bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis of murine megakaryocyte colony size and ploidy: Effects of interleukin‐3
Authors: Segal Gerald M. , Stueve Terri , Adamson John W. 
Published: February 2005

AbstractMegakaryocytes develop from diploid precursor cells that, after variable numbers of mitoses, cease cell division and then undergo synchronous nuclear endoreduplication (endomitosis). Megakaryocyte colony formation represents an in‐vitro model of these processes in which the number and ploidy of colony cells reflect the activity of the mitotic and endomitotic pathways, respectively. We have analyzed the size and ploidization of murine megakaryocyte colonies grown in agar and examined the influence of interleukin‐3 (IL‐3) on these parameters. Colonies were identified in situ by staining for acetylcholinesterase and the ploidy of colony cells was determined by fluorescence cytophotometry. More than 98% of the megakaryocytes that developed in culture could be analyzed. In cultures of unfractionated marrow cells stimulated by either pokeweed mitogen‐stimulated spleen cell‐conditioned medium (PWM‐SCM, a crude source of megakaryocyte colony‐stimulating activity) or IL‐3, the modal ploidy of day‐5 colony megakaryocytes was 16N (range 2N‐128N). The distribution of colony size was described by an inverse exponential relationship. Colony size and geometric mean ploidy were inversely correlated under conditions of maximal stimulation with PWM‐SCM and at all concentrations of IL‐3 tested. Increasing concentrations of IL‐3 in cultures of either unfractionated marrow cells or nonadherent T‐lymphocyte‐depleted (NATD) marrow cells stimulated similar dose‐dependent increases in the mean size and numbers of megakaryocyte colonies but did not significantly alter their ploidy distribution. However, the mean ploidy of colony megakaryocytes in IL‐3‐stimulated cultures of NATD marrow cells was significantly less (P < 0.001) than the mean ploidy of megakaryocytes in IL‐3‐stimulated cultures of unfractionated marrow cells. The mean ploidy of megakaryocytes, which developed in PWM‐SCM‐stimulated cultures, was not affected by initial accessory cell depletion. We conclude that the size and ploidy characteristics of day‐5 murine megakaryocyte colonies are structured as continua and that IL‐3 stimulates an increase in mean colony size and numbers without affecting ploidization. T‐lymphocytes and adherent cells elaborate an activity which promotes endomitosis in vitro; factors in PWM‐SCM can substitute for this activity.
Platelet‐derived growth factor or basic fibroblast growth factor induce anchorage‐independent growth of human fibroblasts
Authors: Palmer Helen , Maher Veronica M. , McCormick J. Justin 
Published: February 2005

AbstractAnchorage‐independent growth, i.e., growth in semi‐solid medium is considered a marker of cellular transformation of fibroblast cells. Diploid human fibroblasts ordinarily do not exhibit such growth but can grow transiently when medium contains high concentrations of fetal bovine serum. This suggests that some growth factor(s) in serum is responsible for anchorage‐independent growth. Much work has been done to characterize the peptide growth factor requirements of various rodent fibroblast cells for anchorage‐independent growth; however, the requirements of human fibroblasts are not known. To determine the peptide growth factor requirements of human fibroblasts for anchorage‐independent growth, we used medium containing serum that had had its peptide growth factors inactivated. We found that either platelet‐derived growth factor (PDGF) or the basic form of fibroblast growth factor (bFGF) induced anchorage‐independent growth. Epidermal growth factor (EGF) did not enhance the growth induced by PDGF, or did so only slightly. Transforming growth factor beta (TGF‐β) decreased the growth induced by PDGF. EGF combined with TGF‐β induced colony formation in semi‐solid medium at concentrations at which neither growth factor by itself was effective, but the combination was much less effective in stimulating anchorage‐independent growth than PDGF or bFGF. This work showed that PDGF, or bFGF, or EGF combined with TGF‐β can stimulate anchorage‐independent growth of nontransformed human fibroblasts. The results support the idea that cellular transformation may reduce or eliminate the need for exogenous PDGF or bFGF.
Superoxide dismutase activities of differentiating clones from an immortal cell line
Authors: Oberley Larry W. , Ridnour Lisa A. , Sierra‐Rivera Elaine , Oberley Terry D. , Guernsey Duane L. 
Published: February 2005

AbstractWe have studied superoxide dismutase (SOD) levels in the X‐REF‐23 rat embryo fibroblast cell line. X‐REF‐23 is an immortal cell line that maintains a nontransformed phenotype throughout its known lifespan. Low‐passage X‐REF‐23 cells undergo spontaneous differentiation into muscle and adipose cells, while high‐passage X‐REF‐23 cells undergo little or no differentiation. SOD activities were measured in subclones of X‐REF‐23, which differentiate into muscle (AMC subclone) or adipose (AMB‐J) cells, as well as the parental nondifferentiating X‐REF‐23 cells. Total SOD activity increased in all three cell lines with time in culture. Cu‐ZnSOD was induced in the AMB‐J and the X‐REF‐23 cells with time in culture, whereas the AMC cells showed no induction. MnSOD activity was induced during the time period in which differentiation occurred in the two differentiating clones. In contrast, MnSOD was not induced in this time period in the nondifferentiating X‐REF‐23 cell line. However, MnSOD activity was induced in the latter cell line at a much later time. Levels of immunoreactive MnSOD correlated quite well with MnSOD activity in all three cell lines. The nondifferentiating X‐REF‐23 cells, but not the two differentiating cells lines, showed a large increase in cell organelles with time in culture. In particular, an increase in very small mitochondria was observed; these mitochondria often showed evidence of disorganization.
Simultaneous stimulation of urokinase and tissue‐type plasminogen activators by phorbol esters in human ovarian carcinoma cells
Authors: Band Vimla , Karlan Beth Y. , Zurawski Vincent R. , Littlefield Bruce A. 
Published: February 2005

AbstractOVCA 433 human ovarian carcinoma cells secrete both mammalian plasminogen activators (PAs) urokinase (UK) and tissue‐type PA (tPA). Treatment of cells with 4β‐phorbol‐12‐myristate‐13‐acetate (PMA), a stimulator of protein kinase C (PKC), leads to large increases in the secretion rates of both PA types. PA stimulation by PMA is time‐ and concentration‐dependent, with maximal effects occurring between 12 and 24 h at PMA concentrations of 1–10 ng/ml. The PMA effect is mimicked by mezerein, another known PKC stimulator, but not by 4α‐phorbol or 4α‐phorbol‐12,13‐didecanoate, two phorbol compounds that do not stimulate PKC. PA activity is virtually unaffected by 1‐oleoyl‐2‐acetylglycerol (OAG), a synthetic diacylglycerol that stimulates PKC in vitro but has variable effects on whole cells. PMA stimulation of PA activity is blocked by both actinomycin D and cycloheximide, indicating requirements for new RNA and protein synthesis. When analyzed individually, the relative PMA‐induced increases in UK and tPA activities are identical. Increased UK activity is fully accounted for by increased UK antigen secretion, whereas increased tPA secretion accounts for only about one‐half of the increased tPA activity. Similarly, PMA induces large increases in steady‐state UK mRNA levels, while its effects on tPA mRNA levels are only modest. Thus, while increases in secretion rates and mRNA levels can completely account for UK stimulation, other mechanisms augmenting these processes must exist specifically for tPA. Since the relative increases in UK and tPA activities are identical despite the probable existence of multiple mechanisms contributing to tPA regulation, our data suggest the possibility of interrelationships between the two pathways such that equivalent degrees of UK and tPA activity stimulation are ultimately achieved.
Adenovirus infection retards ribosomal RNA processing
Authors: Lawler Susan H. , Jones Robert W. , Eliceiri Brian P. , Eliceiri George L. 
Published: February 2005

AbstractEight hours after infection of KB cells with adenovirus type 12, the rate of conversion from the 32S ribosomal RNA (rRNA) precursor to mature 28S and 5.8S rRNA decreased. An additional RNA species was detected, which appears to be novel, on the basis of its estimated size (about 6.5 kilobases) and its high level of radiolabeling early after infection at low multiplicity.
Selective release from cultured mammalian cells of heat‐shock (stress) proteins that resemble glia‐axon transfer proteins
Authors: Hightower Lawrence E. , Guidon Peter T. 
Published: February 2005

AbstractCultured rat embryo cells were stimulated to rapidly release a small group of proteins that included several heat‐shock proteins (hsp110, hsp71, hscp73) and nonmuscle actin. The extracellular proteins were analyzed by two‐dimensional polyacrylamide gel electrophoresis. Heat‐shocked cells released the same set of proteins as control cells with the addition of the stress‐inducible hspllO and hsp71. Release of these proteins was not blocked by either monensin or colchicine, inhibitors ofthe common secretory pathway. A small amount of the glucose‐regulated protein grp78 was externalized by this pathway. The extracel‐lular accumulation of these proteins was inhibited after they were synthesized in the presence of the lysine analogue aminoethyl cysteine. It is likely that the analogue‐substituted proteins were misfolded and could not be released from cells, supporting our conclusion that a selective release mechanism is involved. Remarkably, actin and the squid heat‐shock proteins homologous to rat hsp71 and hsp110 are also among a select group of proteins transferred from glial cells to the squid giant axon, where they have been implicated in neuronal stress responses (Tytell et al.:Brain Res., 363:161‐164, 1986). Based in part on the similarities between these two sets of proteins, we hypothesized that these proteins were released from labile cortical regions of animal cells in response to perturbations of homeostasis in cells as evolutionarily distinct as cultured rat embryo cells and squid glial cells.
Interleukin 3‐stimulated proliferation is sensitive to pertussis toxin: Evidence for a guanyl nucleotide regulatory protein‐mediated signal transduction mechanism
Authors: Kelvin David J. , Shreeve M. , McAuley C. , McLeod D. L. , Simard G. , Connolly J. A. 
Published: February 2005

AbstractInterleukin 3 (IL‐3) stimulates several biochemical and biological responses in IL‐3‐dependent tissue culture cells. We examined the possibility that guanyl nucleotide regulatory (G) proteins may transduce signals from IL‐3 receptors. We report here that pertussis toxin (PT), which can covalently modify a subclass of G proteins, is capable of inhibiting IL‐3‐stimulated proliferation in a dose‐dependent fashion. PT inhibiton of IL‐3‐stimulated proliferation could be overcome by using the Ca+ + ionophore A23187 in conjunction with TPA. PT could also inhibit IL‐3‐stimulated hexose transport. In the absence of IL‐3, hexose transport could be stimulated by introducing GTPγS into intact cells. From these data we propose that IL‐3 receptors transduce signals via a PT‐sensitive G protein(s).
Regulation of expression of the cell adhesion receptors, integrins, by recombinant human interleukin‐1β in human osteosarcoma cells: Inhibition of cell proliferation and stimulation of alkaline phosphatase activity
Authors: Dedhar Shoukat 
Published: February 2005

AbstractRecombinant human interleukin‐1β, a mediator of osteoblastic cell function, was found to regulate the expression of the cell adhesion receptors, integrins, on human osteosarcoma cells. Interleukin‐1β (IL‐1β) at picomolar concentrations, specifically elevated approximately six‐ to tenfold the expression of the β1 subunit and its associated α subunits, but not the related vitronectin receptor, within 20 hours. Integrin β1 messenger RNA levels were elevated within 6 hours and peaked to tenfold higher levels after 20 hours exposure to IL‐1β in two human osteosarcoma cell lines. The increase in the cell‐surface β1 integrins resulted in a stronger binding of the IL‐1β‐treated cells to fibronectin. Cell growth was also inhibited by IL‐1β cell morphology was altered, and IL‐1β‐treated cells expressed an approximately two‐ to threefold higher alkaline phosphatase. This increase in alkaline phosphatase activity was found to be independent of the inhibition of cell proliferation. These data indicate that the β1 integrin family of cell surface receptors is a target for regulation by IL‐1β which also regulates cell proliferation and the expression of the osteoblastic phenotype in human osteosarcoma cells.
Analysis of the multiple 5′ and 3′ termini of poly(A) + and poly(A)− deficient thymidylate synthase mRNA in growth‐stimulated mouse fibroblasts
Authors: DeWille James W. , Harendza Christopher J. , Jenh Chung‐Her , Johnson Lee F. 
Published: February 2005

AbstractThymidylate synthase (TS) mRNA content increases about 20‐fold when growth‐stimulated mouse cells progress from the GO/G1 phase into the S phase of the cell cycle. Previous studies, using a cell line in which the TS gene is amplified (LU3‐7), indicated that transcriptional initiation as well as polyadenylation of the mRNA occur at several locations in unsynchronized cells. In the present study, we have used S1 nuclease protection assays to analyze the possible significance of the multiple transcriptional initiation and polyadenylation sites. We found that the same pattern of 5′ and 3′ termini were detected with RNA isolated from the overproducing cells as with RNA isolated from the parental mouse 3T6 cell line, demonstrating that the heterogeneous termini are not a consequence of gene amplification. There was no change in the pattern of 5′ or 3′ termini with either cell line during the progression from G1 phase through S phase in serum‐stimulated cells. Therefore, the increase in TS mRNA content is not the result of differential utilization of the various transcriptional initiation or polyadenylation sites. Analyses of poly(A)− deficient cytoplasmic TS RNA showed that the 5′ termini were the same as those found in poly(A)+ mRNA. However, the 3′ termini were extremely heterogeneous in length. Although some of the poly(A)− deficient RNA extended beyond the normal site of polyadenylation, most of it was shorter than full‐length TS mRNA.
Identification of a 15,000‐molecular‐weight form of immunoreactive transforming growth factor α in extracts of porcine pituitary
Authors: Riss Terry L. , Sirbasku David A. 
Published: February 2005

AbstractTwo different mitogenic activities were identified from extracts of porcine pituitary by using COMMA‐D mouse mammary epithelial cells in a serum‐free 3H‐thymidine incorporation assay. Porcine pituitaries were extracted in phosphate‐buffered saline (pH 7.4) and 25–80% (NH4)2SO4 pellets were dialyzed and chromatographed by using DEAE‐Sepharose Chromatography (pH 8.0), resulting in two peaks (I and II) of mitogenic activity. Peak I represented a recovery of 73% of the units of mitogenic activity present in crude extract of pituitary while only 1.25% of the activity was recovered in peak I was further purified by using CM‐Sephadex and heparin‐Sepharose chromatographies and yieled a mitogen that was able to elicit one‐half‐maximal stimulation of 3H‐thymidine incorporation by COMM‐D cells at 48 pg/ml. As expected with pituitary as the tissue source, peak I was confirmed to be basic fibroblast growth factor (bFGF) by using specific antibodies in enzyme‐linked immunosorbent assay and Western immunoblotting pocedures. Peak II was futher purified by using chromatorofocusing (pH 7.3–5.0), reverse‐phase, and action‐exchange HPLCs. The mitogenic activity eluted at pH 6.3 from chromatofocusing, migrated as a 13‐kDa molecule on gel filtration HPLC, and did not bind to heparin‐Sepharose under conditions which bound fibroblast growth factors. The material purified from peak II and rat synthetic transforming growth factor α (TGFα) competed in a parallel fashion with 125I‐epidermal growth factor for receptor binding with A431 human epidermal carcinoma cells. In addition, the mitogen purified from peak II showed a single immunoreactive band migrating at 15 kDa when specific antiserum against TGfα was used in a Western immunoblotting procedure. The data suggest that in addition to the well‐documented presence of bFGF, normal adult porcine pituitaries contain a 15‐kDa form of immunoreactive TGFα that binds to EGF receptors and is mitogenic for mammary epithelial cells.
Developmental regulation of calcium‐binding proteins (calelectrins and calpactin I) in mammary glands
Authors: Lozano Joseph J. , Silberstein Gary B. , Hwang Soo‐In , Haindl Alfred H. , Rocha Victor 
Published: February 2005

AbstractWe recently showed that mammary glands contain a novel class of calcium‐binding proteins (CBPs) that bind to membranes in a calcium‐dependent manner. We have also established that these mammary CBPs are equivalent to the calelectrins and calpactin I/p36. Since it has been suggested that these proteins might be involved in exocytosis, we examined mammary glands for these CBPs during secretory differentiation. Immunohistochemical examination showed, glands from virgin animals to be rich in calelectrins and calpactin I/p36, while glands from lactating animals contained little immunoreactive material. In addition, silver‐staining and immunoblot estimation of the CBPs in lysates from collagenase harvested secretory epithelia showed these proteins to be significantly reduced compared to nonsecretory epithelia. Close examination of the CBP immunoreactive cells of the mammary gland shows that ductal cells are prominent in their staining and that the immunoreactive material is associated with the cell surface. Also, in juvenile glands the.myoepithelial stem cells (cap cells) of the elongating end bud are devoid of the CBPs. In contrast to the in vivo data, epithelia cultivated on collagen gels demonstrate comparable levels of the CBPs in both nonsecretory and secretory monolayers. The in vivo data indicate that the CBPs are developmentally regulated during mammary gland differentiation such that secretory epithelia are essentially devoid of these novel proteins. Furthermore, a role for calelectrin and calpactin I/p36 in exocytotic casein secretion is questioned.
Effect of macrophages on the translocation of protein kinase C in concanavalin a‐stimulated lymphocytes
Authors: Grove Deborah S. , Mastro Andrea M. 
Published: February 2005

AbstractThe concanavalin A (Con A)‐induced proliferation of lymph node lymphocytes is dependent on the presence of macrophages. When lymphocytes are depleted of macrophages, Con A is no longer mitogenic. Either 12‐0‐tetradecanoylphorbol‐13‐acetae (TPA), interleukin 1 (IL1), or macrophages in combination with Con A can restore proliferation. To establish where the proliferation process is blocked in the absence of macrophages, an early step in the signalling pathway, the activation of protein kinase C, was examined. It was found that although Con A caused translocation of protein kinase C from the cytosol to the membrane of lymph node cells, when the lymph node cells were depleted of macrophages and exposed to Con A, this translocation of protein kinase C did not occur. Instead, protein kinase C activity decreased in the membrane fraction and increased in the cytosol. On the other hand, TPA caused translocation of protein kinase C (PKC) from the cytosol to the membrane regardless of the presence of macrophages. However, the macrophage product, IL1, alone or in combination with Con A did not cause translocation of protein kinase C. In a reconstitution experiment, in which lymph node cells were depleted of macrophages and then macrophages were added back, the addition of Con A again lead to translocation of protein kinase C from the cytosol to the membrane. This combination also restored cell proliferation. Therefore, the Con A induced PKC translocation in T lymphocytes is macrophage mediated. TPA overcomes the macrophage requirement by directly activaing PKC, while IL1 appears to act at a different step in proliferation.
Alteration of microtubule physiology in hepatocytes by insulin
Authors: Caron Joan M. 
Published: February 2005

AbstractIncubation of primary cultures of rat hepatocytes with insulin caused an alteration in several parameters of microtubule physiology. There was a transient decrease in total cellular tubulin levels and, at the same time, an apparent stabilization of microtubule polymers which was also transient. The level of microtubule polymer, however, was not altered, which indicated that an insulin‐inducible decrease in the level of nonpolymerized tubulin was responsible for the decrease in total cellular tubulin. These changes were followed by an increase in tubulin mRNA levels and, shortly thereafter, by an increase in tubulin protein synthesis. This study demonstrates that microtubule physiology in hepatocytes is modulated by insulin and, further, suggests a model by which insulin‐induced changes in microtubule physiology may play a role in regulating other insulin responses.
Mutual contact of murine erythroleukemia cells activates depolarizing cation channels, whereas contact with extracellular substrata activates hyperpolarizing Ca2+‐dependent K+ channels
Authors: Arcangeli Annarosa , Del Bene Maria Riccarda , Poli Riccardo , Ricupero Letizia , Olivotto Massimo 
Published: February 2005

AbstractThis study deals with the modulation of the plasma membrane potential (Δψp) of murine erythroleukemia (MEL) cells by cell‐substratum or cell‐cell contact. Δψp was determined by measuring the distribution of tetraphenylphosphonium (TPP+) across the plasma membrane; it appeared strongly, and inversely, influenced by the two types of cell contacts. Contact with the culture surface produced a Δψp hyperpolarization directly proportional to average distance among the ideal centers of the cells on this surface (d) within the range 10–80 μm. A detailed mathematical analysis of the function Δψp = f(d) is presented, as well as experiments involving the use of ionophores (valinomycin and A23187) and the conditioning of the culture surface. We concluded that the d‐dependent hyperpolarization (dDH) was the result of a complex interplay between the activating properties of substratum on Ca2+‐dependent K+ channels (KCa) and some substratum‐adherent factors that are shed by MEL cells and antagonize KCa activation (substratum‐attached cellular factors = SACF), By contrast, contact of the cells with each other, obtained by incubating MEL cells at d smaller than the average cell diameter (Φ = 10 μm), produced a marked Δψp depolarization. This intercellular contact‐dependent depolarization (ICDD) was unaffected by valinomycin; it was abolished by substituting Na+ in the external medium with a nondiffusible cation (choline), which shows that ICDD was sustained by Na+ influxes, probably mediated by stretch‐activated (s.a.) cation channels.