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  • 1
    Electronic Resource
    Electronic Resource
    Relationship between culture conditions and the dependency on mitochondrial function of mammalian cell proliferation (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 152 (1992), S. 632-638 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In cultured mammalian cells, the relationship was investigated between mitochondrial function and proliferation under various culture conditions. Continuous inhibition of the expression of the mitochondrial genome was used to reduce the activity of enzymes involved in oxidative phosphorylation by 50% at every cell division. Under these conditions, culturing in relatively poor media resulted in arrest of the proliferation of most cell lines after 1 cell division. This was preceded by decreasing levels of ATP and increasing levels of ADP, suggesting that the ATP-generating capacity of the cells was limiting. Culturing in richer media led to arrest of the proliferation after 5 to 6 divisions, but accumulation of ADP was not observed. Addition of pyruvate to rich culture media and, at least for 1 cell line, increasing the CO2 levels, completely prevented proliferation arrest. Inability to synthesise metabolic precursors via mitochondrial intermediary metabolism probably explains growth arrest of cells cultured in rich media. Pyruvate and CO2 were, however, without effect on the proliferation arrest of cells cultured in relatively poor media. Therefore, pyruvate dependency for growth of cells without functional mitochondria holds true only under culture conditions where the ATP-generating capacity of the cells is not limiting. © 1992 Wiley-Liss, Inc.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041520323
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  • 2
    Electronic Resource
    Electronic Resource
    Adaptation of EGF receptor signal transduction to three-dimensional culture conditions: Changes in surface receptor expression and protein tyrosine phosphorylation (1994)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 161 (1994), S. 374-382 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A431 cells grown as three-dimensional spheroids show growth stimulation in response to nanomolar concentrations of EGF in contrast to monolayer cultures that show inhibition. In investigating the alterations in EGF signal transduction that underlie this modification of the proliferative response, we have compared the expression of EGF receptors on A431 cells under these conditions and related our findings to tyrosine phosphorylation and the growth response. EGF receptors were measured by 125I-EGF binding to trypsin-dispersed cells. Unexpectedly, dispersion of the monolayers caused an 80% decrease in surface EGF receptor, although, after dispersion, EGF receptor was digested by trypsin with a half-life of 69 ± 32 min. No evidence for a comparable loss of cellular EGF receptor was seen on trypsin dispersion of spheroids. After allowing for this effect, we found that the receptor density on nondispersed monolayers (5 × 106 per cell) was twentyfold greater than that on spheroids (0.25 × 106 per cell). EGF-induced tyrosine phosphorylation was confined to the outermost cells of the spheroid, although the presence of surface-expressed EGF pinding sites could be demonstrated throughout the structure and the number of EGF receptors/cell on dispersed spheroid cells showed a single distribution peak by flow cytometry, with no evidence for more than one population. Using RCM-lysozyme as a substrate, tyrosine phosphatase activity in spheroids lay within the range observed in monolayer cultures. Autophosphorylation of the EGF receptor following EGF stimulation in monolayer cultures of A431 cells rose rapidly in the first 10 seconds and then slowly increased for at least 3 h. In spheroids, it reached a maximum within 10 seconds and then declined over 3 h. Since the microenvironment within a tumor resembles that in a spheroid, a similar reduction in surface EGF receptor expression may be expected in tumors relative to monolayer cultures, together with corresponding growth stimulation in response to EGF. © 1994 Wiley-Liss, Inc.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041610223
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  • 3
    Electronic Resource
    Electronic Resource
    Dog platelets accumulate intracellular Fibrinogen as they age (1994)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 161 (1994), S. 23-30 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The alpha granules of circulating platelets are dynamic structures that acquire endogenous and exogenous components by synthesis and uptake, respectively. The uptake of exogenous components is a result of either receptor-mediated endocytosis or fluid-phase pinocytosis. Despite many detailed studies on the function and content of α-granules, little is known of the impact of platelet age on these organelles. In this report, we describe the use of platelet biotinylation to identify and isolate aged platelets for the analysis of α-granule contents. When aged platelets were permeabilized and examined by flow cytometry utilizing fluorescently labeled antibodies, two exogenously acquired proteins, fibrinogen and immunoglobulin G, were found to increase significantly with platelet age. The levels of intracellular fibrinogen were found to be elevated relative to control, 114 ± 2% and 119 ± 5% on days 4 and 5 postbiotinylation, respectively; the life span of dog platelets is 6.0 days. Intracellular immunoglobulin G content increased similarly. Levels of two endogenously synthesized proteins, thrombosponding and P-selectin, were not elevated in aged platelets. Confirmation of the flow cytometric data was obtained by isolating aged, biotinylated platelets by fluorescence-activated cell sorting and quantitating the fibrinogen levels with an ELISA assay. For platelets averaging 4.6 days of age, the fibrinogen level was elevated to 128 ± 23% of the level for the entire platelet population. These data demonstrate that age-dependent changes in exogenously acquired α-granule proteins do occur and that the uptake mechanism for these proteins is active through out the platelet life span. © 1994 Wiley-Liss, Inc.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041610104
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  • 4
    Electronic Resource
    Electronic Resource
    Ultraviolet stimulated melanogenesis by human melanocytes is augmented by di-acyl glycerol but not TPA (1990)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 142 (1990), S. 334-341 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Epidermal melanocytes (MC) synthesise melanin in response to ultraviolet radiation (UVR). The mechanisms mediating the UV-induced activation of melano-genesis are unknown but since UVR induces turnover of membrane phospholipids generating prostaglandins (PGs) and other products, it is possible that one of these might provide the activating signal. We have examined the effects of prostaglandins (PGs) E1, E2, D2, F2α, and di-acyl glycerol upon the UV-induced responses of cultured human MC and the Cloudman S91 melanoma cell line. The PGs had little effect on unirradliated cells and did not alter the response to UVR in either human MC or S91 melanoma cells. However, a synthetic analogue of di-acyl glycerol, 1-oleyl 2-acetyl glycerol (OAG), caused a significant (P〈0.0001), dose-related augmentation of melanin content both in human MC (seven-fold) and S91 cells (three-fold). UVR caused a significant augmentation of the OAG-induced melanognesis of both human MC and S91 cells. Since OAG is known to activate protein kinase C, it was possible that the observed modulation of the UVR signal could be via that pathway. Di-octanoyl glycerol, another di-acyl glycerol, which activates kinase C, caused a small (70%) increase in melanogenesis in MC which was not altered by UVR. However, 12-0 tetradecanoyl phorbol 13-acetate (TPA), a potent activator of protein kinase C, had no significant effect on either basal or UV-induced melanin synthesis in either cell type. These data suggest that the UV-induced signal activating melanogenesis could be mediated by di-acyl glycerol. Furthermore, they imply that the signal is transduced via an alternative, pathway that might be independent of protein kinase C.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041420216
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  • 5
    Electronic Resource
    Electronic Resource
    Rapid inverse changes in α1B-and β2-adrenergic receptors and gene transcripts in acutely isolated rat liver cells (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 152 (1992), S. 79-86 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In vitro incubation of hepatocytes acutely isolated from adult male rats leads to a rapid conversion of the adrenergic activation of glycogenolysis from an α1-receptor (α1AR) to a β2-receptor (β2AR) mediated response within 4 h. In order to understand the underlying mechanism, we examined time-dependent changes in α1- and β2-adrenergic activation of glycogenolysis and second messenger systems, the cellular density and affinity of α1AR and β2AR, and the steady state levels of α1BAR and β2AR mRNAs. Incubation of hepatocytes for 4 h resulted in a decrease in phosphorylase activation and inositol 1,4,5 trisphosphate accumulation in response to phenylephrine, a 40% decrease in α1AR density, and a 70% decrease in α1BAR mRNA levels. Incubation of hepatocytes for 4 h also resulted in the emergence of a phosphorylase response to isoproterenol, an increase in isoproterenol-induced but not in glucagon- or forskolin-induced cAMP accumulation, no significant change in β2AR density, and a twofold increase in β2AR mRNA levels. Exposure of cells to cycloheximide, 2 μM throughout the 4 h incubation, prevented the emergence of the phosphorylase response to isoproterenol and reduced β2AR densities, while the decrease in α1AR density was not affected and the decrease in phosphorylase activation by phenylephrine was attenuated. The results indicate that dissociation of rat liver cells triggers a rapidly developing decrease in α1BAR mRNA and increase in β2AR mRNA levels and corresponding inverse changes in the synthesis of α1BAR and β2AR which account, at least in part, for the rapid conversion from α1- to β2-adrenergic glycogenolysis. © 1992 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041520111
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  • 6
    Electronic Resource
    Electronic Resource
    The effect of warming velocity on motility and acrosomal integrity of boar sperm as influenced by the rate of freezing and glycerol level (1993)
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 34 (1993), S. 190-195 
    Details
    ISSN: 1040-452X
    Keywords: Thawing velocity ; Freezing rate ; Glycerol concentration ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The effect of thawing velocities ranging from 10°C/min to 1.800°C/min on the motility and acrosomal integrity of boar spermatozoa frozen at 1°C/min (suboptimal), 5°C/min, and 30°C/min (optimal) rate was studied with the sperm suspended for freezing in diluent containing 2, 4, or 6% of glycerol (v/v). The influence of thawing on sperm survival depends on the rate at which the sperm had been frozen. In semen frozen at a suboptimal rate of 1°C/min, the percentage of motile sperm (FMP) initially fell to 3.5-4.0% when the thawing rose to 200°C/ min, but, with further increases in thawing rate, increased and reached peak values (10.3-11.0% FMP) after thawing at 1,800°C/min. The percentage of sperm with normal apical ridge (NAR) also increased moderately with thawing rate, but the degree of improvement decreased as the glycerol level was increased. In semen frozen at 1°C/min, acrosomal integrity (NAR) was best maintained in 2% glycerol, reaching 22.9% NAR after thawing at 1,800°C/min. In semen frozen at the optimal rate of 30°C/min, the increases in thawing rates above 200°C/min substantially improved motility. Motility was generally higher in semen protected by 4 or 6% glycerol, with the peak values of 44 or 46% FMP, respectively, after thawing at 1,200°C/min. The proportion of sperm with NAR also increased with thawing rate, but as in the case of suboptimally frozen sperm it was influenced negatively by the glycerol concentration. The peak value 53% NAR was recorded in semen protected by 2% glycerol, frozen at 30°C/min, and thawed at 1,200°C/min. In view of the inverse relationship between FMP and NAR, selection of optimal conditions from among the interacting variables, freezing rate, glycerol concentration, and thawing rate requires compromising between maximal FMP and maximal NAR. Accordingly, we have adopted as optimal a protocol with a thawing rate of 1,200°C/min, a freezing rate of 30°C/min and concentrations of 3% glycerol. © 1993 Wiley-Liss, Inc.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1080340211
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  • 7
    Electronic Resource
    Electronic Resource
    Instrumentation, techniques, and applications of electron microscopy in the solid state physics group at Glasgow university (1993)
    New York, NY [u.a.] : Wiley-Blackwell
    Microscopy Research and Technique 24 (1993), S. 316-332 
    Details
    ISSN: 1059-910X
    Keywords: TEM ; CTEM ; STEM ; Electron energy loss spectroscopy (EELS) ; Energy dispersive X-ray spectroscopy (EDX) ; Radiation damage ; Mass loss ; Lorentz microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: This review discusses some of the work performed by the Solid State Physics Group at Glasgow University. A major aim of the group is to obtain quantitative information with high spatial resolution and to do this reliably requires a thorough understanding of both the instrumentation and the interactions between the electron beam and the specimen. Thus the first part of the review discusses those aspects of instrumentation and techniques that the group has considered in detail while the final part deals with applications which involve the study of a wide range of materials covering metallurgical, semiconductor, organic, and magnetic systems. In all these applications, the results from a range of techniques have been required to provide as complete a picture of the material as possible. © 1993 Wiley-Liss, Inc.
    Additional Material: 14 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jemt.1070240405
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  • 8
    Electronic Resource
    Electronic Resource
    Efficient transfection of chicken cells by lipofection, and introduction of transfected blastodermal cells into the embryo (1991)
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 30 (1991), S. 304-312 
    Details
    ISSN: 1040-452X
    Keywords: Transfection ; Chimeras ; β-galactosidase ; lacZ ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Chicken blastodermal cells (CBCs) and primary chicken fibroblasts (PCFs) have been lipofected with a variety of lacZ constructs encoding Escherichia coli β-galactosidase (β-gal). A reporter construct (phspPTlacZpA) containing a mouse heat-shock protein 68 gene (hsp 68) promoter was used to establish conditions for efficient lipofection. The construct, in circular or linear plasmid form or as reporter sequences alone, was transferred efficiently by incubating the cells for 3.5 h in a mixture of 6.2 μg LipofectinTM (a cationic liposome preparation from Bethesda Research Laboratories) and 1.55-3.1 μg DNA per mL DMEM. These lipofection conditions were used to transfer a reporter construct (pCBcMtlacZ) containing a Zn2+-inducible chicken metallothionein (cMt) promoter, and constructs showing constitutive expression due to Rous sarcoma virus plus chicken β-actin (pmiwZ) or cytomegalovirus (pMaori3) promoters.Endogenous chicken β-gal and transferred bacterial β-gal activity could be distinguished clearly by incubating the cells with the substrate, Xgal, at pH 4.3 or 7.4, respectively. Expression of phspPTlacZpA in chicken cells did not appear to require specific induction of the mouse hsp68 promoter, whereas expression of pCBcMtlacZ required treatment of the cells for 6-12 h with 150 μM ZnCl2. Bacterial β-gal activity was observed following lipofection of CBCs that were cultured in suspension or plated. The efficiency of lipofection was at least 1 in 25 for CBCs, judging by the proportion of cells shown to have b̃-gal activity 16-24 h after lipofection treatment began; these events could represent transient or stable incorporation of the construct. Plated PCFs were lipofected as well, with stable incorporation of the gene construct indicated in 10% of positive events.Lipofected CBCs were injected into the subgerminal cavity of stage X (Eyal-Giladi and Kochav, 1976) chicken embryos in a manner that has been shown to produce somatic and germline chimeras for untreated CBCs (Petitte et al., 1990). After 65 h of incubation, cells expressing β-gal activity were observed in the prosencephalon, head ectoderm, and ventricle of the heart of a stage 11 (Hamburger and Hamilton, 1951) embryo. In other cases, bacterial β-gal activity was detected extraembryonically, both in individual cells and in foci of expressing cells, 24, 48, and 65 h after injection. Few, if any, single expressing cells and no foci were detected following injection of the LipofectinTM:DNA mixture directly into the embryo.Refinement of these procedures could contribute to the development of transgenic poultry, without reliance on retroviral vectors for DNA transmission or incorporation.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1080300404
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  • 9
    Electronic Resource
    Electronic Resource
    Effect of microinjection and two types of electrical stimuli on bovine sperm-hamster egg penetration (1990)
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 27 (1990), S. 163-167 
    Details
    ISSN: 1040-452X
    Keywords: Electrofusion ; Electroporation ; In vitro fertilization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: These experiments were designed to test the effects of an electrofusion and an electroporation pulse on bovine sperm-hamster egg development. In experiment 1, single motile sperm were injected into the perivitelline space of each egg. A 4,500 V/cm, 30 μsec fusion pulse (FP) was applied while sperm-egg membrane contact was maintained. It was observed that single motile sperm were rendered immotile immediately after FP application whereas nonpulsed single motile sperm remained motile for up to 36 h postinjection. In addition, both motile and sanicated spermatozoa were injected directly into the ooplasm prior to receiving an FP to determine whether the FP was detrimental to sperm viability. In experiment 2, to induce the acrosome reaction, an 1,150 V/cm electroporation pulse was applied to washed bovine sperm suspended in TALP medium containing 5 mM Ca2+. Treated and nontreated sperm were coincubated with zona-free hamster ova, and sperm-penetrating ability was measured. Results from experiment 1 indicate that FP failed to induce sperm-egg fusion (0/69). FP did not, however, inhibit decondensation or pronuclear formation of sperm injected into hamster egg ooplasm. Single motile sperm injected into the ooplasm resulted in development of both pulsed (19/28) and nonpulsed (21/28) groups. Sonicated tail-free sperm heads injected into the ooplasm resulted in no detectable difference between treated (18/30) and nontreated (19/30) groups. In experiment 2, treatment of sperm with electroporation pulse +5 mM Ca2+ increased zona-free hamster ova penetration scores over nontreated sperm within bulls (P 〈 .05). These results indicate that an electroporation pulse in conjunction with high Ca2+ rather than an electrofusion pulse facilitates sperm penetration and early development.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1080270212
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  • 10
    Electronic Resource
    Electronic Resource
    Expression and release of phosphatidylinositol anchored cell surface molecules by a cell line derived from sensory neurons (1992)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 48 (1992), S. 61-72 
    Details
    ISSN: 0730-2312
    Keywords: differentiation ; neuronal cells ; GPI-anchored molecules ; metabolism ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Early postnatal mouse dorsal root ganglion neurons were found to express several glycosylphosphatidylinositol-anchored (GPI) molecules from the immunoglobulin superfamily (neural cell adhesion molecule 120 kD isoform, F3, Thy1) whose expression is developmentally regulated. A hybrid cell line (ND26), made by fusing postmitotic rat dorsal root ganglion (DRG) neurons with the mouse neuroblastoma N18Tg2, could be induced to differentiate by manipulating the composition of the culture medium and expressed similar GPI molecules to DRG neurons. We used this model system to investigate the metabolism of GPI-anchored molecules. We found that neural cell adhesion molecule 120 Kd isoform expression decreased upon differentiation, whereas the level of F3 and Thy1 increased, suggesting a role in neurite outgrowth processes.The ratio of molecules cleavable by exogenous phosphatidylinositol phospholipase C (PI-PLC) was similar for all the GPI-anchored molecules, which could mean that cell-specific modifications of the basic anchoring structure determine the level of potentially releasable molecules. Measurements of spontaneous release indicated that this reflected the overall level of expression of these molecules by the ND26 cell line.Finally, we observed an effect of dibutyryl cAMP on the level of expression of F3 and Thy1 but not of N-CAM. However, we could not detect any significant effect of nerve growth factor (NGF) either on the level of expression or on the amount of spontaneously released molecules.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240480110
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