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  • 1
    ISSN: 1615-6102
    Keywords: Physarum polycephalum ; DAPI ; Fluorescence microscopy ; Centrosome ; Comigration ; Centrosome migration ; Cell-nuclear migration ; Actin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The mechanism of cell-nuclear migration during the amoebo-flagellate transformation inPhysarum polycephalum was examined by fluorescence microscopy after staining with a tubulinspecific antibody, rhodamine-conjugated phalloidin and 4′,6-diamidino-2-phenylindole (DAPI). While the round amoeba cells changed to comma-shaped swarm cells within 20min after suspension in buffer, the cell nuclei moved from the central region of each cell to the periphery, each forming a sharp projection in the direction of movement. A centrosome also migrated from the center of the cell to the cell periphery. Since the centrosome was in close contact with the tip that protruded from the cell nucleus throughout the cellnuclear migration, the migration of the cell nucleus and the centrosome could be recognized as comigration. Then the flagella began to elongate from the centrosome and the cells became slender and polarized, adopting the so-called “comma-shape”. On the basis of these observations, the transformation process was classified into three steps: cell-nuclear migration, flagella formation and swarm maturation. The comigration of the cell nucleus and the centrosome was not inhibited by the anti-microtubule drug nocodazole (4 μM) but it was inhibited by the anti-microfilament drug cytochalasin A (4 μM), suggesting that the force of migration is generated by microfilaments. To investigate the role of the centrosome in this comigration in detail, we identified two aberrant strains, defective in swimming ability, from among various laboratory strains. The two strains, TM 4 and J, were found to have defects in cell-nuclear migration. Strain TM 4 had two types of irregular swarm cells: in one, only a part of the cell nucleus projected a thin filamentous structure; and in the other, no cell-nuclear migration occurred. Strain J had two centrosomes per cell and such swarm cells exhibited an attempt of cell-nuclear migration at two sites which corresponded to the position of the centrosome. The characteristics of these two strains indicate that the centrosome is essential for cell-nuclear migration. Our observations suggest that the cell-nuclear migration is mediated by actin-generated forces that act on the centrosome rather than on the cell nucleus itself.
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  • 2
    ISSN: 1615-6102
    Keywords: Mitochondrial nuclei ; Mitochondrial division ; Mitochondrial replicon cluster ; Physarum polycephalum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We investigated the manner of mitochondrial DNA (mtDNA) replication and distribution during the culture ofPhysarum polycephalum amoebae cells by microphotometry, anti-BrdU immunofluorescence microscopy, and quantitative hybridization analysis. In amoebae cells ofP. polycephalum, the number of mitochondria per cell and the shape of both mitochondria and mitochondrial nuclei (mt-nuclei) noticeably changed over the culture period. At the time of transfer, about 27 short ellipsoidal shaped mitochondria, which each contained a small amount of DNA, were observed in each cell. The number of mitochondria per cell decreased gradually, while the amount of mtDNA in an mt-nucleus and the length of mt-nuclei increased gradually. Midway through the middle logarithmic growth phase, the number of mitochondria per cell reached a minimum (about 10 mitochondria per cell), but most mtnuclei assumed an elongated shape and contained a large amount of mtDNA. During the late log- and stationary-growth phase, the number of mitochondria per cell increased gradually, while the amount of DNA in an mt-nucleus and mt-nuclei length decreased gradually. Upon completion of the stationary phase, the number and condition of mitochondria within cells returned to that first observed at the time of transfer. The total amount of mtDNA in a cell increased about 1.6-fold the first day, decreased immediately, then maintained a constant level ranging from 130 to 160 T. Except for the fact that mtDNA synthesis began earlier than synthesis of cell nuclei, the rate of increase in mtDNA paralleled that of cell-nuclear DNA throughout the culture. These results indicate that mtDNA is continuously replicated in pace with cell proliferation and the rate of mitochondrial division varies during culture; this mitochondrial division does not synchronize with either mtDNA replication or cell division. Furthermore, we observed the spatial distribution of DNA replication sites along mt-nuclei. Replication began at several sites scattered along an mt-nucleus, and the number of replication sites increased as the length of mt-nuclei increased. These results indicate that mtDNA replication progresses in adjacent replicons, which are collectively termed a mitochondrial replicon cluster.
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  • 3
    ISSN: 1615-6102
    Keywords: Chlamydomonas reinhardtii ; DAPI ; Basal bodies ; Absence of DNA ; Fluorimetry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A search was made for DNA in both the basal bodies (BBs) in situ and BBs isolated from cells ofChlamydomonas reinhardtii by high-resolution epifluorescence microscopy after staining with 4′-6-diamidino-2-phenylindole (DAPI), by fluorimetry using a video-intensified microscope photon-counting system (VIMPICS) and by immunofluorescence microscopy after staining with a monoclonal tubulin-specific antibody. The flagella and intracellular microtubules radiate from the BBs. The BBs in young vegetative cells, gametes and young zygotes do not emit fluorescence after staining with DAPI but the spherical cell nucleus, the ovoid chloroplast nuclei and the tiny mitochondrial nuclei emit bright, blue-white fluorescence. Thus, it appears that BBs do not contain larger amounts of DNA than do the other organelles. To avoid the halation effects of fluorescence from the cell debris and cytoplasm and to measure carefully any extremely low levels of DNA that might be present in the organelles, a complex, composed of two flagella, a pair of BBs and the cell nucleus, was isolated from the gametes by treatment with autolysin and 0.1% Triton X-100. After staining with DAPI, the BBs of such complexes exhibit faint fluorescence while the cell nucleus emits strong fluorescence. The point and total intensities of the fluorescence emitted from each portion of the complex were measured with the VIMPICS. When the fluorescence intensity “T” of T 4 phage is taken as a standard, the fluorescence intensities of the flagella, the pair of BBs, the cell nucleus and the nucleus ofEscherichia coli are respectively 0.2 T, 0.40 T, 1452.2 T and 20.4 T. The slight fluorescence emitted from the BB seems to be due to the halation of the fluorescence emitted from the cell nucleus. The intensity of the fluorescence from the BBs is reduced to the intensity of the fluorescence of the flagella when the cell nucleus is removed from the complex. From these results, we conclude that the BBs do not contain DNA. Discrepancies related to the reported presence of DNA in the BBs are discussed.
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  • 4
    ISSN: 1615-6102
    Keywords: Kinesin-like motor ; Mammalian cell cycle ; Centrosomes ; Mitotic spindles ; Nuclear antigen
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A 69 kDa protein present in the interphase centrosome and mitotic spindle/pole was identified with the CHO2 monoclonal antibodies raised against mitotic spindles isolated from Chinese hamster ovary (CHO) cells. Isolation and characterization of antigen-specific cDNAs and recombinant proteins demonstrated that the protein is a minus-end-directed microtubule motor with the motor domain located at the C terminus. Affinity-purified polyclonal antibodies prepared to bacterially expressed fusion proteins revealed the presence of the antigen in interphase nuclei, and the degree of nuclear immunostaining intensity varied among cells at different cell cycle stages. In order to examine the change of antigen expression during the cell cycle, we prepared synchronized populations of CHO cells, double stained with CHO2 and PCNA (proliferating cell nuclear antigen) antibodies, and quantitated the amounts of nuclear fluorescence using the MetaMorpho image analysis software package. Cells right after cell division contained nuclei with the lowest level of CHO2 immunofluorescence. The immunofluorescence intensity progressively increases through G1 to S, reaching a maximum level by the end of G2. The antibody uniformly stained the entire nuclear region, and the total amount of fluorescence detected in G2 cells was greater than three times that of G1 cells. Cell cycle dependent accumulation of the CHO2 antigen was further confirmed by immunoblot analysis of the protein included in whole cell extracts and nuclei isolated from synchronized CHO cells. Northern blot analysis showed that, although the CHO2-transcript accumulated during later stages of the cell cycle, its abundance declined through G1 to S, and was lowest in cells at the early S phase. The difference in the expression pattern of the antigen protein and its transcript may suggest the presence of multiple mechanisms controlling the level of CHO2 antigen during the course of the cell cycle.
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  • 5
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Affinity chromatography ; Anhydrotrypsin ; C-terminal Arg- or Lys-containing peptides ; Diol silica
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary Anhydrotrypsin (AHT), a catalytically inert derivative of trypsin in which the active site serine residue was converted to dehydroalanine residue by chemical modification, was immobilized onto diol silica through the activation with trifluoroethanesulfonyl chloride, and an AHT-diol-silica column was used for high-performance affinity chromatography separation of peptides containing arginine or lysine at their C-termini from the others. Improved separation in terms of speed was accomplished.
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  • 6
    ISSN: 1612-1112
    Keywords: Ion chroamtography ; Affinity chroamtography ; Tresyl groups ; 2,2,2-Trifluoroethanesulfonic acid
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary A simple and sensitive ion chromatographic method for the estimation of tresyl groups on several activated supports (tresylated diol silica, tresylated Sepharose 4B and Tresyl-5PW) has been developed. The method involves the hydrolysis of tresyl groups on the supports followed by ion chromatographic determination of the liberated 2,2,2-trifluoroethanesulfonic acid. It is useful for the prediction of the immobilization efficiency of proteins.
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  • 7
    ISSN: 1612-1112
    Keywords: Affinity chromatography ; Anhydrochymotrypsin ; Peptides with aromatic amino acids at C-termini ; Diol silica
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary Anhydrochymotrypsin (AHC), a catalytically inert derivative of chymotrypsin in which the serine-residue active site has been converted chemically to a dehydroalanine residue, was immobilized on diol silica by activation with trifluoroethanesulfonyl chloride. A AHC-diol-silica column was used for high-performance affinity chromatographic separation of peptides with aromatic amino acids at their C-termini from other peptides. Faster separations were achieved.
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  • 8
    ISSN: 1615-6102
    Keywords: Giant mitochondria ; Mitochondrial nuclei ; Three-dimensional reconstruction ; Megasporogenesis ; Megagametogenesis ; Egg cell
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The preferential development of giant mitochondria and their nuclei (nucleoids) in the egg cells ofPelargonium zonale Ait. during megasporogenesis and megagametogenesis was examined by fluorescence microscopy, after Technovit embedding and 4′,6-diamidino-2-phenylindole (DAPI) staining, fluorimetry for DNA content, using a video-intensified microscope photon-counting system (VIMPICS), and by three-dimensional reconstruction of mitochondrial nuclei (mt-nuclei). Reproductive cells during the megaspore mother cell, meiosis, tetrad, and functioning megaspore stages contained many small mitochondria with characteristic, uniformly DAPI-stained mt-nuclei about 0.3 μm in diameter, containing a small amount of DNA (0.3 Mbp). During formation of the 2-, 4-, and 8-nucleate embryo sac, mt-nuclei did not markedly change in shape or DNA content. When the embryo sac formed and differentiation of each cell began, mitochondria and their nuclei in the egg cell took on a small ring or string-like shape. Accompanying the maturation of the embryo sac, they underwent progressive enlargement and gradually altered to long thick strings, or stacks of concentric or half concentric rings. By flower opening, they have developed to an extremely large size. One of these stacks of mt-nuclei was reconstructed in three dimensions; each ring in the stack was cup- or plate-shaped; 5 to 10 rings made up the stack, though each remained discontinuous from the others. From serial sections, we counted 44 mitochondria in one egg cell. Fluorometry using VIMPICS revealed that DNA amount within the stacked mitochondrion increased to 40 times that of the megaspore mother cell stage; a single stack of mitochondria contained 340–1700 Mbp DNA; which means that one egg cell contains at least 15000 Mbp mt-DNA, a value greater than the cell-nuclear DNA content.
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  • 9
    ISSN: 1573-4838
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine , Technology
    Notes: Abstract The usefulness of collagen fibers and the YIGSR sequence (Tyr-lle-Gly-Ser-Arg) of laminin for nerve regeneration were examined in vivo. Type I collagen gel (G-group), Type I collagen fibers (F-group), Type I collagen fibers coated with laminin (L-group) or the YIGSR sequence (Y-group) were packed into silicone tubes, 15 mm long, and transplanted to the sciatic nerves of Wistar rats. Empty silicone tubes were used as the control. The animals were sacrificed 8 weeks after transplantation. Bridging of the nerve was confirmed in the F-(7/12), Y-(7/10) and L-group (6/10), but no bridging was observed in any of the animals of the G- and control group. Nerve regeneration among the space of collagen fibers was observed, and it was suggested that fibroblasts infiltrated the gap in the substance of the degenerated collagen fibers were followed by Schwann cells on the basis of immunocytochemistry. The number of myelinated axons per regenerated tissue in the tube (density), and total area of myelinated axons per measured regenerated tissue in the tube (% axon area) in each the L- and Y-group were significantly higher than that in the F-group (P 〈 0.05). These results suggest the possibility of obtaining adequate nerve regeneration with new artificial materials only. © 1999 Kluwer Academic Publishers
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  • 10
    Electronic Resource
    Electronic Resource
    Springer
    Journal of materials science 19 (1984), S. 1699-1709 
    ISSN: 1573-4803
    Source: Springer Online Journal Archives 1860-2000
    Topics: Mechanical Engineering, Materials Science, Production Engineering, Mining and Metallurgy, Traffic Engineering, Precision Mechanics
    Notes: Abstract Polycarbonate and its blends with ABS and SAN were tested to study the energy absorbing properties and the deformation mechanism. In tension and impact tests, these blends were found to possess a high energy absorbing capability, high yield strength and large rupture elongation. ABS and SAN particles in the blends deform in a ductile manner to an elongation of more than 100% under a tensile stress. In an elongation test of ABS under a hydraulic pressure of 90×105 N m−2, the transformation from crazing to a cold-drawing mechanism was found to occur. The large elongation of ABS and SAN in the blends is attributed to the cold drawing which occurs under the influence of the pressure acting on the dispersed ABS and SAN, caused by the difference between the elastic moduli of the dispersoid and the matrix.
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