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  • 1
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We previously demonstrated that neurotransferrin (NTF), a transferrin extracted from adult chicken peripheral nerves, promotes growth of primary chick muscle cells in the absence of embryo extract. NTF was shown to stimulate DNA synthesis and cell proliferation. In the present study, we demonstrate that NTF is a mitogen using two independent methods; counts of orcein-stained mitotic figures and analysis of cell cycle kinetics with a fluorescence-activated cell sorter. In low-density cultures mitotic activity increases with increasing doses of NTF followed by a plateau at concentrations greater than 6 μg/ml. Residual, embryonic mitotic activity progressively declines with time after plating muscle cells in the absence of NTF. Absence of NTF for 2 days causes cells to lose irreversibly their myogenic potential. In the presence of NTF, mitotic activity increases for 2 days followed by a decline concurrent with myoblast fusion and formation of myotubes. Cell cycle analysis showed that NTF addition causes cell populations to shift from Gt to S and G2 + M within 18.5 hr. Muscle cells, plated at high densities in the absence of NTF, show mitotic activities similar to those plated at low densities in the presence of NTF. Addition of NTF to high-density cultures is ineffective in stimulating mitosis. These studies show that at typical cell plating densities, NTF is a required mitogen for primary chick muscle cell cultures.
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  • 2
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Prior studies have shown that vitamin D regulation of protein kinase C activity (PKC) in the cell layer of chondrocyte cultures is cell maturation-dependent. In the present study, we examined the membrane distribution of PKC and whether 1α,25-(OH)2D3 and 24R,25-(OH)2D3 can directly regulate enzyme activity in isolated plasma membranes and extracellular matrix vesicles. Matrix vesicle PKC was activated by bryostatin-1 and inhibited by a PKC-specific pseudosubstrate inhibitor peptide. Depletion of membrane PKC activity using isoform-specific anti-PKC antibodies suggested that PKCα is the major isoform in cell layer lysates as well as in plasma membranes isolated from both cell types; PKCζ is the predominant form in matrix vesicles. This was confirmed in Western blots of immunoprecipitates as well as in studies using control peptides to block binding of the isoform specific antibody to the enzyme and using a PKCζ-specific pseudosubstrate inhibitor peptide. The presence of PKCζ in matrix vesicles was further verified by immunoelectron microscopy. Enzyme activity in the matrix vesicle was insensitive to exogenous lipid, whereas that in the plasma membrane required lipid for full activity. 1,25-(OH)2D3 and 24,25-(OH)2D3 inhibited matrix vesicle PKC, but stimulated plasma membrane PKC when added directly to the isolated membrane fractions. PKC activity in the matrix vesicle was calcium-independent, whereas that in the plasma membrane required calcium. Moreover, the vitamin D-sensitive PKC in matrix vesicles was not dependent on calcium, whereas the vitamin D-sensitive enzyme in plasma membranes was calcium-dependent. It is concluded that PKC isoforms are differentially distributed between matrix vesicles and plasma membranes and that enzyme activity is regulated in a membrane-specific manner. This suggests the existence of a nongenomic mechanism whereby the effects of 1,25-(OH)2D3 and 24,25-(OH)2D3 may be mediated via PKC. Further, PKCζ may be important in nongenomic, autocrine signal transduction at sites distal from the cell. © 1996 Wiley-Liss, Inc.
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  • 3
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Tryptophan transport has been examined in A9 and in mutants resistant to 5-fluorotryptophan (5-FT). Evidence indicates that in A9 cells two systems are present for tryptophan transport, which are analogous to the A and L systems found in Ehrlich ascites cells differing, however, in terms of amino acid specificity. Tryptophan uptake via the L system, a high affinity, low capacity system, is Na+ independent and occurs by a counter transport mechanism, while uptake via the A system, a low affinity, high capacity system, is Na+ dependent. Alanine, arginine, lysine, proline, asparagine, and aspartate (listed in order of decreasing inhibitory effect) inhibit tryptophan uptake via the A system from approximately 80-50% while having no inhibitory effect on the L system. In addition, glutamine which inhibits tryptophan uptake by 80% via the L system only inhibits to the extent of 20% via the A system. Previous kinetic studies of 5FT resistant clone FTr37 indicated system A was altered while the analysis of the effects of the mutation on system L was inconclusive. However, in these studies Na+ independent uptake was not altered in FTr 37 indicating system L was not affected. Amino acid competition studies confirmed this observation and suggested that a change in the specificity of system A had occurred in FTr 37. The amino acid competition studies in FTr 23, indicated that the specificities of both systems differed from A9. The possibility that this change may be due to a single mutational event is discussed.
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  • 4
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Results of hemacytometer cell counts and of tyrosinase measurements made by the Pomerantz method demonstrate that imidazole added to the medium of cultured B 16 mouse melanoma cells can stimulate tyrosinase specific activity and inhibit cell division. These effects are greater than with adenosine 3′,5′ cyclic monophosphate (cAMP) or the cAMP-phosphodiesterase inhibitor theophylline. The effects of imidazole on cell division and tyrosinase are enhanced by theophylline and antagonized by cAMP. Cyclic AMP-phosphodiesterase activity in cell-free extracts can be inhibited by theophyllne and stimulated by imidazole. However, imidazole does not affect cAMP-phosphodiesterase specific activity in vivo, nor does it affect intracellular cAMP concentrations as determined by competitive protein-binding assays. In contrast, the specific activity of cAMP-phosphodiesterase in vivo is stimulated by cAMP and theophylline, supporting the hypothesis that cAMP and agents which increase intracellular cAMP concentrations induce the synthesis of cAMP-phosphodiesterase. Studies with actinomycin-D and cycloheximide support the hypothesis that cAMP can also mediate posttranslational activation of tyrosinase. Similar experiments suggest that imidazole, or a derivative therof, can induce the synthesis of tyrosinase at the pretranslational level of control. We hypothesize that this type of regulation (pretranslational) by imidazole may define a role for the concept of “Metabolite Gene Regulation” (MGR), in mammalian cells.
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  • 5
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The effect of inhibitors of RNA synthesis on 1,25(OH)2 vitamin D3-induced monocytic differentiation was studied in a well-differentiating clone AB 47 of HL 60 cells. The concentrations of these inhibitors were chosen to permit the maintenance of cell viability for at least 48 hours, and resulted in 40-60% inhibition of total cellular RNA synthesis. No impairment of 1,25(OH)2 vitamin D3-induced monocytic differentiation was observed with all inhibitors tested, and the presence of cordycepin actually enhanced differentiation. The phenotypic evidence of monocytic differentiation correlated with the increased levels of mRNA for c-fos and c-fms measured by hybridization to appropriate nick-translated cDNA probes. In contrast, nuclear run-on experiments showed the expected inhibition of transcription of these genes by the compounds used. The data suggest that interference by external agents with transcription of genes essential for a differentiation program brings into play compensatory mechanisms which permit the program to continue. Thus, differentiation appears to have a high priority among various competing intracellular pathways in 1,25(OH)2 vitamin D3-treated HL 60 cells. Stabilization of messenger RNA levels evident in this study may therefore represent a general cellular mechanism for the correction of unwanted effects of xenobiotics on the cell.
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  • 6
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Endothelial cell differentiation into capillary structures is a complex process that requires the concerted effects of several extracellular matrix proteases, including plasminogen activators. Here, the role of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) was evaluated in an in vitro model of endothelial morphogenesis involving organization of human umbilical vein endothelial cells into tubular structures when they are cultured on the basement membrane preparation, Matrigel. Both uPA and tPA were detected in HUVEC cultures on Matrigel, and inhibitors of plasminogen activators or of serine proteases decreased the extent of the tube network formed by the cells. The decrease resulting from serine protease inhibitors was additive to that from matrix metalloproteinase inhibitors which have previously been shown to decrease tube formation in this model, suggesting that the two classes of proteases modulate tube formation by distinct mechanisms. Plasminogen activator inhibitor (PAl)-1 decreased tube formation by 50% when added up to 4.5 h after the initiation of an 18 h assay and caused 25% inhibition when added 9.5 h after culture initiation, indicating that the effects of plasminogen activators are not limited to an early event in the differentiation process. Steady-state expression of mRNA for uPA increased during the first several hours of culture on Matrigel, further supporting a role for PA activity throughout the process of tube formation. These findings suggested that PAs may affect multiple events during tube-forming activity. A fucosylated peptide comprising the amino-terminal domain of uPA that binds to the uPA receptor (uPAR) but lacking proteolytic activity enhanced tube formation. In contrast, a defucosylated form of the same peptide had no effect. Since fucosylation of this fragment has been shown to be essential in other models of cell stimulation by uPA-uPAR interaction, these data support the hypothesis that uPA enhances endothelial morphogenesis both through proteolytic activity and via uPAR occupancy. Plasminogen activators could facilitate angiogenesis in vivo. © 1995 Wiley-Liss Inc.
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  • 7
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: CHO-K1 requires proline for growth. Two proline-independent revertants were isolated - K1-J and K1-6. CHO-K1 pro- is much more sensitive than the pro+ cell lines to inhibition of growth by addition to the medium of amino acids and amino acid analogues that are transported through the A system. In contrast, pro+ cells are as sensitive as, or in some cases slightly more sensitive than, pro- cells to glycine, basic amino acids, and to amino acids that are mainly transported by the L system. The A system analogue α(methylamino) isobutyric acid (MAIB) in low concentrations reacts competitively with proline to regulate the growth of pro- cells, yielding a Ki for MAIB of 0.56 mM. CHO-K1 and K1-6 transport proline at the same initial rate and are equally sensitive to the inhibition of proline transport by alanine. Alanine and MAIB inhibit proline transport strongly and similarly in CHO-K1. Thus although these compounds inhibit the transport of proline by both cell types to the same extent, pro+ cells are immune to the effect of this starvation since they are able to synthesize their own proline. We also describe a secondary inhibition caused by high A system amino acid concentrations that affects both pro- and pro+ cells.
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  • 8
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The involvement of soluble growth-promoting factors in stretch-induced hypertrophy of the Patagilis muscle (PAT) in the chicken wing was investigated. Soluble extracts were prepared from young chicken PAT muscle made hypertrophic by passive stretch and from unstretched contralateral controls. Extracts were tested for their ability to stimulate cell proliferation and creatine phosphokinase (CPK) activity in primary monolayer cultures of chick embryo muscle cells.Factors were present in muscle extracts which showed a dose-dependent stimulation of cell proliferation and CPK activity in vitro. Passive stretch for 5 days produced a rapid hypertrophy of the PAT which was accompanied by a dramatic increase in the activity of the growth factor(s). Release of stretch resulted in an arrest of growth and an immediate fall in growth factor activity.The difference in growth-stimulating activity between control and stretched PAT extracts could be demonstrated in chicken transferrin-sensitive chick myoblast cultures. Stretch thus induces an increase in a class-specific growth factor, possibly Transferrin, in the PAT.Stretched PAT extracts stimulated: (a) chick myoblast proliferation to a greater extent than an optimum concentration of chick embryo extract, and (b) CPK activity in vitro to a greater extent than excess Transferrin. Both control and stretched PAT extracts supported the growth of rat myoblasts. We conclude that PAT muscle extracts also contain unknown growth factor(s) which are different from Transferrin.
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  • 9
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: When amino acids that are generally transported through the A system are added to derepressed cultures of CHO-K1 cells or to cultures that are undergoing starvation-derepression, as in the co-repressor (co-r), co-inactivator (co-i), (co-ri) assay, the A system undergoes trans-inhibition, inactivation, and repression. The effect of inactivation and repression is not related to the ability of amino acids to bind to the A system transporter but supports a model in which these amino acids act as co-r's/co-i's, and by binding to a aporepressor/inactivator (apo-ri), the product of gene R1, convert it into a repressor/inactivator (ri). For example, β-alanine acts as a strong co-r but does not inhibit proline transport through the A system. Hydroxyproline and histidine, although poor inhibitors of proline transport, are very effective as co-ri's. Diaminobutyrate, phenylalanine α-keto glutarate, pyro-glutamate, isoleucine, and valine, compounds that inhibit A system transport, listed in decreasing order of effectiveness, are all equally poor as co-ri's. Also the Km for the transport of 2-(methylamino)isobutyric acid (MeAIB) through the A system is two times the concentration of MeAIB required to produce one-half inactivation. Amino acid effectors and mutation can modify the conversion of the apo-ri to repressor (r) and inactivator (i). The apo-ri is converted by alanine, serine, proline, and MeAIB to ri, by β-alanine and tryptophane to r, and by hydroxyproline to r and reduced i. The full constitutive and partial constitutive mutants alar4 and alar2, respectively, are in the same complementation group. Alar4 has no active apo-ri while the rate of derepression of alar2 is twice and the inactivation rate is equal to that of the parent culture.
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  • 10
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The effect of estrogens on tyrosinase (EC 1.14.18.1) activity was studied in B16/C3 melanoma cultures. Estradiol, estriol, and other related steroids failed to influence tyrosinase activity when added to the medium of proliferating cultures. Imidazole (10 mM), on the other hand, induced the activity of that enzyme 3-fold, as reported previously. Estradiol and estriol blocked imidazole induction, however, unlike the other estrogenic compounds. The blockade occurred within 15 min of hormone addition and was reversible. Dose-response studies revealed that the maximal estradiol effect occurred at 0.75 nM and the half-maximal effect occurred at 0.5 nM. Estriol was more potent, with the maximal blockade occurring at ∼ 0.5 nM and half-maximal effect at 0.25 nM. The induction of tyrosinase by imidazole and the blockade of this induction by estradiol and estriol could not be demonstrated in broken cell preparations, suggesting that direct enzyme activation-inactivation was not involved. Studies utilizing inhibitors of protein and RNA synthesis suggest that this effect is mediated at a pre-translational level and is independent of mRNA destabilization.
    Additional Material: 6 Ill.
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