models, Animal; Female; Inflammation; Leukocytes, Mononuclear; Locomotion; Male; Mice; Mice, Inbred C57BL; Microglia; Myeloid Cells; Pregnancy; Psychomotor Performance; Recovery of Function; Stroke
PubMed: 26997513
DOI: 10.1007/s12975-016-0462-x -
PloS One 2016Organization of the plasma membrane into specialized substructures in different blood lineages facilitates important biological functions including proper localization...
Organization of the plasma membrane into specialized substructures in different blood lineages facilitates important biological functions including proper localization of receptors at the plasma membrane as well as the initiation of crucial intracellular signaling cascades. The eukaryotic plasma membrane is a lipid bilayer that consists of asymmetrically distributed phospholipids. This asymmetry is actively maintained by membrane-embedded lipid transporters, but there is only limited data available about the molecular identity of the predominantly active transporters and their substrate specificity in different leukocyte subsets. We demonstrate here that the P4-type ATPase ATP11C mediates significant flippase activity in all murine leukocyte subsets. Loss of ATP11C resulted in a defective internalization of phosphatidylserine (PS) and phosphatidylethanolamine (PE) in comparison to control cells. The diminished flippase activity caused increased PS exposure on 7-aminoactinomycin D- (7-AAD-) viable pro-B cells freshly isolated from the bone marrow of ATP11C-deficient mice, which was corrected upon a 2-hour resting period in vitro. Despite the impaired flippase activity in all immune cell subsets, the only other blood cell type with an accumulation of PS on the surface were viable 7-AAD- developing T cells but this did not result in any discernable effect on their development in the thymus. These findings show that all leukocyte lineages exhibit flippase activity, and identify ATP11C as an aminophospholipid translocase in immune cells.
Topics: Adenosine Triphosphatases; Animals; B-Lymphocytes; Biological Transport, Active; Cells, Cultured; Dactinomycin; Leukocytes; Mice; Mice, Inbred C57BL; Phosphatidylethanolamines; Phosphatidylserines; Phospholipid Transfer Proteins; T-Lymphocytes
PubMed: 26799398
DOI: 10.1371/journal.pone.0146774 -
Journal of Veterinary Internal Medicine 1994Fifty dogs with advanced malignancies were treated with actinomycin D at doses ranging from 0.5 to 1.1 mg/m2 every 3 weeks. The greatest number of responses was noted in...
Fifty dogs with advanced malignancies were treated with actinomycin D at doses ranging from 0.5 to 1.1 mg/m2 every 3 weeks. The greatest number of responses was noted in dogs with lymphoma, including dogs that had received prior chemotherapy. Other responding tumor types included anal sac adenocarcinoma, perianal adenocarcinoma, squamous cell carcinoma, thyroid carcinoma, and transitional cell carcinoma. The median time to maximum response for dogs with lymphoma was 7 days, with a median duration of 42 days. Gastrointestinal toxicity was the most frequently observed side effect. A dose of 0.6 to 0.7 mg/m2 appears to be appropriate for treating various malignancies in dogs.
Topics: Animals; Carcinoma; Dactinomycin; Dog Diseases; Dogs; Female; Lymphoma; Male; Neoplasms; Treatment Outcome
PubMed: 8064663
DOI: 10.1111/j.1939-1676.1994.tb03224.x -
Journal of Bacteriology Aug 1970The specific activity of alpha-mannosidase (EC 3.2.1.24) has been found to increase more than a thousandfold during development of the cellular slime mold, Dictyostelium...
The specific activity of alpha-mannosidase (EC 3.2.1.24) has been found to increase more than a thousandfold during development of the cellular slime mold, Dictyostelium discoideum. The enzyme accumulates in both spore and stalk cells. Studies with preferential inhibitors of macromolecular synthesis indicate that accumulation of alpha-mannosidase requires concomitant protein synthesis and prior ribonucleic acid synthesis. Control of the period of synthesis by the overall developmental program is demonstrated in two temporally deranged morphological mutants. alpha-Mannosidase is found in lysosomes of D. discoideum in association with other acid hydrolases which may be involved in metabolism of extracellular polysaccharide.
Topics: Chromatography, Thin Layer; Dactinomycin; Electrophoresis, Disc; Hydrogen-Ion Concentration; Hydrolases; Mutation; Myxomycetes; RNA
PubMed: 5464526
DOI: 10.1128/jb.103.2.375-381.1970 -
Journal of Bacteriology Sep 1988Recent studies have suggested that the onset of synthesis of actinomycin D in Streptomyces parvulus is due to a release from L-glutamate catabolic repression. In the...
Metabolic regulation in Streptomyces parvulus during actinomycin D synthesis, studied with 13C- and 15N-labeled precursors by 13C and 15N nuclear magnetic resonance spectroscopy and by gas chromatography-mass spectrometry.
Recent studies have suggested that the onset of synthesis of actinomycin D in Streptomyces parvulus is due to a release from L-glutamate catabolic repression. In the present investigation we showed that S. parvulus has the capacity to maintain high levels of intracellular glutamate during the synthesis of actinomycin D. The results seem contradictory, since actinomycin D synthesis cannot start before a release from L-glutamate catabolic repression, but a relatively high intracellular pool of glutamate is needed for the synthesis of actinomycin D. Utilizing different labeled precursors, D-[U-13C]fructose and 13C- and 15N-labeled L-glutamate, and nuclear magnetic resonance techniques, we showed that carbon atoms of an intracellular glutamate pool of S. parvulus were not derived biosynthetically from the culture medium glutamate source but rather from D-fructose catabolism. A new intracellular pyrimidine derivative whose nitrogen and carbon skeletons were derived from exogenous L-glutamate was obtained as the main glutamate metabolite. Another new pyrimidine derivative that had a significantly reduced intracellular mobility and that was derived from D-fructose catabolism was identified in the cell extracts of S. parvulus during actinomycin D synthesis. These pyrimidine derivatives may serve as a nitrogen store for actinomycin D synthesis. In the present study, the N-trimethyl group of a choline derivative was observed by 13C nuclear magnetic resonance spectroscopy in growing S. parvulus cells. The choline group, as well as the N-methyl groups of sarcosine, N-methyl-valine, and the methyl groups of an actinomycin D chromophore, arose from D-fructose catabolism. The 13C enrichments found in the peptide moieties of actinomycin D were in accordance with a mechanism of actinomycin D synthesis from L-glutamate and D-fructose.
Topics: Chemical Phenomena; Chemistry; Dactinomycin; Fructose; Gas Chromatography-Mass Spectrometry; Glutamates; Magnetic Resonance Spectroscopy; Streptomyces
PubMed: 3410824
DOI: 10.1128/jb.170.9.4055-4064.1988 -
Cytometry Jan 1999Techniques to measure apoptosis are used to study a wide spectrum of conditions, from acquired immune deficiency syndrome (AIDS) to cancer to autoimmune diseases.... (Comparative Study)
Comparative Study
BACKGROUND
Techniques to measure apoptosis are used to study a wide spectrum of conditions, from acquired immune deficiency syndrome (AIDS) to cancer to autoimmune diseases. Therefore, a critical comparison of common assays for apoptosis is warranted.
METHODS
The kinetics of apoptosis induction in dexamethasone (DEX)-exposed thymocytes was examined after 2, 4, 8, 12, 26-28, and 48-50 h of culture. An additional aim was to ascertain whether a similar thymic atrophy-inducing hormone, diethylstilbestrol (DES), also directly induces thymocyte apoptosis. Apoptosis was evaluated by flow cytometric examination of cells stained with propidium iodide (PI), 7-aminoactinomycin D (7-AAD), or fluorescein isothiocyante (FITC)-annexin; by forward-and side-scatter (FS, SS) analysis, cell-size analyzer; and through cytopathologic examination.
RESULTS
After 4 h of DEX exposure, apoptosis was evident by 7-AAD, annexin, and cytopathological assays, but no cells with sub-diploid DNA content were evident by PI analysis. Maximal apoptosis was evident by all the above flow cytometric techniques at 12 h after DEX exposure. The 7-AAD and annexin assays, which allow discrimination between early apoptosis and late apoptosis/necrosis, were comparable and identified similar apoptotic populations. Appearance of a FSlow/SSincreased population was evident only after 12 h of DEX exposure. Apoptosis could not be detected by any of the above assays in thymocytes exposed to various doses of DES.
CONCLUSION
Two of the six assays, 7-AAD and annexin, were similar in detecting apoptosis at an early kinetic time point. Results of both assays were comparable at all time points studied. Our studies imply that DEX and DES induce thymic atrophy, in vivo, by different mechanisms.
Topics: Animals; Annexin A5; Apoptosis; Carcinogens; Cells, Cultured; DNA; Dactinomycin; Dexamethasone; Diethylstilbestrol; Flow Cytometry; Fluorescent Dyes; Glucocorticoids; Kinetics; Male; Mice; Mice, Inbred C57BL; Thymus Gland
PubMed: 10554184
DOI: 10.1002/(sici)1097-0320(19990101)35:1<80::aid-cyto11>3.3.co;2-#