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PloS One 2014Demecolcine (DEM) treatment of oocytes induces formation of a membrane protrusion containing a mass of condensed maternal chromosomes, which can be removed with minimal...
Demecolcine (DEM) treatment of oocytes induces formation of a membrane protrusion containing a mass of condensed maternal chromosomes, which can be removed with minimal damage prior to somatic cell nuclear transfer (SCNT). However, the effect of this method on the distribution of maturation-promoting factor (MPF) in porcine oocytes has not been reported. Here, the level of MPF and the distribution of cyclin B1 were assessed in porcine oocytes following DEM treatment. In addition, the efficiencies of DEM-assisted and mechanical enucleation were compared, as were the development (in vitro and in vivo) of these oocytes following SCNT. MPF was uniformly distributed in oocytes that had been treated with 0.4 μg/ml DEM for 1 h. Immunofluorescence microscopy showed that in untreated oocytes, cyclin B1, the regulatory subunit of MPF, accumulated around the spindle, and was lowly detected in the cytoplasm. DEM treatment disrupted spindle microtubules, induced chromosome condensation, and reduced the level of cyclin B1 in the nuclear region. Cyclin B1 was uniformly distributed in DEM-treated oocytes and the level of MPF was increased. The potential of embryos generated from DEM-treated oocytes to develop in vivo was significantly greater than that of embryos generated from mechanically enucleated oocytes. This is the first study to report the effects of DEM-assisted enucleation of porcine oocytes on the distribution of cyclin B1. MPF in mature oocytes is important for the development of reconstructed embryos and for efficient SCNT.
Topics: Animals; Cyclin B1; Cytoplasm; Demecolcine; Ear; Embryo Transfer; Fibroblasts; Gene Expression Regulation, Developmental; Microscopy, Fluorescence; Microtubules; Nuclear Transfer Techniques; Oocytes; Spindle Apparatus; Swine; Swine, Miniature; Tubulin Modulators
PubMed: 24626152
DOI: 10.1371/journal.pone.0091483 -
Infection and Immunity Jul 1997The mechanisms which enable entry into cultured human epithelial cells by Klebsiella pneumoniae were compared with those of Salmonella typhi Ty2. K. pneumoniae 3091,... (Comparative Study)
Comparative Study
The mechanisms which enable entry into cultured human epithelial cells by Klebsiella pneumoniae were compared with those of Salmonella typhi Ty2. K. pneumoniae 3091, isolated from a urine sample of a patient with a urinary tract infection, invaded human epithelial cells from the bladder and ileocecum and persisted for days in vitro. Electron microscopic studies demonstrated that K. pneumoniae was always contained in endosomes. The internalization mechanism(s) triggered by K. pneumoniae was studied by invasion assays conducted with different inhibitors that act on prokaryotic and eukaryotic cell structures and processes. Chloramphenicol inhibition of bacterial uptake revealed that bacterial de novo protein synthesis was essential for efficient invasion by K. pneumoniae and S. typhi. Interference with receptor-mediated endocytosis by g-strophanthin or monodansylcadaverine and inhibition of endosome acidification by monensin reduced the number of viable intracellular K. pneumoniae cells, but not S. typhi cells. The depolymerization of microfilaments by cytochalasin D inhibited the uptake of both bacteria. Microtubule depolymerization caused by colchicine, demecolcine, or nocodazole and the stabilization of microtubules with taxol reduced only the invasion ability of K. pneumoniae. S. typhi invasion was unaffected by microtubule depolymerization or stabilization. These data suggest that the internalization mechanism triggered by K. pneumoniae 3091 is strikingly different from the solely microfilament-dependent invasion mechanism exhibited by many of the well-studied enteric bacteria, such as enteroinvasive Escherichia coli, Salmonella, Shigella, and Yersinia strains.
Topics: Bacterial Proteins; Cadaverine; Cells, Cultured; Chloramphenicol; Colchicine; Cytochalasin D; Demecolcine; Endocytosis; Epithelial Cells; Epithelium; Humans; Ionophores; Klebsiella pneumoniae; Microtubules; Monensin; Nocodazole; Nucleic Acid Synthesis Inhibitors; Ouabain; Paclitaxel; Protein Synthesis Inhibitors; Salmonella typhi; Urinary Tract; Urinary Tract Infections
PubMed: 9199471
DOI: 10.1128/iai.65.7.2950-2958.1997 -
FEBS Letters Oct 1998Microsomal preparations from immature seeds of Colchicum autumnale L. catalyse the ring expansion reaction of O-methylandrocymbine to demecolcine in the presence of...
Microsomal preparations from immature seeds of Colchicum autumnale L. catalyse the ring expansion reaction of O-methylandrocymbine to demecolcine in the presence of NADPH and O2. In addition evidence is given for further transformation of demecolcine to colchicine in the presence of acetyl-CoA and NADPH.
Topics: Alkaloids; Carbon Monoxide; Chromatography, Thin Layer; Colchicine; Colchicum; Cytochrome P-450 Enzyme System; Demecolcine; Hydrogen-Ion Concentration; Light; Microsomes; NADP; Nitrogen; Oxygen; Plants, Medicinal; Seeds; Solvents
PubMed: 9821969
DOI: 10.1016/s0014-5793(98)01282-4 -
Journal of Visualized Experiments : JoVE Jan 2017Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to be responsible...
Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to be responsible for carcinogenesis in such tissues. Although proliferative polyploid cells are requisite for analyzing chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is rather challenging. Induction of tetraploidy by chemical agents usually results in a mixture of diploid and tetraploid populations, and most studies employed fluorescence-activated cell sorting or cloning by limiting dilution to separate tetraploid from diploid cells. However, these procedures are time-consuming and laborious. The present report describes a relatively simple protocol to induce proliferative tetraploid cells from normal human fibroblasts with minimum contamination by diploid cells. Briefly, the protocol is comprised of the following steps: arresting cells in mitosis by demecolcine (DC), collecting mitotic cells after shaking off, incubating collected cells with DC for an additional 3 days, and incubating cells in drug-free medium (They resume proliferation as tetraploid cells within several days). Depending on cell type, the collection of mitotic cells by shaking off might be omitted. This protocol provides a simple and feasible method to establish proliferative tetraploid cells from normal human fibroblasts. Tetraploid cells established by this method could be a useful model for studying chromosome instability and the oncogenic potential of polyploid human cells.
Topics: Cell Line; Cell Proliferation; Chromosomal Instability; DNA; Demecolcine; Female; Fibroblasts; Flow Cytometry; Fluorescent Dyes; Humans; Karyotyping; Mitosis; Tetraploidy
PubMed: 28117785
DOI: 10.3791/55028 -
Proceedings of the National Academy of... Nov 1982To determine the relationship between thin filaments, Z-bands, microtubules, intermediate filaments (IFs), T-tubules, and sarcoplasmic reticulum (SR) during...
To determine the relationship between thin filaments, Z-bands, microtubules, intermediate filaments (IFs), T-tubules, and sarcoplasmic reticulum (SR) during myofibrillogenesis, myotubes were selectively depleted of their myofibrils with 12-tetradecanoylphorbol 13-acetate (TPA) and then were allowed to regenerate in (i) normal medium, (ii) taxol, and (iii) Colcemid. Myofibrils assembled in normal medium formed typical A-, I-, Z-, M-, and H-bands and associated IFs, T-tubules, and SR. Myofibrils assembled in taxol formed "A-bands" of aligned thick filaments interdigitating with long microtubules and "I-bands" consisting only of microtubules. These unprecedented sarcomeres lacked thin filaments, Z-bands, and associated IFs and SR. "Solitary A-bands," consisting exclusively of laterally aligned bipolar thick filaments 1.6 microM in length without either thin filaments or microtubules, were observed. Myofibrils assembled in Colcemid formed all myofibrillar components in the absence of microtubules but these did not achieve rigorous lateral alignment. Colcemid and taxol induced the formation of patchy Z-bands that invariably served as insertion sites for thin filaments, irrespective of the presence or absence of adjacent thick filaments. Z-bands may function as actin-organizing centers for each sarcomere.
Topics: Actins; Alkaloids; Animals; Cell Differentiation; Cells, Cultured; Chick Embryo; Cytoskeleton; Demecolcine; Microtubules; Myofibrils; Myosins; Paclitaxel; Protein Binding
PubMed: 6128733
DOI: 10.1073/pnas.79.21.6556 -
Bioinformation 2020It is known that beta-catenin is associated with fibromatosis, sarcoma and mesenchymal tumor. Therefore, it is of interest to design an effective inhibtitor to the...
It is known that beta-catenin is associated with fibromatosis, sarcoma and mesenchymal tumor. Therefore, it is of interest to design an effective inhibtitor to the target protein beta-catenin. In this study, we report the molecular docking analysis of alkaloid compounds (aristolochicacid, cryptopleurine, demecolcine, fagaronine and thalicarpine) with beta-catenin for further consideration towards the design and development of potential inhintors for the treatmnet of colon cancer.
PubMed: 32308271
DOI: 10.6026/97320630016283 -
Cell Proliferation Apr 2011DNA content of diploid H1 (ES) cells (2H1 cells) has been shown to be stable in long-term culture; however, tetraploid and octaploid H1 (ES) cells (4H1 and 8H1 cells,...
OBJECTIVES
DNA content of diploid H1 (ES) cells (2H1 cells) has been shown to be stable in long-term culture; however, tetraploid and octaploid H1 (ES) cells (4H1 and 8H1 cells, respectively) were DNA-unstable. Pentaploid H1 (ES) cells (5H1 cells) established recently have been found to be DNA-stable; how, then is cell DNA stability determined? To discuss ploidy stability, decaploid H1 (ES) cells (10H1 cells) were established from 5H1 cells and examined for DNA stability.
MATERIALS AND METHODS
5H1 cells were polyploidized using demecolcine (DC) and 10H1 cells were obtained by one-cell cloning.
RESULTS
Number of chromosomes of 10H1 cells was 180 and durations of their G(1), S, and G(2)/M phases were 3, 7 and 6 h respectively. Volume of 10H1 cells was double that of 5H1 cells and morphology of 10H1 cells was flagstone-like in shape. 10H1 cells exhibited alkaline phosphatase activity and their DNA content decayed in 91 days of culture. 10H1 cells injected into mouse abdomen formed solid tumours that contained several kinds of differentiated cells with lower DNA content, suggesting that 10H1 cells were pluripotent and DNA-unstable. Loss of DNA stability was explained using a hypothesis concerning DNA structure of polyploid cells as DNA reconstructed through ploidy doubling was arranged in mirror symmetry in a new configuration.
CONCLUSION
In the pentaploid-decaploid transition of H1 cells, cell cycle parameters and pluripotency were retained, but morphology and DNA stability were altered.
Topics: Animals; Antineoplastic Agents, Phytogenic; Cell Division; Chromosomal Instability; DNA; Demecolcine; Embryonic Stem Cells; G1 Phase; G2 Phase; Mice; Polyploidy; S Phase
PubMed: 21401752
DOI: 10.1111/j.1365-2184.2011.00734.x -
The Journal of Cell Biology Sep 2011The correlation between stress-induced nucleolar disruption and abrogation of p53 degradation is evident after a wide variety of cellular stresses. This link may be...
The correlation between stress-induced nucleolar disruption and abrogation of p53 degradation is evident after a wide variety of cellular stresses. This link may be caused by steps in p53 regulation occurring in nucleoli, as suggested by some biochemical evidence. Alternatively, nucleolar disruption also causes redistribution of nucleolar proteins, potentially altering their interactions with p53 and/or MDM2. This raises the fundamental question of whether the nucleolus controls p53 directly, i.e., as a site where p53 regulatory processes occur, or indirectly, i.e., by determining the cellular localization of p53/MDM2-interacting factors. In this work, transport experiments based on heterokaryons, photobleaching, and micronucleation demonstrate that p53 regulatory events are directly regulated by nucleoli and are dependent on intact nucleolar structure and function. Subcellular fractionation and nucleolar isolation revealed a distribution of ubiquitylated p53 that supports these findings. In addition, our results indicate that p53 is exported by two pathways: one stress sensitive and one stress insensitive, the latter being regulated by activities present in the nucleolus.
Topics: 3T3 Cells; Active Transport, Cell Nucleus; Animals; Cell Fusion; Cell Line, Tumor; Cell Nucleolus; Cell Nucleus; Cyclin-Dependent Kinase Inhibitor p21; Cycloheximide; Cytoplasm; DNA Damage; Demecolcine; Fatty Acids, Unsaturated; Green Fluorescent Proteins; Humans; Intranuclear Space; Leupeptins; Mice; Models, Biological; Nucleolus Organizer Region; Photobleaching; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-mdm2; Recombinant Fusion Proteins; Ribosomal Proteins; Subcellular Fractions; Tumor Suppressor Protein p53; Ubiquitin; Ubiquitination
PubMed: 21893597
DOI: 10.1083/jcb.201105143 -
Proceedings of the National Academy of... Feb 1979The phosphorylation of the subunit proteins of intermediate (10-nm) filaments has been investigated in chicken muscle and nonmuscle cells by using a two-dimensional gel...
The phosphorylation of the subunit proteins of intermediate (10-nm) filaments has been investigated in chicken muscle and nonmuscle cells by using a two-dimensional gel electrophoresis system. Desmin, the 50,000-dalton subunit protein of the intermediate filaments of muscle, had previously been shown to exist as two major isoelectric variants-alpha and beta-in smooth, skeletal, and cardiac chicken muscle. Incubation of skeletal and smooth muscle tissue with (32)PO(4) (3-) reveals that the acidic variant, alpha-desmin, and three other desmin variants are phosphorylated in vivo and in vitro. Under the same conditions, minor components of alpha- and beta-tropomyosin from skeletal muscle, but not smooth muscle, are also phosphorylated. Both the phosphorylated desmin variants and the nonphosphorylated beta-desmin variant remain insoluble under conditions that solubilize actin and myosin filaments, but leave Z-discs and intermediate filaments insoluble. Primary cultures of embryonic chicken muscle labeled with (32)PO(4) (3-) possess, in addition to the desmin variants described above, a major nonphosphorylated and multiple phosphorylated variants of the 52,000-dalton, fibroblast-type intermediate filament protein (IFP). Filamentous cytoskeletons, prepared from primary myogenic cultures by Triton X-100 extraction, contain actin and all of the phosphorylated and nonphosphorylated variants of both desmin and the IFP. Similarly, these proteins are the major components of the caps of aggregated 10-nm filaments isolated from the same cell cultures previously exposed to Colcemid. These results demonstrate that a nonphosphorylated and several phosphorylated variants of desmin and IFP are present in assembled structures in muscle and nonmuscle cells.
Topics: Animals; Cells, Cultured; Chickens; Contractile Proteins; Demecolcine; Isoelectric Point; Muscle Proteins; Muscle, Smooth; Muscles; Phosphorylation
PubMed: 284403
DOI: 10.1073/pnas.76.2.819 -
Cell Structure and Function Dec 1994Demecolcine (Colcemid), an inhibitor of spindle fiber formation in M phase, induced apoptosis in V79 cells. At a concentration of 0.01 microgram/ml demecolcine, V79...
Demecolcine (Colcemid), an inhibitor of spindle fiber formation in M phase, induced apoptosis in V79 cells. At a concentration of 0.01 microgram/ml demecolcine, V79 cells proliferated exponentially as well as controls, although temporal M phase accumulation occurred 6 h after the addition of demecolcine. At 0.1 microgram/ml, the cells became hyperploid after remaining in the M phase for some time. Apoptosis occurred in V79 cells exposed to demecolcine at a concentration of 0.03 microgram/ml. Apoptosis was defined as the appearance of a sub-G1 peak in DNA histograms and a ladder pattern of fragmented DNA in gelelectrophoresis.
Topics: Animals; Apoptosis; Cell Cycle; Cell Line; Cricetinae; Cricetulus; DNA Damage; Demecolcine; Dose-Response Relationship, Drug; Lung; Ploidies
PubMed: 7720099
DOI: 10.1247/csf.19.391