bluuebunny/crossref_metadata_2025_split huggingface dataset:
Homologous and heterologous desensitization of proto‐oncogene cfos expression in murine peritoneal macrophages
Authors: Introna Martino , Bast Robert C. , Johnston Paul A. , Adams Dolph O. , Hamilton Thomas A. 
Published: February 2005

AbstractTreatment of murine peritoneal macrophages for 30 min with lipopolysaccharide (LPS) resulted in a transient increase in c‐fos proto‐oncogene mRNA levels (Introna et al., 1986). After 2 h from the initial treatment, c‐fos mRNA could no longer be detected and its expression could not be restimulated either by LPS or by other signals including colony stimulating factor‐1 (CSF‐1) and phorbol myristate acetate (PMA), both of which are able to induce expression of the c‐fos gene in unstimulated macrophages. When LPS was removed after an initial 30 min incubation, responsiveness to a second exposure to LPS began to reappear after 3 h and was completely restored by 20 h. The same pattern of desensitization of c‐fos induction was observed when CSF‐1 stimulated macrophages were subsequently exposed to LPS. The loss of sensitivity to PMA following pretreatment with LPS was selective for c‐fos expression as LPS treated macrophages remained responsive to PMA with respect to the ability to stimulate secretion of H2O2. The mechanism of desensitization was localized, at least in part, at the level of transcription as demonstrated by analysis of c‐fos transcripts in nuclei isolated from macrophages pretreated and restimulated with LPS.
Magnesium deprivation inhibits the expression of differentiation‐related phenotypes in human promyelocytic leukemia HL‐60 cells
Authors: Okazaki Toshiro , Mochizuki Toshihiro , Tashima Masaro , Sawada Hiroyoshi , Uchino Haruto 
Published: February 2005

AbstractThe role of magnesium ions in the differentiation of human promyelocytic leukemia HL‐60 cells was investigated. When HL‐60 extracellular magnesium was deficient (less than 0.01 mM), the total intracellular magnesium content and [3H] leucine incorporation rates decreased to 61 and 28%, respectively, on day 3. When the cells were treated with various inducers (100 nM 1α, 25 dihydroxyitamine D3 (1,25(OH)2D3), 100 nM β‐all‐trans retinoic acid (RA), 20 nM 12‐o‐tetradecanoyl phorbol‐13‐acetate (TPA), 1.25% dimethylsulfoxide (DMSO) and 30 nM aclacinomycin (AcM)) in magnesium‐deficient medium, the expression of differentiation‐related phenotypes (nitroblue tetrazolium (NBT) reducing ability, nonspecific esterase (NSE) activity and monoclonal antibody, OKM1 binding activity) was almost completely inhibited. After a 2‐day treatment with 100 nM 1,25(OH)2D3 in magnesium‐deficient medium, the expression of differentiation‐related phenotypes was restored by further incubation in the absence of inducer in standard magnesium medium (0.4 mM). These results suggested that magnesium deprivation inhibited the expression of HL‐60 differentiation‐related phenotypes but not their commitment to differentiation. These phenotypes were expressed without inducer in standard magnesium medium after a 2‐day simultaneous treatment with 1,25(OH)2D3 and cyclohexamide (protein synthesis inhibitor) in magnesium‐deficient medium, but not after simultaneous pretreatment with 1,25(OH)2D3 and α ‐amanitin (RNA synthesis inhibitor). Thus, it was suggested that the magnesium‐requiring step in HL‐60 cell differentiation is in protein but not mRNA synthesis. This conclusion is supported by the findings that changes in c‐myc and c‐fms mRNA levels in HL‐60 cells treated with 100 nM 1,25(OH)2D3 in magnesium‐deficient medium and those in standard magnesium medium were the same. In addition, dibutyryl cyclic adenosine monophosphate (dbc AMP) could restore expression of differentiation‐related phenotypes inhibited by magnesium deprivation but not those inhibited by cyclohexamide, even though magnesium deprivation inhibited protein synthesis as much as did cyclohexamide. This suggests that magnesium‐requiring step in HL‐60 cell differentiation is different from that inhibited by cyclohexamide.
Exposure to caffeine and suppression of DNA replication combine to stabilize the proteins and RNA required for premature mitotic events
Authors: Schlegel Robert , Croy Robert G. , Pardee Arthur B. 
Published: February 2005

AbstractCaffeine had been shown to induce mitotic events in Syrian hamster fibroblast (BHK) cells that were arrested during DNA replication (Schlegel and Pardee, Science 232:1264–1266, 1986). Inhibition of protein synthesis blocked these caffeine‐induced events, while inhibition of RNA synthesis showed little effect. We now report that the protein(s) that are required for inducing mitosis in these cells were synthesized shortly after caffeine addition, the activity was very labile in the absence of caffeine, and the activity was lost through an ATP‐dependent mechanism. Caffeine dramatically increased the stability of these putative proteins while having no effect on overall protein degradation. Experiments with an inhibitor of RNA synthesis indicated that mitosis‐related RNA had accumulated during the suppression of DNA replication, and this RNA was unstable when replication was allowed to resume. These results suggest that the stability of RNA needed for mitosis is regulated by the DNA replicative state of the cell and that caffeine selectively stabilizes the protein product(s) of this RNA. Conditions can therefore be selected that permit mitotic factors to accumulate in cells at inappropriate times in the cell cycle. Two‐dimensional gel electrophoresis has demonstrated several protein changes resulting from caffeine treatment; their relevance to mitosis‐inducing activity remains to be determined.
Heparin modulation of the neurotropic effects of acidic and basic fibroblast growth factors and nerve growth factor on PC12 cells
Authors: Neufeld Gera , Gospodarowicz Denis , Dodge Linda , Fujii Dennis K. 
Published: February 2005

AbstractNerve growth factor (NGF) and acidic or basic fibroblast growth factor (aFGF and bFGF, respectively) induce neurite outgrowth from the rat pheochromocytoma cell line, PC12. The neurites induced by these three factors are stable for up to a month in cell culture in the continued presence of any of the above growth factors. bFGF (ED50 = 30 pg/ml) is 800 fold more potent in stimulating neurite outgrowth than aFGF (ED50 = 25 ng/ml) and 260 fold more potent than NGF (ED50 = 8 ng/ml). While the neurotropic activities of aFGF and NGF are potentiated by heparin, that of bFGF is both partially inhibited or stimulated, depending upon the concentration of bFGF. Radioreceptor binding experiments show that aFGF and bFGF bind to a common binding site on the PC12 cell surface. Affinity labeling studies demonstrate a single receptor with an apparent molecular weight of 145,000 daltons, which corresponds to the high molecular weight receptor identified in BHK‐21 cells. NGF does not appear to compete with aFGF or bFGF for binding to the receptor. Heparin blocked the binding of bFGF to the receptor but had only a small inhibitory effect on the binding of aFGF to the receptor. Thus, it appears that heparin inhibition of the neurotropic effects of bFGF occurs, at least in part, by impairing the interaction of bFGF with the receptor, while having little effect on that of aFGF. The stimulatory effects of heparin on the neurotropic activity of aFGF, bFGF, and NGF may occur through a site not associated with the respective cellular receptor for the growth factors.
Temporal requirement for epidermal growth factor and insulin in the stimulation of hepatocyte DNA synthesis
Authors: Sand Tor‐Erik , Christoffersen Thoralf 
Published: February 2005

AbstractPrimary monolayer cultures of adult rat hepatocytes were used to study the temporal interaction of epidermal growth factor (EGF) and insulin in their stimulation of DNA synthesis. The hepatocytes were cultured both under defined conditions and with serum. EGF and insulin interacted synergistically. The entry into S phase (G1 exit) followed first‐order kinetics both in untreated and hormone‐stimulated cells. Addition of EGF and insulin at the time of plating did not alter the lag period before the DNA synthesis started (25–26 h), but the rate constant for the S phase entry increased five‐ to sixfold. Experiments where the time of hormone addition was varied indicated that insulin exerted its strongest effect at the time of plating, whereas the cells became more responsive to EGF after being cultured for up to 40–50 h. The responsiveness to EGF at these later stages required an early exposure of the hepatocytes to insulin. When the administration of EGF to insulin‐pretreated hepatocytes was postponed for 44 h after plating in serum‐free medium, the cellular sensitivity was increased as compared to EGF treatment at 0 h (a one‐log shift of the dose‐effect curve), the rate of S phase entry was more rapid, and the lag period for the onset of the EGF effect (i.e., shift of rate constant) was shortened (6–7 h vs. 26 h).
Resolution of multiple endosomal compartments associated with the internalization of epidermal growth factor and transferrin
Authors: Gorman Regina M. , Poretz R. D. 
Published: February 2005

AbstractMorphological studies have indicated divergent pathways for the endocytosis of epidermal growth factor (EGF) and transferrin (Tf). In order to obtain biochemical evidence for the pathways associated with the endocytosis of EGF and Tf, a series of Percoll density gradients were employed to separate individual cellular components. Subcellular fractionation of murine fibro‐blasts exposed to a 2‐min pulse of either 125l‐Tf or 125I‐EGF results in the detection of a total of six cellular compartments related to the internalization process of these ligands. The results of kinetic analysis of the entry of EGF into five membranous fractions is consistent with a model in which ligand is transferred sequentially from the plasma membrane through three distinct prelysosomal environments prior to reaching secondary lysosomes. Each prelysosomal compartment exhibits distinct density and temporal properties in a Percoll density gradient and may represent preexisting endocytic vesicles and/or specific domains of a continuous tubular structure, vesicularized during the process of cell disruption. In addition, the observed differential migration on Percoll density gradients of Tf and EGF containing compartments indicates that the majority of cell bound Tf segregates from EGF and enters a compartment lacking EGF within 5 min of internalization.
Fibronectin‐associated transforming growth factor
Authors: Fava R. A. , McClure D. B. 
Published: February 2005

AbstractWe have studied the ability of fibronectins to induce anchorage‐independent growth of NRK‐49F cells in serum‐free medium. Cells were seeded in soft agar in the presence of various concentrations of plasma fibronectins, and colonies were counted after 10 days. It was found that, with some exceptions, human plasma fibronectins induced anchorage‐independent growth at concentrations in 20–100 μg/ml range. The ability of exogenously supplied fibronectins to promote anchorage‐independent growth of NRK cells is attributed to a transforming growth factor (TGF) activity associated with gelatin‐agarose affinity purified plasma fibronectins. This TGF activity required epidermal growth factor (EGF) in our serum‐free assay system. The TGF‐like activity appears to either co‐purify or to be associated with fibronectin at neutral pH during molecular sieve chromatography and during ultracentrifugation through sucrose density gradients. The TGF activity “dissociates” from fibronectin at extremes of pH, however, and can be separated from fibronectin by molecular sieve chromatography in 1 M acetic acid. Under these conditions, the TGF‐like activity chromatographed as a single peak with an apparent molecular weight of approximately 30 kDa. The physical‐chemical properties, chromatographic behavior, and biological activity of this TGF suggest that it is type‐β transforming growth factor/growth inhibitor (β‐TGF/GI). The TGF activity has been observed in fibronectin isolated from fresh human plasma as well as in fibronectins from several other species obtained from commercial suppliers. Our results would suggest that caution be applied in the interpretation of experiments in which gelatin affinity purified fibronectins are used at μg/ml concentrations.
Functional differentiation of virgin mouse mammary epithelium in explant culture is dependent upon extracellular proline
Authors: Smith Gilbert H. 
Published: February 2005

AbstractDepletion of proline from insulin, hydrocortisone, and prolactin‐containing medium prior to incubating virgin mouse mammary explants prevents both DNA synthesis and functional differentiation in the mammary epithelial cells; however, DNA synthesis in the mammary stroma and total incorporation of radioactive amino acids into total protein appears to continue without hindrance. Removal of glycine instead of proline had no deleterious effect on either DNA replication in the hormone‐stimulated epithelium or in its functional differentiation. Functional differentiation was determined by the induction of casein and α‐lactalbumin synthesis in the insulin, hydrocortisone, and prolactin (IFPrl)‐treated explant cultures. As a control, the induction of mouse mammary tumor virus (MMTV) gene expression, a cortiocosteroid‐regulated function, was also measured. Neither the absence of proline or glycine prevented the glucocorticoid stimulation of MMTV gene expression. In contrast to mammary tissue from virgin mice, explants from nonpregnant primiparous mice responded fully to IFPrl stimulation with respect to DNA, casein, and α‐lactalbumin synthesis in medium depleted of proline. These data suggest that the uncommitted epithelium of virgin mouse mammary glands requires the presence of exogenous proline in order to respond to lactogenic hormonal signals. We have demonstrated earlier that DNA synthesis is a prerequisite of functional differentiation in virgin mouse mammary explants (Smith and Vonderhaar, 1981, Dev. Biol., 88:167–179; Vonderhaar and Smith, 1982, J. Cell Sci, 53:97–114), although cytological differentiation proceeded unencumbered in explants prevented from synthesizing DNA. Here, without proline, neither cytological nor functional differentiation can be induced; this suggests that proline provides an essential metabolic interlock in the acquisition of lactogenic hormone responsiveness in uncommitted mouse mammary tissue.
The role of benzodiazepine receptors in the induction of differentiation of HL‐60 leukemia cells by benzodiazepines and purines
Authors: Ishiguro Kimiko , Taft William C. , DeLorenzo Robert J. , Sartorelli Alan C. 
Published: February 2005

AbstractA series of benzodiazepines was evaluated for their capacity to induce the differentiation of HL‐60 acute promyelocytic leukemia cells. Benzodiazepines were effective initiators of maturation in the concentration range of 50 to 150 μM. The possible involvement of benzodiazepine receptors in mediating the differentiation induced by these agents was investigated. The presence of high affinity, peripheral type benzodiazepine binding sites (KD = 7.3 nM, TB = 14.5 pmol/mg protein with Ro5–4864) was demonstrated in HL‐60 membranes. The occupancy of peripheral type high affinity benzodiazepine receptors by various benzodiazepines showed some correlation (r = 0.76) with their differentiation‐inducing capabilities, but binding potencies were 1,000‐fold higher than the concentrations required to produce differentiation. A class of benzodiazepine receptors with lower binding affinity was also detected in HL‐60 membranes (KD = 28.6 μM; TB = 199 pmol/mg protein with diazepam). A higher level of correlation (r = 0.88) was demonstrated between benzodiazepine occupancy of these lower affinity receptors and the capacity to induce maturation. Significantly, benzodiazepine concentrations needed for low affinity binding and induction of differentiation were the same (25–200 μM), suggesting that low affinity benzodiazepine receptors may be involved in the induction process. We have shown that the molecular form responsible for the induction of the differentiation of HL‐60 cells to mature forms by 6‐thioguanine (TGua) is the free base, TGua, itself [Ishiguro, Schwartz, and Sartorelli (1984) J. Cell. Physiol., 121:383–390]. Since hypoxanthine (Hyp) and inosine (Ino) have been identified as putative endogenous ligands for high affinity benzodiazepine receptors in brain tissue, the potential involvement of benzodiazepine receptors in the differentiation of HL‐60 cells by the purines was investigated. Physiological purines such as Hyp and Ino were inactive in displacing the benzodiazepines from their high and low affinity binding sites in HL‐60 membranes. In contrast, TGua caused inhibition of benzodiazepine binding to high and low affinity sites. The inhibition of Ro5–4864 binding to high affinity binding sites by TGua appeared to be due to the binding of TGua to membranes through the formation of a mixed disulfide between the 6‐thiopurine and protein thiols, since the inhibition was reversed by the presence of 2‐mercaptoethanol. The findings suggest a possible relationship between the occupancy of benzodiazepine receptors by TGua and the induction of leukemic cell differentiation.
γ‐glutamyl transpeptidase (γ‐GT) and maintenance of thiol pools in tumor cells resistant to alkylating agents
Authors: Ahmad Shakeel , Okine Laud , Wood Robert , Aljian John , Vistica David T. 
Published: February 2005

AbstractPrevious studies from this laboratory have established that acquired resistance of murine L1210 leukemia cells to L‐phenylalanine mustard (L‐PAM) and other alkylating agents is accompanied by a two‐to threefold elevation in their glutathione (GSH) concentration (Biochem. Pharm. 31:121). In an attempt to gain insight into the mechanism by which resistant tumor cells maintain their increased GSH content, we have assessed the possible role of γ‐glutamyl transpeptidase (γ‐GT), a membrane bound enzyme involved in GSH metabolism. These results indicate that the enzyme is present in both sensitive and resistant murine L1210 leukemia cells but that the cellular content of γ‐GT is elevated two‐to threefold in L‐PAM resistant cells as compared to their sensitive counterparts. This elevation in enzymatic activity correlates well with the increased cellular GSH content in resistant cells. The results of a detailed kinetic analysis of γ‐GT activity indicate that there is no difference, between cell types, in the apparent Km of the enzyme for the γ‐glutamyl donor (L‐γ‐glutamyl‐p‐nitroanilide) or the acceptor (glycylglycine). However, the apparent Vmax is increased two‐to threefold in L‐PAM resistant tumor cells. Investigation into the role of γ‐GT in the extracellular metabolism of GSH indicates that resistant tumor cells metabolize two‐fold more GSH than do sensitive cells and that such metabolism results in a similar difference in the intracellular concentration of cysteine. Results of studies with cellular lysates also indicate a role for the enzyme in the supply of cysteine to the glutathione precursor pool of the tumor cell and in the maintenance of elevated GSH concentrations in cells resistant to alkylating agents.
Modulation of cellular heat sensitivity by specific amino acids
Authors: Vidair Charles A. , Dewey William C. 
Published: February 2005

AbstractWhen either plateau‐phase or exponentially growing Chinese hamster ovary (CHO) cells are incubated in amino acid‐free medium, the cells become sensitized to killing by heat. For cells deprived of amino acids for 12 h survival decreases from 1 × 10−2 for controls to 1 × 10−6 for the deprived cells, following heating at 45°C for 38 min. The survival of these sensitized cells is rapidly increased by the addition of a single amino acid just prior to heating. Of the 21 amino acids which are added in purified form to make McCoy's 5a medium, 12 show no protective effect, four have a small protective effect, and either alanine, asparagine, glutamine, serine, or theronine raise survival to a level similar to that of the control cells. The nonmetabolizable alanine analogue, 2‐aminoisobutyric acid (AIB), increases survival of amino acid‐deprived cells as effectively as each member of the group of five listed above, suggesting that metabolic conversion of the amino acids is not required for their protective effect. The data suggest that an increase in the intracellular concentrations of specific amino acids, independent of any change in cellular ATP content or the rate of protein synthesis, enables these cells to become quickly more resistant to killing by heat. We also conclude that the amino acid concentrations in poorly vascularized regions of some tumors should be considered, along with the oxygen, glucose, and proton concentrations, as factors which determine cellular survival following hyperthermia.
Characterization of the receptor for protease nexin‐I: Protease complexes on human fibroblasts
Authors: Howard Eric W. , Knauer Daniel J. 
Published: February 2005

AbstractFibroblasts as well as several other cell types, secrete a number of protease inhibitors into their culture media. Among these inhibitors are the protease nexins, a class of proteins which covalently bind serine proteases, thereby inactivating their specific targets. Protease nexin‐l, first discovered in human foreskin fibroblasts, binds thrombin, plasmin, and urokinase with high affinity, forming covalently linked complexes. Human fibroblasts bind complexes of protease nexin‐l and its target protease via a cell‐surface, high‐affinity receptor. We have analyzed a number of characteristics of this receptor, and found them to be typical of class II receptors in general. At 4°C binding of PN‐l:protease complexes was competable by heparin. In addition, binding was independent of the particular protease bound to the PN‐l; purified complexes of PN‐l with thrombin or urokinase competed equipotently for [125]l‐thrombin:PN‐l binding. As the pH of the binding buffer was lowered, binding to cells increased. A twofold increase in binding was attained by lowering the pH from 7.5 to 4.5. This phenomenon was not due to irreversible, pH‐induced changes to either the cell surface or the labeled complexes. At 37°C, the removal of labeled complexes from culture medium was rapid; approximately 80% was removed by 4 hours under given conditions. The internalization of complexes was also very rapid, with an estimated ke (endocytic rate cor stant) of 1.0 min−1 At neutral pH, fibroblasts bind complexes in a saturable manner. Scatchard analysis yields areceptor number of 250,000 per cell and a Kd of 1 nM.
Differential control of mRNA levels for Thy‐1 antigen and laminin in rat mammary epithelial and myoepithelial‐like cells
Authors: Barraclough Roger , Kimbell Rosemary , Rudland Philip S. 
Published: February 2005

AbstractThy‐1 antigen and laminin are two components often associated with the basement membrane of the rat mammary gland and are thought to be synthesized, at least in part, by the adjacent myoepithelial cells in vivo. The relative levels of Thy‐1 mRNA and laminin mRNA are compared in a rat mammary cuboidal epithelial cell line and a derivative elongated myoepithelial‐like cell line by hybridizing cloned cDNAs to cellular mRNA isolated from these cell types. Although the elongated myoepithelial‐like cells synthesize four times as much laminin protein as the cuboidal epithelial cells, there is only a 1.7‐fold increase in laminin mRNA between the two cell types. In contrast the 17‐fold increase in Thy‐1 antigen between the elongated cells and the cuboidal cells can be accounted for completely by a 14–18‐fold increase in Thy‐1 mRNA, suggesting that changes in the steady‐state levels of Thy‐1 mRNA in these cell lines are modulated at either a transcriptional or a post‐transcriptional level. Run‐off transcription by nuclei isolated from the cell lines does not distinguish between these two possibilities. The comparative results on Thy‐1 antigen and laminin show that the enhanced production of two proteins often associated with the basement membrane of the rat mammary gland can be controlled at different levels in the elongated myoepithelial‐like cells.