bluuebunny/crossref_metadata_2025_split huggingface dataset:
Bombesin and phorbol ester stimulate phosphatidylcholine hydrolysis by phospholipase C: Evidence for a role of protein kinase C
Authors: Muir Jane G. , Murray Andrew W. 
Published: February 2005

AbstractBombesin caused a marked stimulation of 32Pi into phosphatidylinositol (Pl), with no apparent lag, and into phosphatidylcholine (PC), after a lag of about 20 min. Stimualtion was blocked by the bombesin receptor antagonist, [D‐Arg1, D‐Pro2, D‐Trp7,9, Leu11] substance P, indicating that the effects on both Pl and PC were mediated through the same receptor. The tumor‐promoting phorbol ester 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA) and dioctanoylglycerol (diC8) both directly activate protein kinase C and in this report were shown to stimulate 32Pi incorporation into PC but not into Pl. In addition, TPA stimulated the release of [3H]choline and [3H]phosphocholine and the accumulation of [3H]diacylglycerol from prelabelled cells. These results strongly suggest that TPA activates a phospholipase C specific for PC. Pretreatment of cells with phorbol‐12, 13‐dibutyrate (PDBu) for 24 h depleted cellular protein kinase C activity and inhibited the ability of TPA to induce these effects suggesting a direct involvement of protein kinase C. Similarly the bombesin stimulation of 32Pi into PC and of [3H]choline and [3H]phosphocholine release was inhibited by PDBu pretreatment. DiC8 and, to a lesser extent, TPA stimulated the translocation of CTP:phosphocholine cytidylyltransferase from the cytosolic to the particulate fraction. DiC8 also stimulated this translocation in cells depleted of protein kinase C. It was concluded that both bombesin and TPA activated protein kinase C leading to activation of a phospholipase C specific for PC.
Effects of temperature, pH elevators, and energy production inhibitors on horseradish peroxidase transport through endocytic vesicles
Authors: Sullivan Peter C. , Ferris Andrea L. , Storrie Brian 
Published: February 2005

AbstractWe investigated the effects of reduced temperature, the pH elevators NH4 CI, monensin, and HEPES (N‐2‐hydroxy‐ethylpiperazine‐N'‐2‐ethanesulfonic acid) buffer, as well as the metabolic poisons NaF/KCN on transport of the fluid phase pinocytic marker, horseradish peroxidase (HRP), to lysosomes in Chinese hamster ovary (CHO) cells. In cell fractionation experiments, these agents appeared to block HRP transit at specific point(s) from “early” to “late” (i.e., low to high density) prelysosomal vesicles and lysosomes. Reduced temperature (17°C) most strongly inhibited HRP transport from low density, early endosomes to lysosomes. In long‐term HRP uptakes at 17°C, marked peroxidase accumulation occurred both in early endosomes and in lysosomes. Loss (reversible pinocytosis) of HRP from “very early” endosomes occurred at 17°C. All three pH elevators including the common media supplement HEPES buffer inhibited transit of internalized HRP into lysosomes. For all three pH elevators, inhibition was most pronounced at the “early” endosome stage. The respiratory inhibitors NaF/KCN also inhibited transport most strongly at the early endosome stage. Together these results suggest that “early” steps in the endocytic transport of HRP are the most sensitive and that the conditions tested may exert direct effects on the processing of endocytic vesicles.
The role of aggregation in embryonal carcinoma cell differentiation
Authors: Smith Steven C. , Reuhl Kenneth R. , Craig Jane , McBurney Michael W. 
Published: February 2005

AbstractCultures of the P19 line of embryonal carcinoma cells differentiate into various cell types including cardiac muscle when aggregated and exposed to medium containing 1% dimethylsulfoxide (DMSO). DMSO‐treated aggregates became completely covered with an epithelial cell type 3 to 4 days following drug exposure. This epithelial cell was tentatively identified as primitive extraembryonic endoderm by its ultrastructural appearance and its possession of cytokeratin intermediate filaments. Muscle cells developed within the interior of DMSO‐treated aggregates. They first became apparent 5 to 6 days after DMSO exposure and were characterized by the presence of striated muscle‐specific myosin, immature myofibrils, and intercalated discs. We determined the proportion of cells developing into epithelium and muscle in aggregates of various sizes and showed that the proportion of epithelium was highest in small aggregates whereas muscle cells developed only in aggregates of relatively large size. The muscle was usually associated with necrotic areas which developed within the interior of large aggregates. Our results suggest that cardiac muscle differentiation in the aggregates requires both the DMSO‐induced formation of an epithelial cell coat and one other condition which may be the proximity to necrotic areas.
Enhanced synthesis of a Mr = 55,000 dalton peptide by senescent human fibroblasts
Authors: Sottile Jane , Hoyle Marian , Millis Albert J. T. 
Published: February 2005

AbstractThe secretory protein profiles of early and late passage cultures of human fibroblasts were compared using polyacrylamide gel electrophoresis. In comparison with early passage cell cultures (40–50% lifespan completed), late passage (> 80% of lifespan completed) cell cultures exhibited enhanced production of several peptides in the Mr range 55–60,000. One of those peptides had an apparent molecular weight of Mr = 55,000 and was constitutively present in the late passage cell conditioned medium. Late passage cell cultures synthesized the Mr = 55,000 peptide in the presence or absence of fetal bovine serum. Serum did not enhance its production by early passage cells. Further, production of the peptide was not induced in early passage cell cultures whose proliferation was arrested either by serum starvation or by contact inhibition. Pulse chase studies demonstrated that the peptide appears in the culture medium within 60 min of labeling. There was no evidence that it is derived via degradation of other proteins present either in early passage or late passage cell conditioned media. Further, the production of the 55,000 dalton peptide did not appear to be regulated by factors present in conditioned media. The peptide was detected in the conditioned media produced by late passage cultures of several different cell strains.
Bombesin induction of c‐fos and c‐myc proto‐oncogenes in Swiss 3T3 cells: Significance for the mitogenic response
Authors: Rozengurt Enrique , Sinnett‐Smith James W. 
Published: February 2005

AbstractBombesin is a potent mitogen for Swiss 3T3 cells and acts synergistically with insulin and other growth factors. We show here that addition of bombesin to quiescent Swiss 3T3 cells causes a striking increase in the levels of c‐fos and c‐myc mRNAs. Enhanced expression of c‐fos (122 ± 14‐fold) occurred within minutes of peptide addition followed by increased expression of c‐myc (82 ± 16‐fold). The concentrations of peptide required for half‐maximal increase in the levels of c‐fos and c‐myc mRNAs were 1.0 and 0.9 nM, respectively. The peptide [D‐Arg1, D‐Pro2, D‐Trp7,9, Leu11] substance P which inhibits the binding of bombesin to its receptor and bombesin‐stimulated DNA synthesis in Swiss 3T3 cells blocked the increase in c‐fos and c‐myc mRNA levels promoted by bombesin. Down‐regulation of protein kinase C by long‐term exposure to phorbol esters prevented c‐fos and c‐myc induction by bombesin. This and other results indicate that the induction of these proto‐oncogenes by bombesin could be mediated by the coordinated effects of protein kinase C activation and Ca2+ mobilization. The marked synergistic effect between bombesin and insulin was used to assess whether the increase in the induction of c‐fos and c‐myc is an obligatory event in cell activation. In the presence of insulin, bombesin stimulated DNA synthesis at subnanomolar concentrations but had only a small effect on c‐fos and c‐myc mRNA levels. This apparent dissociation of mitogenesis from proto‐oncogene induction was even more dramatic in 3T3 cells with down‐regulated protein kinase C. In these cells bombesin stimulated DNA synthesis in the presence of insulin but failed to enhance c‐fos and c‐myc mRNA levels at comparable concentrations. Thus, the induction of c‐fos and c‐myc may be a necessary step in the mitogenic response initiated by ligands that act through activation of protein kinase C but the expression of these proto‐oncogenes may not be an obligatory event in the stimulation of mitogenesis in 3T3 cells by mitogens that utilise other signalling pathways.
Effect of acute serum depletion on Na+ ‐K+ homeostasis in cultured human skin fibroblasts
Authors: Hopp L. , Kuriyama S. , Kino M. , Aviv A. 
Published: February 2005

AbstractIn order to elucidate changes in cell transport behavior of cultured human skin fibroblasts in response to acute serum depletion, we performed uptake and washout of 22Na+ and 86Rb+ as well as measurements of the intracellular Na+ and K+ levels in the presence and absence of ouabain. Pronounced and lasting increase in cellular Na+ and decrease in K+ were observed after removal of fetal bovine serum (FBS) from the medium. The sum of the Na+ and K+ contents (nEq/105 cells) was lower in FBS‐free medium (mean ± SD; 17.3 ± 2.2) than in FBS‐containing medium (26.2 ± 3.8; P < .02). Simultaneously, a decrease in cellular water volume was detected in the FBS‐free medium. The cation uptake and washout data suggest that FBS removal primarily renders the cells more permeable to Na+ and K+ with a secondary stimulation of the ouabain‐sensitive Na+ extrusion mechanism. FBS at a concentration of 0.2% prevented approximately 50% of the maximal increase in the 86Rb+ washout rate constant associated with FBS depletion. Ouabain (2 μM) produced an increase in the 86Rb+ washout rate constant. This effect was substantially larger in cells subjected to medium without FBS (from 0.0303 to 0.2500 min−1) than in fibroblasts incubated in medium with FBS (from 0.0107 to 0.0487 min−1). The cellular K+ content was drastically reduced by ouabain to a level not different in medium with or without FBS (33.9 ± 4.5 to 1.75 ± 0.38 and 16.7 ± 1.4 to 1.4 ± 0.13 nEq/105 cells, respectively). The 22Na+ washout data exhibited a three‐exponential pattern. Analytical solutions of the washout data by means of two models (serial and parallel) with three compartments showed that FBS depletion resulted in increase of the size of all three compartments. It is concluded that in cultured human skin fibroblasts, FBS is essential to the maintenance of a normal Na+ and K+ homeoatasis. The removal of FBS results in dramatic permutation of this homeostasis that develops within minutes and lasts for hours.
Down‐modulation of EGF receptors in cells transformed by the src oncogene
Authors: Wasilenko William J. , Shawver Laura K. , Weber Michael J. 
Published: February 2005

AbstractThe effects of src oncogene expression on epidermal growth factor (EGF) receptors have been investigated in mouse 3T3 and rat‐1 fibroblasts. Transformation of both cell types with src resulted in marked reductions in cellular EGF receptor levels, as assayed by either 125I‐EGF binding or immunoprecipitation of receptor protein from radiolabeled cell lysates. In contrast to cells transformed by other types of retroviral oncogenes, the loss of EGF receptors in the src‐transformed cells did not appear to be due to secreted transforming growth factor‐α (TGF‐α), since such factors were undetectable in culture fluids from the src‐transformed cells. By several criteria of transformation, an EGF‐receptorless cell line infected with src was shown to be transformed, suggesting that EGF receptors themselves are not obligatory to the src transformation process. We suggest that pp60src down‐modulates EGF receptors by an intracellular mechanism and that the loss of the receptors is symptomatic of more general effects of pp60src on the machinery of growth regulation.
Calcium and calmodulin in cellular intoxication with Clostridium difficile toxin B
Authors: Caspar Maria , Florin Inger , Thelestam Monica 
Published: February 2005

AbstractIn cultured human lung fibroblasts treated with Clostridium difficile toxin B, the development of the cytopathogenic effect was inhibited by the proton ionophore monensin but was not affected by some other ionophores. The calcium channel blockers verapamil and LaCl3 protected the cells against intoxication, as did the calmodulin antagonists trifluoperazine, amitriptylin, R 24571, and dansylcadaverine. Since these agents could not prevent intoxication when added after toxin internalization was completed, we suggest that calmodulin and uptake of extracellular calcium are needed for the internalization but not for the cytosolic action of the toxin.
Colcemid effects on B16 melanoma cell progression and aberrant mitotic division
Authors: Bhuyan B. K. , Adams E. G. , Badiner G. J. , Trzos R. J. 
Published: February 2005

AbstractMitotic cells selectively harvested after several h of colcemid treatment are routinely used to obtain synchronized cell cultures. DNA flow cytometry shows that when colcemid‐treated B16 mitotic cells divide, they give rise to daughter cells in G1, some of which contain abnormal amounts of DNA. Two subpopulations appear to exist, one having a DNA content distribution expected of G1 cells, another having a mean DNA content about 0.8 of expected and an SD of DNA content more than 5 times expected. The effect was dependent on dose and duration of exposure to colcemid. Colcemid was more cytotoxic to cells in G2 + M than to G1 + S phase cells, and it slowed the progression of G1 cells to S. These effects of colcemid were much greater in aneuploid B16 melanoma cells than in pseudodiploid Chinese hamster ovary (CHO) cells.
Iron‐transferrin‐induced increase in protein kinase C activity in CCRF‐CEM cells
Authors: Phillips Jerry L. , Boldt David H. , Harper Jamie 
Published: February 2005

AbstractIron transferrin has been found to induce a mean 10‐fold increase in the activity of protein kinase C in CCRF‐CEM cells. This increase was not detectable up to 45 min after treatment of cells with iron transferrin, although after 60 min, a maximal increase in enzyme activity was observed. Similarly, iron transferrin at concentrations of 0.1–0.5 μg/ml did not alter protein kinase C activity, while concentrations of iron transferrin of 1–100 μg/ml induced a maximal increase in enzyme activity. Apotransferrin and iron in the form of ferric citrate, as well as complexes of transferin with copper, nickel, zinc, manganese, and cobalt did not increase protein kinase C activity. Additionally, CCRF‐CEM cells pretreated with either actinomycin D or cycloheximide and then incubated with iron transferrin did not exhibit increased enzyme activity. Treatment with iron transferrin was found to have no effect on protein kinase C activity in normal human peripheral blood lymphocytes and in HL60, Daudi, and U937 cells. However, normal lymphocytes stimulated with phytohemagglutinin for 48 hr exhibited a 2‐fold increase in protein kinase C activity following treatment with iron transferrin. These results indicate a specific effect of iron transferrin on protein kinase C activity in CCRF‐CEM cells and in mitogen‐stimulated human lymphocytes that may occur through increased synthesis of the enzyme.
Commitment to differentiation of murine erythroleukemia cells involves a modulated plasma membrane depolarization through Ca2+‐activated K+ channels
Authors: Arcangeli Annarosa , Ricupero Letizia , Olivotto M. 
Published: February 2005

AbstractThe role of the plasma membrane potential (Δψp) in the commitment to differentiation of murine erythroleukemia (MEL) cells has been studied by analyzing the ionic basis and the time course of this potential in the absence or the presence of different types of inducers. Δψp was determined by measuring the distribution of tetraphenylphosphonium (TPP+) across the plasma membrane and displayed a 22‐hour depolarization phase (from −28 to +5 mV) triggered by factors contained in foetal calf serum (FCS) and followed by a nearly symmetrical repolarization phase. After measuring the electrochemical equilibrium potential of Na+, K+, and Cl−, the relative contribution of these ions to Δψp was evaluated by means of ion substitution experiments and by the addition of ion flux inhibitors (tetrodotoxin [TTX], 4‐acetoamide‐4′‐isothiocyanostilbene‐2,2′‐disulfonate [SITS]) and ionophores (Valinomycin, A23187). The Na+ contribution to Δψp appeared negligible, the potential being essentially generated by K+ and Cl− fluxes. When evaluated by a new mathematical approach, the effects of Valinomycin and A23187 at different times of incubation provided evidence that both the depolarization and the repolarization phase were due to variations of the K+ permeability across the plasma membrane (Pk) mediated by Ca2+‐activated K+ channels. All the inducers tested (dimethylsulfoxide [DMSO], hexamethylen‐bis‐acetamide [HMBA], diazepam), although they did not modify the ionic basis of Δψp, strongly attenuated the depolarization rate of this potential. This attenuation was not brought about when the inducers were added to noninducible MEL cell clonal sublines. Cell commitment occurred only during the depolarization phase and increased proportionally to the attenuation of this phase up to a threshold beyond which the further increase of the attenuation was associated with the inhibition of commitment. The major role of the inducers apparently consisted of the stabilization of the Ca2+‐activated K+ channels, suggesting that a properly modulated Δψp depolarization through these channels is primarily involved in the signal generation for MEL cell commitment to differentiation.
Changes in protein kinase C and cAMP‐dependent kinase in lymphocytes after treatment with 12‐O‐tetradecanoylphorbol‐13‐acetate or concanavalin A: Quantitation of activities with an in situ gel assay
Authors: Grove Deborah S. , Mastro Andrea M. 
Published: February 2005

AbstractPrimary lymphocytes can be stimulated to proliferate by mitogenic lectins such as concanavalin A (Con A). While the phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) alone is not mitogenic for these cells, it can enhance the response to Con A. Previously, protein kinases and phosphorylation have been reported to be important in lymphocyte proliferation. More recently TPA has been found to bind and activate protein kinase C. Therefore, we examined kinase activity in lymphocytes stimulated with the complete mitogen Con A and the comitogen TPA. In order to monitor more than one kinase we used an in situ gel assay and developed the system to compare both protein kinase C and cAMP‐dependent kinases. When total cell extracts were assayed in the presence of histone five major bands of activity were detected by autoradiography of the gel. The bands corresponding to protein kinase C and to cAMP‐dependent kinases were identified by partial purification of the enzymes, by binding of [20‐3H(N)]7‐phorbol‐12, 13‐dibutyrate (3H‐PDBU), and by photoaffinity labelling with 8‐azidoadenosine‐3′:5′‐cyclic monophosphate (8‐N3‐[32P]cAMP). Differential extraction of cell lysate allowed comparison of soluble and particulate kinases.We found that when the preparations from either TPA‐ or Con A‐treated lymphocytes were assayed, protein kinase C activity increased three‐ to four‐fold in the particulate fraction within 5 min after treatment. A concurrent decrease of 30–50% occurred in the cytosol. In contrast, cytosolic cAMP‐dependent protein kinase II increased 1.4‐fold in the same period with Con A. PKI and PKII showed the most significant changes after 24 h of stimulation by Con A when the activity of the holoenzyme decreased to half that of the unstimulated cells. Therefore, although TPA and Con A separately can affect protein kinase C this alone is not sufficient for proliferation to occur.
Cytogenetic evaluation of human endothelial cell cultures
Authors: Nichols Warren W. , Buynak Eugene B. , Bradt Carole , Hill Rosina , Aronson Margaret , Jarrell Bruce E. , Mueller Stephen N. , Levine Elliot M. 
Published: February 2005

AbstractCytogenetic evaluation of serially subcultivated human endothelial cells revealed significant differences between cultures derived from fetal umbilical cords and cultures derived from various vessel sites in adults. A rapid increase in the prevalence of polyploid cells, to levels of 100% in many cases, was detected in human umbilical vein endothelial cell cultures but not in endothelial cell cultures from adult vessels. Because the development of polyploidy has been viewed as one signpost of in vitro senescence, it may be that these in vitro observations of high levels of polyploidy are a reflection of the fact that umbilical tissue is at the end of its in vivo developmental lifespan when studied. Consistent karyotypic alterations also were observed in two clones from adult human abdominal aorta, even though these cultures exhibited low percentages of polyploid cells. Cultures of one clone exhibited a trisomy of chromosome 11, on which there are at least three onc gene loci, and a deletion of chromosome 13 through band q14. A loss of band 13q14 is a prezygotic chromosomal lesion known to predispose to retinoblastoma. In the other clone, two cell populations were observed, and each displayed a chromosomal abnormality A trisomy of the long arm of chromosome 2 was noted in one cell population via a marker chromosome involving 2 and 14. The other cell population exhibited an abnormality of chromosome 2. Neither of these karyotypic alterations was detected in the parent culture from which the clones were derived. The results reported in this study have both practical and theoretical implications. The high incidence of polyploidy in serially cultivated umbilical cultures as well as the occurrence of chromosomal changes in umbilical and aortic cultures testify to the need for cytogenetic monitoring of cell cultures even though they are derived from presumably normal tissue. Cytogenetic changes in the endothelium may be important in atherogenesis and other pathologic states. The conversion of diploid endothelial cells into polyploid endothelial cells may provide a convenient model cell system for studying mechanisms of the development of polyploidy in cells and their relationship to in vitro senscence.
Membrane blebbing is associated with Ca2+‐activated hyperpolarizations induced by serum and α2‐macroglobulin
Authors: Dixon S. J. , Pitaru S. , Bhargava U. , Aubin J. E. 
Published: February 2005

AbstractWe have reported previously that serum and α2‐macroglobulin (α2M) induce Ca2+‐activated hyperpolarizations in the membrane potential of a clonal rat osteosarcoma cell line (ROS 17/2) (Dixon and Aubin, J. Cell. Physiol., 132:215–225, 1987). In this report, we describe morphological changes that accompany these hyperpolarizations. Both cell surface blebbing (zeiosis) and transient hyperpolarizations were induced by application of 10% fetal bovine serum (FBS) or α2M; neither was induced by serum‐free medium, a suspension of latex beads, or purified bovine serum albumin. Following a brief application of FBS or α2M at time 0, electrical activity typically occurred between 7–40 s and was always followed by blebbing activity that began at 30 s and persisted for 3–5 min. In contrast, continuous exposure to FBS resulted in the persistence of both blebbing activity and transient hyperpolarizations for periods of at least several hours. Scanning electron microscopy (SEM) revealed that the blebs appeared concomitantly with the disappearance of microvilli and the appearance of surface pits that measured 100–300 nm in diameter. Coated pits and vesicles, similar in size to the pits observed by SEM, were observed using transmission electron microscopy (TEM). By TEM, blebs were found to contain few organelles other than centrally located free ribosomes. Fluorescence microscopy of nitrobenzooxadizole‐phallacidin‐labeled cells indicated that blebs contained filamentous actin and that microfilament bundles remained primarily on the substratum side of blebbed cells. We propose that blebbing results from a dynamic local reorganization of microfilaments initiated by ligand‐induced transient increases in intracellular Ca2+.