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  • 1
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 12 (1989), S. 33-41 
    ISSN: 0886-1544
    Keywords: phosphorylation ; MPM-2 ; mitotic spindle ; microtubule-associated protein ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Mitotic spindles isolated from the diatom Stephanopyxis turris become thiophosphorylated in the presence of ATPγS at specific locations within the mitotic apparatus, resulting in a stimulation of ATP-dependent spindle elongation in vitro. Here, using indirect immunofluorescence, we compare the staining pattern of an antibody against thiophosphorylated proteins to that of MPM-2, an antibody against mitosis-specific phosphoproteins, in isolated spindles. Both antibodies label spindle poles, kinetochores, and the midzone. Neither antibody exhibits reduced labeling in salt-extracted spindles, although prior salt extraction inhibits thiophosphorylation in ATPγS. Furthermore, both antibodies recognize a 205 kd band on immunoblots of spindle extracts. Microtubule-organizing centers and mitotic spindles label brightly with the MPM-2 antibody in intact cells. These results show that functional mitotic spindles isolated from S. turris are phosphorylated both in vivo and in vitro. We discuss the possible role of phosphorylated cytoskeletal proteins in the control of mitotic spindle function.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 4 (1984), S. 7-23 
    ISSN: 0886-1544
    Keywords: axoplasm ; elastic modulus ; viscosity ; motility ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A magnetic sphere viscoelastometer has been developed to peform rheological experiments in living axoplasm of Loligo pealei. The technique includes the use of a calibrated magnetic sphere viscoelastometer on surgically implanted ferro-magnetic spheres in intact squid giant axons. The axoplasm was discerned to be “living” by the biological criterion of tubulovesicular organelle motility, which was observed before and after experimentation. From these in vivo experiments, new structural characteristics of the axoplasm have been identified. First, analysis of magnetic sphere trajectories has shown the axoplasm to be a complex viscoelastic fluid. Directional experimentation showed that this material is structurally anisotropic, with a greater elastic modulus in the direction parallel to the axon long axis. Second, both magnetic sphere and in vivo capillary experiments suggested that the axoplasm is tenaciously anchored to the axolemma. Third, it was found that axoplasm could be modelled as a linear viscoelastic material in the low shear rate range of 0.0001 to 0.004 s-1. The simplest mechanical model incorporating the discovered properties of the material in this range is Burger's model.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 37 (1994), S. 398-406 
    ISSN: 1040-452X
    Keywords: Preimplantation embryo ; Insulin ; IGFs ; Protein synthesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Previously constructed protein databases for two stages of preimplantation mouse embryogenesis, the compacted eight-cell stage and the fully expanded blastocyst stage, have been used to analyze the effects of insulin, IGF-I, and IGF-II on protein synthesis in these developmental stages. Proteins were labeled by placing, for 2 hr, synchronous cohorts of 35-50 embryos into human tubal fluid (HTF) medium containing L-[35S]-methionine (1 mCi/ml) in the presence or absence of one of the growth factors. The embryos were then washed with medium and lysed. Samples were processed for 2-D gel analysis. For each embryonic stage and each growth factor, four or five experimental replicates were done and the gel images were compared using the PDQUEST system. Using the computer-assisted analysis, we were able to identify proteins that showed a statistically significant (P 〈 0.05) change in synthesis. At the eight-cell stage of development insulin caused increased synthesis of two proteins and decreased synthesis in three proteins. Insulin-treated blastocyst stage embryos exhibited an increased synthesis in eight proteins and decreased synthesis for one protein. The effect of IGF-I at the eight-cell stage of development was mostly inhibitory; the synthesis of only one protein increased and the synthesis of five proteins showed a decrease. Similar results were obtained with blastocyst stage embryos; four proteins demonstrated an increase in synthesis while 14 proteins showed a decrease. Eight-cell stage embryos incubated with IGF-II had seven proteins with a decreased synthesis, although in blastocyst stage embryos, nine proteins showed increased synthesis. However, seven IGF response proteins were found to be proteins that showed significant changes in isotope incorporation during the eight-cell to blastocyst stage of development (Shi et al., 1993). In all, 54 proteins were affected, and these were unique; thus, protein synthesis in preimplantation mouse embryos is influenced by insulin and the IGFs, and further, each growth factor affects specific proteins. © 1994 Wiley-Liss, Inc.
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  • 4
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Microscopy Research and Technique 20 (1992), S. 390-405 
    ISSN: 1059-910X
    Keywords: Reflection electron microscopy ; Secondary electron imaging ; Field emission gun ; In situ REM ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: A review is given on the techniques and applications of high-energy reflection electron energy-loss spectroscopy (REELS) and reflection electron microscopy (REM) for surface studies in scanning transmission electron microscopes (STEM) and conventional transmission electron microscopes (TEM). A diffraction method is introduced to identify a surface orientation in the geometry of REM. The surface dielectric response theory is presented and applied for studying α-alumina surfaces. Domains of the α-alumina (012) surface initially terminated with oxygen can be reduced by an intense electron beam to produce Al metal; the resistance to beam damage of surface domains initially terminated with Al+3 ions is attributed to the screening effect of adsorbed oxygen. Surface energy-loss near-edge structure (ELNES), extended energy-loss fine structure (EXELFS), and microanalysis using REELS are illustrated based on the studies of TiO2 and MgO. Effects of surface resonances (or channeling) on the REELS signal-to-background ratio are described. The REELS detection of a monolayer of oxygen adsorption on diamond (111) surfaces is reported.It is shown that phase contrast REM image content can be significantly increased with the use of a field emission gun (FEG). Phase contrast effects close to the core of a screw dislocation are discussed and the associated Fresnel fringes around a surface step are observed. Finally, an in situ REM experiment is described for studying atomic desorption and diffusion processes on α-alumina surfaces at temperatures of 1,300 - 1,400°C.
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  • 5
    ISSN: 1040-452X
    Keywords: Microtubles ; microfilaments ; Microtublel-organizing centers ; Parthenogenetic activation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In metaphase II arrested rat oocytes (M il), microtubles were found in the taper-shaped meiotic spindle and in the cytoplasm as asters and free microtubules. Whereas spindle microtubules were acetylated, those located in the cytoplasm were not. Cytoplasmic microtubules were also labile as assessed by mild cooling. In contast to mouse oocytes, rat microtubule organizing centers (MTOCs) did not react with MPM-2 antibody by immunofluorescence despite the fact that this antibody reacts with several proteins as shown by immunoblot. However, cytoplasmic MTOCs in M II-arrested rat oocytes could be detected by their nucleating capacity in the presence of taxol, a drug that induced the formation of numerous cytoplasmic asters. In addition, taxol caused a change in the spindle shape and the formation of astral microtubules at the spindle poles. Meiotic spindles (as well as chromosomes devoid of microtubules after nocodazoletreatment) were overlaid by an actin-rich domain. Spontaneous abortive activation led to the extrusion of the second polar body followed by another metaphase arrest -  metaphase III; however, normal spindles did not form and dispersed chromosomes surrounded by microtubles were observed. Electron microscopic studies confirmed these observations and revealed that the kinetochores are located deep within the chromosomes in contrast to mouse kinetochores, and this might be responsible for the absence of a metaphase III spindle in the rat oocyte. Induced activation caused transition to interphase with the formation of a characteristic microtubule network. This study shows that there are several significant differences in the cytoskeletal organization of rat and mouse oocytes. © 1993 Wiley-Liss, Inc.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 39 (1994), S. 25-29 
    ISSN: 1040-452X
    Keywords: Hemizona assay (HZA) ; Rhesus monkey ; Oocyte ; Sperm ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The hemizona assay (HZA) in Rhesus monkeys was employed to study the correlation of zona-binding ability with sperm motility or with naturally developing oocytes at various maturational stages. Oocytes from unstimulated ovaries were retrieved within 2 hr from monkeys sacrificed for vaccine production (in reproductive season, but with their menstrual cycles not determined). Oocytes were divided into four groups based on their morphological maturation: 1) Oocytes surrounded by more than one cumulus layer (MC); 2) Oocytes retaining intact germinal vesicle nuclei (GV); 3) Oocytes with germinal vesicle breakdown showing distinct perivitelline space (PVS); and 4) Oocytes extruding the first polar body (PBI). The mean numbers of sperm bound to hemizona for PB1, PVS, GV, and MC groups were 132.9 ± 12.0, 71.5 ± 10.1, 36.1 ± 4.0, and 20.1 ± 2.9 (Mean ± SE), respectively. The four groups showed significant differences from each other in sperm/egg binding ability (P 〈 0.01). The number of bound sperm significantly increased with oocyte maturation. The present study also showed that zona-binding ability was also affected by sperm motility. For sperm with 67.7% motility and sperm with 31.2% motility, the average numbers of bound sperm were 43.5 ± 2.2 and 25.3 ± 2.9 (Mean ± SE), respectively. There was significantly higher binding ability for sperm with higher motility (P 〈 0.01). The results suggest that: 1) The rhesus monkey model can serve as a very sensitive model for studying sperm/egg interaction by HZA; 2) Sperm motility positively correlated with sperm/egg binding; and 3) Sperm/egg binding ability increases with oocyte maturation. The binding ability is highest when oocytes matured to the PB1 stage, which is also the best opportunity for fertilization. This is strong evidence for the “zona maturation” hypothesis. © 1994 Wiley-Liss, Inc.
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  • 7
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 42 (1995), S. 58-64 
    ISSN: 1040-452X
    Keywords: Eggs ; Biflagellate spermatozoa ; Electron microscope ; Fish (Zoarcidae) ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Ultrastructure of sperm and eggs of the ocean pout (Macrozoarces americanus L.), an internally fertilizing marine teleost, was examined by scanning and transmission electron microscopy. The results showed that the sperm do not have an acrosome but have a very long mid-piece (one to two times the sperm head length) containing numerous well-developed elongated mitochondria. The sperm also have two tails (is biflagellate) each consisting of nine peripheral and one central pair (9 ± 2) of microtubules. This long mid-piece and the biflagellate nature of the sperm appear to be associated with the long life-span of the sperm and with sperm dispersal in the ovary to fertilize the eggs internally. The ocean pout eggs are enveloped by a porous chorionic membrane similar to that found in other teleosts but have two micropyles, a condition likely related to a mechanism of egg fertilization which increases the egg fertlity in the presence of low sperm numbers. Following insemination, some biochemically undefined excretions appeared on the surface of fertilized eggs and led to the acquisition of adherent capability of the eggs which formed a tightly associated egg mass in sea water. © 1995 wiley-Liss, Inc.
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  • 8
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 37 (1994), S. 34-47 
    ISSN: 1040-452X
    Keywords: Preimplantation development ; Two-dimensional gel electrophoresis ; PDQUEST ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: High-resolution two-dimensional sodium dodecyl sulfate-polyacrylamide (2D-SDS) gel electrophoresis combined with computerized analysis of gel images was used to construct and analyze protein databases for two stages of preimplantation mouse embryogenesis, the compacted eight-cell stage and the fully expanded blastocyst stage. These stages were chosen for their ease in identification of multiple synchronous embryos. Synchronous cohorts of 30-50 embryos were labelled with L-[35S]methionine for 2 hr. The embryos were then lysed in 30 μl hot SDS sample buffer, and the lysates were stored at -80°C until the gels were run. Five replicates were run for eight-cell embryos, and four for blastocyst-stage embryos. The samples were processed for 2D gel electrophoresis and fluorography; multiple exposures were made. Gel images were analyzed using the PDQUEST system, and databases were constructed. Analysis of the databases for both developmental stages showed high reproducibility of protein spots in multiple gel images. Of 1,674 total spots in eight-cell embryo standards, 〉79% of spots had a percentage error (S.E.M./average) 〈50%, and 〉45% had a percentage error 〈30%. Similarly, of 1,653 total spots in blastocyst-stage embryo standards, 74% of spots had a percentage error 〈50%, and approximately 47% of spots had a percentage error 〈30%. Forty-three spots (approximately 3% of the total spots) were found to be detected only in the eight-cell stage, while 75 spots were detected solely in the blastocyst stage. Sixty-nine proteins showed a greater than threefold increase in isotope incorporation from the eight-cell to the blastocyst stage, with a percentage error 〈50% in both the eight-cell and the blastocyst stages. In contrast, 41 of the proteins showed a decrease during this period. Analysis of the protein databases described in this study has allowed us to document the overall quantitative changes in proteins from the compacted eight-cell stage to the blastocyst stage of mouse preimplantation development. These databases provide a valuable tool for further detailed quantitative analysis of specific proteins associated with developmental events. In addition they will permit analysis of the effects of environmental factors, such as growth factors, on early embryo development. © 1994 Wiley-Liss, Inc.
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  • 9
    ISSN: 1040-452X
    Keywords: Alphafetoprotein ; Liver gene expression ; Promoter/enhancer regions ; Transgenic mice ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A truncated mouse alphafetoprotein (AFP) gene promoter/enhancer region was tested for its ability to regulate the expression of the Escherichia coli chloramphenicol acetyltransferase (CAT) reporter gene in the livers of transgenic mice. The AFP regulatory region lacked any AFP gene structural DNA, included one enhancer sequence together with the proximal promoter sequence, and an element believed to be responsible for the postnatal repression of AFP gene transcription. The neonatal livers of AFP/CAT transgenic mice showed a high level of CAT enzyme expression, which was dramatically reduced between 7 and 14 days after birth. The staining of liver sections with anti-CAT antibodies showed that this expression was limited to hepatocytes. In one lineage, reexpression of CAT in the adult liver could be achieved by restitutive proliferation of hepatocytes following partial hepatectomy or CCl4-induced necrosis; reexpression in young animals (3-4 weeks of age) was even greater. These studies show that a truncated AFP promoter/enhancer region functions in a tissue-specific and developmental stage-specific fashion, and may be used to control the expression of other genes in the livers of transgenic mice. © 1995 Wiley-Liss, Inc.
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  • 10
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 26 (1990), S. 361-365 
    ISSN: 1040-452X
    Keywords: Stallion spermatozoa ; lonophore ; IVF ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In vitro fertilizing ability of stallion spermatozoa was assessed using horse follicular oocytes matured in vitro. After collection, stallion spermatozoa were either: 1) washed and incubated in TALP medium with 3 mg/ml bovine serum albumin (BSA) and 10 μg/ml heparin for 4 h, 2) washed and incubated in TALP with 3 mg/ml BSA for 3 h and cultured for a further 1 h with 1 mM caffeine and 5 mM dbcAMP, 3) washed and incubated in TALP medium with 3 mg/ml BSA at pH 7.9-8.2 for 2-4 h, or 4) diluted and incubated in TALP medium with 10 mg/ml BSA and 7.14 μM calcium ionophore A 23187 for 5-10 min followed by washing. After a given pretreatment, suspensions were diluted into B2 medium to a concentration of 5 × 106 sperm/ml and co-incubated with oocytes for 12 h or 24-48 h. In the ionophoretreated group, 18 of 54 oocytes (33%) were fertilized by 12 h, and 11 of 45 (24%) cleaved by 24-48 h. Evidence of fertilization was not found in the oocytes incubated with spermatozoa from other treatment procedures.
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