bluuebunny/crossref_metadata_2025_split huggingface dataset:
Loss of α2‐macroglobulin and epidermal growth factor surface binding induced by phenothiazines and naphthalene sulfonamides
Authors: Dipaola Mario , Keith Charles H. , Feldman David , Tycko Benjamin , Maxfield Frederick R. 
Published: February 2005

AbstractWe have found that certain naphthalenesulfonamides [e.g., N‐6(‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide (W‐7)] and phenothiazines [e.g., trifluoperazine (TFP)] induce a loss of cell‐surface receptors for α2‐macroglobulin and epidermal growthfactor (EGF) in fibroblasts. The loss of α2‐macroglobulin receptors is independent of receptor occupancy and is rapidly reversed upon removal of these agents from the culture medium. The extent of EGF receptor loss is less than for α2‐macroglobulin, and the EGF receptors do not reappear at the surface when W‐7 isremoved. Receptor loss was measured as a change in the capacity for binding iodinated ligands;nochange in affinity of binding was observed. This receptor loss could reflect inactivation of receptors or internalization. W‐7 did not induce a loss of cell surface β2‐microglobulin, a membrane protein which is excluded from coated pits and which isnot internalized, indicating that the effect of W‐7 was specific for membrane receptorsandnot a result of bulk depletion of plasma membrane. The loss of α2‐macroglobulin and EGF receptors occurs at concentrations which do not cause an increase in the pH of endocytic vesicles or the cytoplasm, indicating that these agents act by a mechanism distinct from the effect of other weak bases. Since both TFP and W‐7 are potent inhibitors of calmodulin, we investigated the possibility that inhibition of calmodulin was responsiblefor the loss of receptors. Three lines of evidence suggest that calmodulin inhibition is not responsible for the inhibition of binding and endocytosis: (1) Promethazine, a phenothiazine that is a poor inhibitor of calmodulin, is nearly as effective as TFP at inhibiting endocytosis; calmidazolium, a potent inhibitor of several calmodulin functions, did not cause a loss of binding; (2) the microinjection of calmodulin into cells did not reverse the effects of W‐7; using pressure microinjection, we introduced up to a 100‐fold excess of calmodulin over native levels into individual gerbil fibroma cells; using rho‐damine‐labeled α2‐macroglobulln, we saw that the W‐7 induced inhibition of receptor‐mediated endocytosis was the same in injected and uninjected cells; (3) we injected calcineurin, a calmodulin‐binding protein, into cells (1‐3 pg/ cell) and observed no effect on the receptor‐mediated endocytosis of rhoda‐mine‐labeled α2‐macroglobulin. These data indicated that cell surface receptor numbers can be regulated by a cellular component that is not cytoplasmic calmodulin but that shares some drug sensitivities with calmodulin.
Induction of ornithine decarboxylase, RNA, and protein synthesis in macrophage cell lines stimulated by immunoadjuvants
Authors: Prosser Frederick H. , Schmidt Christopher J. , Nichols Scott V. , Nichols William K. 
Published: February 2005

AbstractEarly biochemical changes associated with adjuvant stimulation of macrophage protein synthesis were studied using two murine macrophage cell lines, PU5‐1.8 and J774.1. An induction of ornithine decarboxylase (ODC) was detected 2 hours after exposure of PU5‐1.8 and J774.1 cells to two crude immunoadjuvants, BCG cell walls (BCGcw) and lipopolysaccharides from Escherichia coli (LPS). The chemically defined immunoadjuvant glycopeptide, N‐acetyl‐muramyl‐L‐alanyl‐D‐isoglutamine (MDPL) also promoted an increase in ODC activity at 2 hours that was maximal after 4 hours, while little or no effect was observed with the D‐alanyl analog (MDPD) that is devoid of adjuvant activity. The increase in ODC activity promoted by BCGcw in PU5‐1.8 and J774.1 cells returned toward control levels by 6 to 8 hours. BCGcw also stimulated RNA and protein synthesis which remained elevated for at least 24 hours and was associated with a decrease in DNA synthesis and cell proliferation. ODC induction by BCGcw and MDPL was enhanced by the addition of PGE2 in both cell lines. Indomethacin slightly depressed the magnitude of ODC stimulation by BCGcw in J774.1 cells but failed to alter the response of PU5‐1.8 cells. Additional observations indicated that the induction of ODC by BCGcw in both cell lines was preceded by an activation of cyclic AMP‐dependent protein kinase. These observations suggest that a cyclic AMP‐mediated induction of ODC may be an early biochemical marker of adjuvant stimulation in macrophages.
Regulation of production of a platelet‐derived growth factor‐like protein by cultured bovine aortic endothelial cells
Authors: Fox Paul L. , Dicorleto Paul E. 
Published: February 2005

AbstractPlatelet‐derived growth factor (PDGF) is a potent mitogen for cultured cells of mesenchymal origin. Known sources of PDGF or PDGF‐like protein are blood platelets, several transformed cell lines, and cultured endothelial cells (EC). We have examined the regulation of production of a PDGF‐like protein in cultures of bovine aortic EC using a specific radioreceptor assay for PDGF. EC constitutively secreted PDGF‐like protein into serum‐containing or serum‐free medium. The rate of production of PDGF‐like protein was constant for at least 3 weeks and was not due to release of an internal store, since cell lysis by repeated freeze/thaw cycles did not relase significant amounts of the protein. Synthesis of PDGF‐like protein was sensitive to changes in the pH of the media and was maximal at pH 8.5. Production of PDGF‐like protein was independent of EC growth rate: rapidly dividing cells and confluent, quiescent cells produced equal amounts per cell. However, sparse, quiescent EC produced more PDGF‐like protein per cell than did confluent, quiescent cells. Several phorbol esters stimulated production of PDGF‐like protein. At a concentration of 10−6 M, a twofold stimulation was observed upon addition of the tumor promoter 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) and nearly a fourfold stimulation upon addition of the nonpromoting analog, methyl TPA. Incubation of EC with endotoxin (10 μ/ml) resulted in a twofold stimulation of PDGF‐like protein production. In all experiments with endotoxin and phorbol esters, an increase in the production of PDGF‐like protein was accompanied by morphological changes in the EC cultures. The cells appeared elongated and fibroblastic and exhibited low viability. A mathematical model was developed in which PDGF‐like protein production was shown to consist of two separate components—production at a constant rate by healthy cells and a large burst of synthesis and secretion by dying cells. These results suggest that injurious agents may be capable of stimulating production of a growth factor by the endothelium.
Characterization of the metabolic forms of 6‐thioguanine responsible for cytotoxicity and induction of differentiation of HL‐60 acute promyelocytic leukemia cells
Authors: Ishiguro Kimiko , Schwartz Edward L. , Sartorelli Alan C. 
Published: February 2005

AbstractHL‐60 human acute promyelocytic leukemia cells that lack hypoxanthineguanine phosphoribosyltransferase (HGPRT) activity have been developed by mutagenization and selection. These cells exhibited markedly decreased sensitivity to the cytotoxic action of 6‐thioguanine (TG) and, in contrast to parental HL‐60 cells, had the capacity to undergo terminal granulocytic differentiation after treatment with this purine antimetabolite. Analysis of extracellular and intracellular metabolites of TG revealed negligible metabolism of TG in these HGPRT− HL‐60 cells. These findings are consistent with the concept that inhibition of cellular replication requires generation of analog nucleotide and suggest that TG itself is capable of initiation of differentiation. 6‐Thioguanosine (TGuo) had limited activity, while β‐2′‐deoxythioguanosine (dTGuo) was inactive, as an inducer of maturation of HGPRT− HL‐60 cells. These cells converted relatively large amounts of the nucleosides to the free base TG; the simultaneous exposure of cells to 8‐aminoguanosine (AGuo), an inhibitor of purine nucleoside phosphorylase activity, decreased the degradation of TGuo and dTGuo to TG and promoted the intracellular accumulation of TG nucleotides, presumably through the action of nucleoside kinase activities. In a double mutant deficient in both HGPRT and deoxycytidine kinase (DCK) activities, dTGuo was devoid of cytotoxicity and was an effective inducer of maturation. The potency of dTGuo as an inducer in this system was not significantly affected by the presence of AGuo. These results suggested that dTGuo itself was also an active initiator of maturation. Thus, induction of differentiation appeared to be due to the free base, TG, as well as its deoxynucleoside form, dTGuo, whereas the formation of TG nucleotides appeared to antagonize maturation and produce cytotoxicity.
Density‐associated loss of functional receptors for somatomedin‐C/insulinlike growth factor I (SM‐C/IGF‐I) on cultured human fibroblast monolayers
Authors: Rosenfeld Ron G. , Dollar Laura A. , Conover Cheryl A. 
Published: February 2005

AbstractThe mitogenic activity of somatomedin‐C/insulinlike growth factor‐I (SM‐C/IGF‐I) appears to be greatly influenced by cell culture conditions, especially the presence of other growth factors and nutrients in the culture medium. To investigate the effect of cell density on SM‐C/IGF‐I activity, we have evaluated SM‐C/IGF‐I binding and stimulation of DNA synthesis and cell replication as a function of cell density in cultured human fibroblast monolayers. At fibroblast concentrations of 2.7 × 105 and 1.48 × 106 cells per 60‐mm dish, specific binding of [125I]SM‐C/IGF‐I per 106 cells was 170% higher in sparse than dense monolayers (9.3% vs. 3.4%). Increased binding in sparse monolayers was attributable to approximately twice as many receptors in sparse as in dense cells (31,000 vs. 16,000 sites per cell), as well as to a modest increase in the affinity constant. Similarly, half‐maximal stimulation of [methyl‐3H]thymidine incorporation was achieved at SM‐C/IGF‐I concentrations of 2.5 ng/ml in sparse cells but required 20 ng/ml in dense cells. Although this required only 45% occupancy of membrane receptors on sparse cells, and almost 80% occupancy on dense cells, the total number of occupied receptors was similar in both sparse and dense cells (approximately 13,000 receptors/cell for half‐maximal stimulation). The presence of increased numbers of “functional receptors” on sparse fibroblasts thus results in enhanced sensitivity to SM‐C/IGF‐I stimulation of DNA synthesis and cell replication. Progressive decreases in the number of functional receptors, secondary to cell crowding, may contribute to density‐dependent inhibition of fibroblast growth.
Treatment with α‐difluoromethylornithine plus a spermidine analog leads to spermine depletion and growth inhibition in cultured L1210 leukemia cells
Authors: Casero Robert A. , Bergeron Raymond J. , Porter Carl W. 
Published: February 2005

AbstractOf the three biological polyamines, putrescine (Put), spermidine (Spd), and spermine (Spm), the relevance of Spm to cell proliferation has yet to be defined because of our general inability to deplete it selectively in intact cells. In the present study, Spm depletion was accomplished by treating cultured L1210 cells for 96 hr with α‐difluoromethylornithine (DFMO) and an analog of Spd such as aminopropylcadaverine. N4‐methylSpd, N4‐ethylSpd, or homoSpd. DFMO, a specific inhibitor of ornithine decarboxylase, halts continued polyamine biosynthesis and the Spd analog serves as a functional substitute for Spd. Thus, while the Spd analog fulfills the role(s) of Spd in cell proliferation, Spm becomes steadily depleted. In cells treated with DFMO plus the analog, aminopropylcadaverine, Spm pools decline steadily and growth inhibition occurs after 48 hr (when Spm pools decline to 60% of control). By 96 hr, Spm is ∼ 15% of control and growth is < 30%. Prevention studies with exogenous polyamines confirm a causai relationship between Spm depletion and growth inhibition. The critical levels of polyamines for cell proliferation to take place were found to be 30% of control for Spd and 60% for Spm. The use of DFMO plus a Spd analog is proposed as a system for studying the cellular consequences of Spm depletion. Spd depletion can be achieved for comparison purposes by treating cells with DFMO alone.
Urokinase‐type plasminogen activator and its inhibitor secreted by cultured human monocyte‐macrophages
Authors: Saksela Olli , Hovi Tapani , Vaheri Antti 
Published: February 2005

AbstractHuman blood monocytes in culture differentiate to macrophagelike cells within 1 week. Coinciding with this morphological transition the cells started releasing increasing amounts of the serine proteinase plasminogen activator (PA; Mr 56,000) of the urokinase (u‐PA) type and the proteinase inhibitor alpha‐2‐macroglobulin (α2M). Unlike the cell‐associated PA activity, which was also readily detected in fresh monocytes, the activity secreted into the serum‐free culture medium could be measured only after treatment of the samples with sodium dodecyl sulphate. Heat or acid treatment of the medium was not sufficient to reveal the PA activity, suggesting that, apart from α2M, another PA‐inhibiting substance was present in the culture medium. The inhibitor (Mr 65,000) was found to be synthesized by macrophages and specifically inhibited u‐PA activity but not tissue‐type PA (t‐PA) or plasmin activity. Dexamethasone decreased the secretion of PA by differentiated macrophages without affecting the production of α2M or the PA inhibitor. Dexamethasone also inhibited the morphological differentiation of the cells when added to the monocyte‐phase cells.
Quantitation of megakaryocytopoiesis in liquid culture by enzymatic determination of acetylcholinesterase
Authors: Burstein Samuel A. , Boyd Cheryl N. , Dale George L. 
Published: February 2005

AbstractA method has been developed to quantitate megakaryocytopoiesis in culture by measuring acetylcholinesterase synthesized in vitro. Murine marrow cells, treated with diisopropylfluorosphosphate (DFP) to inactivate initial acetylcholinesterase (AchE) present in megakaryocytes and contaminating blood, were set up in Iscove's medium supplemented with 15% DFP‐treated horse serum ± pokeweed mitogen‐stimulated spleen cell conditioned medium (PWM‐SCM) in 96‐well microplates. Following the culture period, Triton X‐100, dithiobisnitrobenzoic acid (DTNB), and acetylthiocholine iodide were added to each well. AchE synthesized in culture cleaved acetylthiocholine to thiocholine, which stochiometrically reduced the colorless indicator DTNB to a highly colored product. Thirty minutes following the addition of substrate, the plates were assayed for activity with a vertical recording photometer. When platelets, freshly prepared bone marrow cells, or cultured marrow were assayed by this method, a linear relationship was observed between optical density (OD) and the number of cells assayed. Moreover, a linear relationship between the number of AchE‐positive megakaryocytes determined histochemically and AchE activity determined spectrophotometrically was observed. Red cells exhibited no activity. Inhibitor studies demonstrated that the activity measured was true AchE. Separation of marrow by density gradient centrifugation showed that the megakaryocyte enriched fraction contained all the AchE while the megakaryocyte depleted fraction contained none. From the data we conclude that this rapid, semiautomated method quantitates megakaryocytic AchE synthesis in culture, and that this method will be a useful assay system for the detection of factors that influence megakaryocytopoiesis.
EGF induces receptor down‐regulation with no receptor recycling in KB cells
Authors: Lyall Raymond M. , Pastan Ira , Willingham Mark C. 
Published: February 2005

AbstractSeveral ligands, including epidermal growth factor (EGF), have been found to negatively modulate or down‐regulate their specific plasma membrane receptors. Using both 125I‐EGF and a monoclonal antibody against the EGF‐receptor (EGF‐R1), we studied the down‐regulation of the EGF‐receptor in the human adenocarcinoma cell line KB. The results presented here demonstrate that incubating KB cells at 37°C with EGF rapidly decreases the number of plasma membrane EGF‐receptors. In addition, there is a concomitant rise of equal magnitude in the number of EGF molecules taken up. The latter result argues strongly that there is negligible recycling of the EGF‐receptor in KB cells and that the major portion of internalized EGF‐receptor complexes are transported to lysosomes and subsequently degraded. The fate of the EGF‐receptor is markedly different from that of receptors not subject to down‐regulation. The biochemical signals that operate to regulate such diverse receptor traffic in cells remains to be elucidated.
Bacterial lipopolysaccharide induction of ornithine decarboxylase in the macrophage‐like cell line RAW264: Requirement of an inducible soluble factor
Authors: Taffet Steven M. , Haddox Mari K. 
Published: February 2005

AbstractOrnithine decarboxylase (ODC, EC 4.1.1.17) activity is induced in the RAW264 macrophage‐like cell line by bacterial lipopolysacharide (LPS). As little as 0.1 ng/ml LPS promoted an increase in ODC activity, while maximal ODC activity (30‐fold above control) was induced with 1.0 μg/ml LPS. An increase in ODC activity was detectable within 90 min of LPS addition. The LPS‐induced increase in ODC activity was prevented by inhibitors of protein and RNA synthesis. The induction of the enzyme by LPS was not dependent on prostaglandin production. However, PGE2(1 μg/ml) and 8‐bromo‐cyclic AMP (1mM), neither of which had an effect on ODC activity when added alone, each acted synergistically to enhance the LPS induction of ODC activity. Enzyme induction was not associated with an alteration in Km for ornithine, which remained constant at 0.04 mM. The extent of the increase in ODC in response to LPS increased with increasing cellular density. This relationship was dependent not on absolute cell density of the monolayer but on the cell number in relation to medium volume, and this dependence could be extrapolated to the origin. Addition of conditioned media from LPS‐stimulated but not unstimulated cultures enhanced the ODC increase in sparsely plated cultures in response to a maximal concentration of LPS. The addition of polymyxin B, a reagent that blocks the effects of LPS, including the increase in ODC activity, did not totally inhibit the conditioned medium stimulation. This data indicates that two signals, LPS and a LPS‐induced mediator, are involved in the induction of ODC activity in RAW264 cells.
Neutrophil degranulation: Evidence pertaining to its mediation by the combined effects of leukotriene B4, platelet‐activating factor, and 5‐HETE
Authors: O'Flaherty Joseph T. 
Published: February 2005

AbstractStimulus‐activated polymorphonuclear neutrophils (PMN) produce leukotriene B4 (LTB4), 5‐hydroxyeicosatetraenoate (5‐HETE), and platelet‐activating factor (PAF). Each of these lipids promotes PMN degranulation; in combination they have additive and potentiating effects that result in prominent degranulation responses at relatively low concentrations. Thus, the combined interactions of LTB4, 5‐HETE, and PAF may mediate responses in PMN activated by other stimuli. This possibility was examined by measuring the responses of PMN made insensitive to one or more of these lipids. Cells were pretreated with LTB4, 5‐HETE, and/or PAF for 8 min; exposed for 2 min to cytochalasin B (which is required for lipid‐induced degranulation); and then challenged. PMN challenged with only buffer released minimal amounts of granulebound enzymes. Furthermore, the lipid‐pretreated cells were hyporesponsive to challenge with (1) various combinations of these same lipids or (2) ionophore A23187. The relative potencies of the lipids in producing hyporesponsiveness to themselves or A23187 were: 5‐HETE < PAF ≤ LTB4 < PAF + LTB4 < PAF + LTB4 + 5‐HETE. For both types of challenge, reduced responsiveness occurred in cells pretreated with > 0.1 nM LTB4 and/or > 0.2 nM PAF, persisted in cells washed after lipid pretreatment, and did not develop in cells pretreated with various combinations of bioinactive structural analogues of the lipids. Thus, PAF, LTB4, and 5‐HETE interacted to desensitize PMN, and the degranulating actions of A23187 required cells that were fully responsive to each of the three lipids. This supports the concept that the lipids act together in mediating certain of the ionophore's effects. However, lipid‐desensitized PMN degranulated fully when challenged with C5a, a formylated oligopeptide, or phorbol myristate acetate. Degranulation responses, therefore, may proceed through various pathways, only some of which involve the lipid products studied here.
Isolation of pituitary fibroblast growth factor by fast protein liquid chromatography (FPLC): Partial chemical and biological characterization
Authors: Gospodarowicz Denis , Massoglia Sharon , Cheng Jannie , Lui Ge‐Ming , Böhlen Peter 
Published: February 2005

AbstractBovine pituitary fibroblast growth factor has been purified 222,000‐fold to homogeneity by a combination of differential salt extraction, gel filtration, and ion exchange chromatography on Mono S column. Pituitary FGF is a single‐chain polypeptide with an apparent molecular mass of 15,800 and an isoelectric point of 9.6. It is highly active in triggering the proliferation of bovine and human vascular endothelial cell [half‐maximal stimulation at 23–40 pg/ml (1.5–2.6 pM) and saturation between 140 and 280 pg/ml (9.3–18.6 pM)]. It displays a similar activity on bovine vascular smooth muscle cells, corneal endothelial cells, granulosa and adrenal cortex cells, and rabbit costal chondrocytes.
Activation of collagen synthesis in primary culture of rat liver parenchymal cells (hepatocytes)
Authors: Hata Ryu‐Ichiro , Ninomiya Yoshifumi , Sano Junjiro , Konomi Hiroshi , Hori Hisae , Sunada Hironobu , Tanaka Shizuko , Kabuki Keiichiro , Nagai Yutaka , Tsukada Yutaka 
Published: February 2005

AbstractAn increase in collagen synthesis by hepatic parenchymal cells (hepatocytes) was observed during 8 days in primary culture by the quantification of total [3H]hydroxyproline as a marker of total collagen synthesis and the ratio of [3H]hydroxyproline in the high‐molecular‐weight fraction to total [3H]hydroxyproline as a marker of collagen degradation after incubation of the cells with [3H]proline for 24 h. Type analysis of the collagen produced by the cells after 8 days in culture showed the presence of type I and type III collagens in addition to the components corresponding to type IV and type V (αA and βB) collagens. Only the latter two types were found in the collagens produced by the cells after 2 days in primary culture. (a) The purity of the hepatocytes inoculated was 97%, and the majority of the contaminating small cells were erythrocytes. (b) The rate of serum albumin synthesis, which is a typical function of the hepatocytes, was constant or increased during the culture period. (c) Immuno‐electron microscopic observation indicated the production of type I collagen by the hepatocytes after 8 days in primary culture. These results are explained only by the activation of collagen synthesis in the day‐8 hepatocytes in primary culture.