bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of chemically and virally transformed variants of Madin‐Darby canine kidney (MDCK) epithelial cells
Authors: U H. S. , Boerner P. , Rindler M. J. , Chuman L. , Saier M. H.
Published: February 2005

Oncogenic derivatives of Madin‐Darby canine kidney (MDCK) cells were isolated in the nude mouse, and nononcogenic anchorage‐independent transformants were isolated in vitro following chemical mutagenesis in vitro. These transformed cell lines as well as a Moloney sarcoma virus (MSV) transformed line were characterized with respect to their serum and anchorage requirements, growth rates, final saturation densities, and sensitivities to contact inhibition. None of these in vitro growth characteristics were found to correlate with tumorigenicity in nude mice. One tumongenic clone, MDCK‐T1, was characterized with respect to serum‐free growth requirements, cAMP production, and ornithine decarboxylase (ODC) activity. These cells exhibited a significant reduction in the PGE1 requirement for growth, they produced higher levels of cAMP, and they expressed a reduced level of ODC activity relative to the parental MDCK cells. These findings may reflect changes in growth control mechanisms which accompany kidney epithelial cell tumori‐genesis and suggest that the study of transformed lines derived in this manner could lead to the identification of in vitro properties which are associated with malignancy.
Clonal heterogeneity in colony stimulating factor production by murine T lymphocytes
Authors: Kelso Anne , Metcalf Donald
Published: February 2005

A panel of 55 alloreactive murine T‐lymphocyte clones was screened for the production of granulocyte‐macrophage colony stimulating factor (GM‐CSF), multilineage CSF (multi‐CSF), human‐active eosinophil CSF (human‐active EOCSF), and interleukin 2 (IL‐2) in response to stimulation with the lectin concanavalin A. Many clones were also characterized for cytolytic specificity and expression of the T‐cell antigen receptor‐associated surface markers Lyt‐2 and L3T4, which reflect their specificity for Class I (H‐2K, H‐2D) or Class II (H‐2I, MIs) histocompatibility antigens, respectively. Eighty percent of the clones secreted detectable quantities of at least one of the four factors measured. Of the factor‐producing clones, all appeared to secrete GM‐CSF and half also secreted multi‐CSF. A subpopulation of multi‐CSF producers also released human‐active EO‐CSF. More than half of the factor‐producing clones secreted detectable IL‐2; whereas the IL‐2 producing clones included some that did not secrete multi‐CSF, IL‐2 production was always associated with concomitant synthesis of GM‐CSF. Comparison of the range and quantities of factors secreted by Lyt‐2+ and L3T4+ clones indicated that more L3T4+ clones produced measurable titers of the four factors; on average, this group also secreted 10‐ to 100‐fold higher titers of both the hemopoietic regulators and IL‐2 than Lyt‐2+ clones. Cells of the L3T4+ phenotype would therefore be expected to account for the majority of CSF and IL‐2 secretion by polyclonal populations of activated T lymphocytes.
Intracellular Ca2+ sequestration in cultured mouse fibroblasts: ATP‐dependent Ca2+ uptake by saponin‐permeabilized Swiss 3T3 cells
Authors: Yamanishi Kiyofumi , Iwashima Akio
Published: February 2005

Intracellular Ca2+ sequestration was studied by determining 45Ca2+ uptake by saponin‐permeabilized Swiss 3T3 mouse fibroblasts. Uptake was enhanced by addition of ATP and was dependent on the concentrations of ATP and saponin. ADP and UTP also enhanced uptake, but less than did ATP. Mitochondrial Ca2+ uptake was distinguished from nonmitochondrial uptake by the use of antimycin A plus oligomycin at various Ca2+ concentrations. Major sites for sequestration at 10−5 M Ca2+ are probably mitochondrial and those at 10−7 M Ca2+ nonmitochondrial, because these drugs inhibited ATP‐dependent Ca2+ uptake at 10−5 M but not at 10−7 M. A23187 completely inhibited uptake, regardless of the Ca2+ concentration. Effects of various agents on sequestration were tested; ruthenium red, 2,4‐dinitrophenol, monovalent cation ionophores, and melittin markedly decreased ATP‐dependent Ca2+ uptake at 10−5 M Ca2+. the calmodulin antagonists trifluoperazine (TFP) and W‐7 inhibited Ca2+ uptake at both 10−5 and 10−7 M Ca2+, TFP moreso than W‐7, especially at 10−5 M. These results suggest that intracellular Ca+2 in Swiss 3T3 cells is regulated by mitochondrial and nonmitochondrial Ca2+ stores in an ATP‐dependent way and that in a physiological resting state the nonmitochondrial Ca2+ store, probably the endoplasmic reticulum, is the major site for the sequestration.
Protein synthesis in differentiating normal and leukemic erythroid cells
Authors: Adkins Becky , Beug Hartmut , Graf Thomas
Published: February 2005

Erythroleukemic cells transformed by the AEV or S13 strains of avian erythroblastosis virus differentiate in vitro either spontaneously (S13) or following a temperature induction (temperature‐sensitive mutants of AEV). To study differentiation in these cells at the molecular level, homogeneous fractions of maturing cells at discrete stages of differentiation were prepared by Percoll density‐gradient centifugation. This method was also used for the fractionation of differentiating normal erythroid cells separated from total bone marrow by an immunological “panning” technique. Total protein synthesis in these cells was then analyzed by two‐dimensional gel electrophoresis. The expression of several proteins was altered in differentiating leukemic cells but not in their normal counterparts. However, in general, the normal and leukemic cells from comparable stages of maturity showed closely related protein synthetic patterns. Similar early and late changes in the synthesis of a number of polypeptides were detected during maturation from early erythroid precursors to terminally differentiated erythrocytes. Further, the leukemic as well as the normal cells appeared to undergo a major switch in total protein synthetic pattern during late differentiation. These results demonstrate that normal and erythroleukemic cells differentiate along similar pathways.
Enhancement of colony‐stimulating‐factor–dependent clonal growth of murine macrophage progenitors and their phagocytic activity by retinoic acid
Authors: Goldman Rachel
Published: February 2005

The effect of retinoic acid (RA) on the colony‐stimulating‐factor‐dependent clonal growth of myeloid progenitors was assessed in semisolid agar cultures of mouse bone marrow cells using L‐cell‐conditioned medium that gave rise to macrophage colonies, granulocyte colonies, and mixed macrophage‐granulocyte colonies and clusters. RA was found to enhance the overall formation of myeloid colonies (about 50%) and clusters in 7‐day cultures. The increase was due to an enhanced formation of macrophage colonies (70–250%) and clusters which reached a maximal value at about 3 μM RA. In 4‐day cultures, the effect of RA on macrophage colony formation was biphasic with a maximal enhancement at 10 nM. RA suppressed granulocyte‐colony formation in macrophages at all stages of differentiation and/or maturation in culture. The Fc‐receptor‐mediated erythrophagocytosis as well as the phagocytosis of heat‐killed yeast cells (HK‐yeast) and starch particles increased by RA treatment in a dose‐dependent manner, reaching an increase of 100–200% of the activity expressed in the absence of RA. Peritoneal exudate macrophages likewise physiological and pharmacological concentrations of RA. Expression of an RA‐mediated increase in phagocytic activity required a prolonged incubation with RA (> 19 hr). The data suggest that RA may be of physiological relevance in the regulation of proliferation and function of hemopoietic cells. Therapeutic doses of RA may potentiate macrophage proliferation and function, elements that are crucial at all phases of the various defense mechanisms that the organism possesses.
Release and properties of endothelium‐derived relaxing factor (EDRF) from endothelial cells in culture
Authors: Cocks T. M. , Angus J. A. , Campbell J. H. , Campbell G. R.
Published: February 2005

Cultured bovine endothelial cells were seeded onto the intimal surface of endothelium‐denuded rings of canine coronary artery. These rings did not previously relax to acetylcholine, substance P, bradykinin, and A23187. After seeding, the same rings relaxed to bradykinin and A23187, but not to acetycholine or substance P. Indomethacin pretreatment did not affect these responses. Cells from the same source were then grown to confluence on microcarrier beads, poured into small columns, and perfused with Krebs+ solution. The perfusate from the columns was bioassayed on endothelium‐denuded rings of coronary artery from either the dog or pig. Challenge of the column in the presence of indomethacin with either bradykinin or A23187 as well as acetylcholine or substance P caused release of a substance that relaxed both types of artery. Its activity half‐life was 6.4 ± 0.4 sec at 37°C and it was hydrophilic and negatively charged. Prostacyclin (PGI2) as a candidate for EDRF was ruled out because (1) indomethacin failed to block its release and (2) the pig coronary artery, although insensitive to PGI2, relaxed to the endothelium‐derived substance. These results show that, in response to a number of dilator drugs, cultured endothelial cells release a vascular relaxing substance (EDRF) that has characteristics similar to the EDRF of normal endothelium. The chemical nature of EDRF awaits clarification.
Dominant and recessive mitogen‐nonproliferative variants of 3T3 cells
Authors: Terwilliger Ernest , Herschman Harvey R.
Published: February 2005

We have previously isolated 3T3 cell variants unable to respond to specific mitogens. In this report we analyze the dominant and/or recessive nature of these variants. (1) Two independently isolated EGF nonproliferative variants are unable to bind EGF. Hybrids between 3T3R5 cells (thymidine kinase deficient, ouabain‐resistant) and these variants express EGF receptors; the “EGF receptorless” phenotype of these variants is recessive. (2) Hybrids between these two variants do not bind EGF; they are defective in a common, non‐complementing function. (3) A TPA nonproliferative 3T3 variant is also recessive; hybrids with 3T3R5 mount a mitogenic response to TPA. (4) In contrast a fourth variant, which can neither bind labeled EGF nor respond to TPA, is dominant for both characteristics. Hybrids between this latter variant and 3T3R5 can neither bind EGF nor mount a mitogenic response to TPA.
Vasoactive amines modulate actin cables (stress fibers) and surface area in cultured bovine endothelium
Authors: Welles Seth L. , Shepro David , Hechtman Herbert B.
Published: February 2005

Cultured bovine aortic endothelial cells were fixed and stained with NBD‐phallicidin and quantitated with a digital image analyzer for changes in actin cables and surface area. Serotonin (5‐HT), norepinephrine (NE), dopamine and histamine (all at 10−4M concentrations) were tested for their ability to induce cytoskeletal changes. Only 5‐HT and NE increased action cables significantly (p < 0.01), 80.7% and 97.9%, respectively. Dopamine and histamine treated cells showed a 67.4% and 80.8% decrease in actin cables respectively (p < 0.01). Stimulated increases of actin cables by 5‐HT were inhibited by Ketanserin, and propranoiol inhibited NE stimulation of actin cables. Treatment of cells with these blockers alone also decreased actin cables below control values (p < 0.01). Pretreatment of cells with diphenhydramine, but not cimetidine, inhibited histamine‐induced decreases in actin cables. Stimulation of surface area by 5‐HT and NE was also observed, with 40.8% and 80.7% increases respectively, when compared with controls (p < 0.01). The increases in actin cables were associated with a lack of ruffled edges that are indicative of motile cells. In contrast, induced decreases in actin cables resulted in cells with ruffled edges. Exogenous 5‐HT and NE have been shown to prevent the increased permeability visible as extravasation of red blood cells from postcapillary venules in thrombocytopenic animals. The present data suggest that 5‐HT and NE may be involved in maintaining the endothelial barrier function by a receptor‐mediated stimulation of actin cables. Also, histamine‐induced decreases in actin cables may be correlated with the amine's action in vivo as a mediator of increased inflammatory permeability.
Effect of endothelial cell conditioned medium on the growth of human bone marrow fibroblasts
Authors: Scarrà Giovanna Bianchi , Fiorentini Paola , Garbarino Giovanni , Bellone Graziella , Sessarego Mario , Ajmar Franco
Published: February 2005

Both endothelial cells (EC) and fibroblasts, two discrete populations of hemopoietic stroma, are known to modulate the proliferation and differentiation of hemopoietic progenitors. Recent reports also demonstrated that EC stimulate the in vitro growth of fibroblasts via a soluble factor. This finding seems to support the hypothesis that EC may play a role in the pathogenesis of bone marrow fibrosis in myeloproliferative disorders (MD).

We have studied the effects of the conditioned medium (CM) from human umbilical vein EC cultures, obtained in serum free conditions, on the growth of bone marrow fibroblasts from normal donors and from patients with MD. The results show that EC derived CM contains a factor which stimulates the proliferation of fibroblasts and that can act as an authentic growth factor by inducing “quiescent” fibroblasts to proliferate.

Moreover, we found that this endothelial derived growth factor (EDGF) equally promotes the proliferation of both normal and pathological progenitors of bone marrow fibroblasts (CFU‐F) by increasing both the number and the size of the colonies.
Synergistic stimulation of alkaline phosphatase activity in bovine aortic endothelial cells grown in the presence of retinoids and glucocorticoids
Authors: Adams Susan E. , Melnykovych George
Published: February 2005

Low concentrations of retinol (10 nM ‐ 10 μM) and dexamethasone (0.1 nM ‐ 1.0 μM) elevated activity of alkaline phosphatase (E.C. 3.1.3.1) in bovine endothelial cells in culture. The effect was apparent after 48 hr of growth in the presence of either of the two compounds, prior to any growth stimulatory effects. A synergistic stimulation of alkaline phosphatase was achieved in the presence of both retinol and dexamethasone implying different mechanisms of induction. Retinoic acid and retinyl acetate also elevated alkaline phosphatase but the retinol analogue reduced in the side chain (perhydroretinol) was inactive. The ability of steroids to elevate alkaline phosphatase activity was associated with the structure commonly required for glucocorticoid activity; however, competitors for the steroid receptor binding failed to prevent the elevation by dexamethasone or the synergism in the presence of retinol and dexamethasone.
Prolongation of duration of G2 arrest delays and finally blocks entry into M phase in contrast to stable and reversible G1 arrest: Study of a G1/G2 temperature‐sensitive mutant of rat 3Y1 fibroblasts
Authors: Zaitsu Hirokazu , Kimura Genki
Published: February 2005

Proliferation of 3Y1tsF121 cells was arrested in G1 and G2 phases after a shift up to 39.8°C (restrictive temperature). Both arrests were reversible: after a shift down to 33.8°C (permissive temperature), these cells effectively entered the next phases. However, the entry into M phase of the G2‐arrested cells was delayed depending on the time in arrest. The G2‐arrested cells finally became incapable of entering M phase with a prolonged incubation at 39.8°C. Under the same condition, G1‐arrested cells did not lose their ability to proliferate, and their delay of entry into S phase was slight. Therefore, cells in G2 phase are, in a sense, more unstable than the cells in G1 phase. These results also suggest that the time required for entry into M phase may depend on the preparedness for the initiation of M phase and, that it may be prolonged under the condition where the preparedness for entry into M phase is diminished.
A Collagen:Glucosyltransferase at the surface of malignant fibroblasts
Authors: Bauvois Brigitte , Roth Stephen
Published: February 2005

3T12 fibroblasts possess glucosyltransferases that catalyze the transfer of glucose from UDP‐Glucose to galactosylhydroxylysyl residues on collagenous acceptors. The presence of the enzyme activity at the cell surface is indicated by the following findings: (a) suspensions of intact cells, as well as intact cell monolayers, glucosylate gelatinized collagen (b) glucose transfer is not due to UDP‐Glucose hydrolysis and subsequent intracellular utilization of the free glucose (c) experiments using cell suspensions with known proportions of broken cells indicate that the glucosyltransferase activity is attributable to intact cells and not to contamination by intracellular enzymes from broken cells. The Km value for UDP‐Glucose is about 20 μM. The enzyme has a pronounced requirement for manganese, and shows highest activity between 2 and 10 mM. The optimal Mn2+ concentration for the intracellular gelatin:glucosyltransferase activity is more restricted (5 to 10 mM). Glucosyltransferase activity is strongly inhibited by diamide and N‐ethylmaleimide (5 mM), suggesting that intact sulfhydryl residues present in the enzyme are essential.
Butyrate enhances the synthesis of interphotoreceptor retinoid‐binding protein (IRBP) by Y‐79 human retinoblastoma cells
Authors: Kyritsis Athanassios P. , Wiggert Barbara , Lee Ling , Chader Gerald J.
Published: February 2005

The synthesis and secretion of interphotoreceptor retinoid‐binding protein (IRBP) from Y‐79 human retinoblastoma cells was investigated using immunocytochemistry and SDS‐polyacrylamide gel electrophoresis. Indirect immunofluorescence of cells growing in monolayer culture for 11 and 13 days showed no significant IRBP staining although by SDS‐polyacrylamide gel electrophoresis, a small amount of IRBP was detected in the culture medium, suggesting synthesis and extracellular secretion. Butyrate (2mM) treatment of cells starting on the eighth day of culture resulted in a dramatic increase of IRBP fluorescence 3–5 days after treatment. Treatment of cells in all conditions with 1 μM monensin for 3 h showed concentration of IRBP in the Golgi apparatus of about 10–20% of cells as proved by a double immunofluorescent technique, employing anti‐IRBP antibody and wheat‐germ agglutinin. Incubation of cells with either radiolabeled amino acids or glucosamine followed by analysis of cell cytosol and culture medium by SDS‐polyacrylamide gel electrophoresis also confirmed that (1) IRBP is synthesized by the Y‐79 cells and secreted into the medium and (2) its production is markedly increased by butyrate treatment. The enhancement of IRBP synthesis by butyrate suggests biochemical differentiation of Y‐79 cells possibly into photoreceptor‐like cells and offers a new system for studying the properties of this unique retinoid‐binding protein and of factors that control its synthesis and secretion.
Binding and phagocytosis of fibronectin‐coated beads by BHK cells: Receptor specificity and dynamics
Authors: McAbee Douglas D. , Grinnell Frederick
Published: February 2005

Studies on the receptor specificity and dynamics involved in fibroblast phagocytosis of latex beads revealed the following: (1) Ligands other than fibronectin such as concanavalin A (ConA) and serum spreading factor, when coated on latex beads, were found to promote phagocytosis of the beads. This indicates that fibroblast phagocytosis, like spreading, is a ligand‐receptor mediated phenomenon not specifically requiring fibronectin (pFN); (2) Anti‐pFN antibodies were found to inhibit the ability of cells to ingest pFN‐coated beads that previously were bound on the cell surfaces. Consequently, binding of beads to the cell surfaces per se is not a sufficient signal to promote ingestion of the beads; (3) Finally, divalent cations protected receptor function necessary for phagocytosis of pFN‐coated beads from proteolysis by trypsin, as previously was found for receptors involved in cell attachment and spreading on pFN‐coated culture dishes. Recovery experiments carried out with cells whose surface receptors had been destroyed indicated that there was an internal (or cryptic cell surface) pool of receptors that amounted to at least 50% of the receptors normally found on the cell surface. After complete destruction of the cell surface and cryptic pools of receptors, reappearance of receptors required for bead binding and phagocytosis required several hours and did not occur in the absence of new protein synthesis.
Receptor‐mediated endocytosis of human transferrin and its cell surface receptor
Authors: Larrick J. W. , Enns C. , Raubitschek A. , Weintraub H.
Published: February 2005

We have studied the process of transferrin endocytosis in human erythromyeloid cell line K562 using fluorescein (FL) and rhodamine (RD) labeled iron‐saturated transferrin (FeTF), and a fluorescein labeled monoclonal antibody to the transferrin receptor (FL‐mAB). Because the antireceptor antibody and FeTF bind to different sites on the TF receptor molecule, it is possible to simultaneously and independently follow receptor and ligand. We have measured the relative amounts of transferrin or antireceptor antibody bound in the presence or absence of proteolytic enzymes using a cell sorter. At 4°C almost all of the FL‐TF and the FL‐mAB is surface bound in a diffuse pattern. Within minutes of elevating the temperature to 37°C surface aggregates form and the FL‐TF is internalized. At this time about one sixth of the transferrin is still surface bound and accessible to papain digestion. The remainder localizes in a perinuclear cluster of vesicles. Monoclonal antibody binds to the cell surface transferrin receptor but is not internalized at 4°C or 37°C. When unlabeled diferric transferrin is added, it promotes the uptake of the FI‐mAB. The addition of goat anti‐mouse immunoglobulin also promotes FL‐mAB uptake. These studies support the concept that a specific trigger is required for transferrin receptor endocytosis.
Respiratory metabolism of L‐929 cells at different water contents and volumes
Authors: Clegg James S. , Gordon Elaine P.
Published: February 2005

Oxygen consumption was measured in mouse L‐929 cells whose volumes and water contents were reduced by adding sorbitol to the medium. The volume of water lost due to a given sorbitol supplement exceeded the loss in apparent cell volume. An explanation is given for this discrepancy. The rate of oxygen uptake in the absence of exogenous respiratory substrate was essentially the same in cells whose total volume was reduced by 45%, amounting to a loss of about 70% of the total cell water, compared to controls at ‘physiological’ volume and water content. Cells under these same conditions responded to added substrates (pyruvate, glucose, and glutamine) and inhibitors (iodoacetate and 2‐deoxyglucose) in nearly the same way as control cells. These observations are in accord with and add to previous work showing that very large fluctuations in cell volume and water content have only modest effects on the rates and directions of a variety of metabolic processes. The results are interpreted in terms of current views on the composition and organization of the aqueous compartments of eucaryotic cells.
Role of phorbol ester receptors in the 12‐0‐tetradecanoyl‐phorbol‐13‐acetate (TPA)‐induced down‐regulation of colony‐stimulating factor (CSF‐1) binding to murine peritoneal exudate macrophages
Authors: Chen Ben D.‐M. , Wilkins Kimberly L.
Published: February 2005

Treatment of murine peritoneal exudate macrophages (PEM) by tumor‐promoting phorbol esters (TPA) results in a rapid loss of binding activity to radioactive‐labeled colony‐stimulating factor ([125I]‐CSF‐1) on the cell surface. The inhibitory effect of TPA on PEM is transient; treated cells recover full [125I]‐CSF‐1 binding activity in less than 6 hr at 37°C either in the presence or after the removal of added TPA. The role of phorbol ester receptors in the induction of [125I]‐CSF‐1 binding inhibition was studied. The biologically active ligand [3H]‐phorbol 12,13‐dibutyrate ([3H]‐PDBu) bound specifically to cultured murine PEM. At 0°C, stable and equilibrium binding occurred after 2–3 hr. Scatchard analysis revealed linear plots with a dissociation constant and receptor number per cell of 20.9 nM and 3.9 × 105/cell, respectively. Treatment of PEM with biologically active phorbol esters at 37°C rapidly inhibited the binding activity of [3‐H]‐PDBu on cell surface (down‐regulation) and rendered these cells refractory to the TPA‐induced [125I]‐CSF‐1 binding inhibition by the subsequent TPA treatment. The inhibition of phorbol ester binding activity on TPA‐treated PEM is caused by a reduction in the total number of available phorbol ester receptors rather than by a decrease in receptor affinity as judged by Scatchard analysis. The disappearance of [3H]‐PDBu binding activity is reversible and transient. However, unlike CSF‐1 receptors the restoration of phorbol ester receptors on TPA‐treated PEM is a very slow process; a prolonged incubation of up to 72 hr after the removal of TPA was required for PEM to regain fully its [3H]‐PDBu binding activity. Furthermore, the degree of TPA‐induced CSF‐1‐receptor down‐regulation is closely associated with the number of available phorbol ester receptors present on PEM at the time of treatment. Thus, the refractoriness to TPA diminished as the phorbol ester receptors on PEM recovered. A 72‐hr incubation time at 37°C was needed for PEM to lose their refractoriness and again become fully sensitive to TPA‐induced CSF‐1‐receptor down‐regulation. This study provides evidence that the loss of CSF‐1‐receptors induced by TPA treatment requires the presence of phorbol ester receptors and proceeds presumbaly via a co‐internalization of both CSF‐1 and phorbol ester receptors; the refractoriness to TPA is thereby induced by a transient loss of available phorbol ester receptors.
Modulation of intracellular potassium and ATP: Effects on coated pit function in fibroblasts and hepatocytes
Authors: Larkin Janet M. , Donzell William C. , Anderson Richard G. W.
Published: February 2005

Previously we reported that cultured human fibroblasts depleted of intracellular potassium (K+) had a reduced number of surface coated pits and were unable to internalize receptor‐bound molecules such as low density lipoprotein (LDL). We have extended these studies in two important ways. First, we have developed a method for modulating the number of coated pits in situ. Human fibroblasts incubated in K+‐free buffer that contains 4μm nigericin rapidly become depleted of K+ and lose the ability to internalize 125I‐LDL. When rat livers are perfused with the same buffer, there is a 75% decrease in the number of surface coated pits in hepatocytes. Secondly, we have explored the possibility that K+‐depletion effects coated pit function by lowering intracellular ATP. We found that although this protocol lowers intracellular ATP by 40–70%, when ATP concentrations are lowered > 95% by metabolic inhibitors, receptor‐mediated endocytosis is unaffected.
Activation and de novo synthesis of transglutaminase in cultured glioma cells
Authors: Korner G. , Bachrach U.
Published: February 2005

The activity of transglutaminase (TGase) was measured in cultured C6 glioma cells after their stimulation by either isoproterenol and isobutyl‐methylxanthine or by a serum‐containing medium. The activity fluctuated in a biphasic manner, with the peaks at 2–3 hr and 7–8 hr poststimulation. The first peak of TGase activity was affected neither by cycloheximide nor by actinomycin D, which inhibited protein synthesis. The second peak, on the other hand, was completely eliminated by cycloheximide and was reduced by actinomycin D. Immunological procedures were employed to find out whether or not the activity of TGase corresponded with the presence of the TGase antigen in the cultured cells. Indirect immunofluorescent staining and radioimmunoblot techniques suggested that unstimulated cells contained an inactive enzyme. This inactive, or cryptic, enzyme had the same molecular weight as its active counterpart. Activation of the enzyme was mediated by cell stimulation, probably by its release from the membrane. This step did not require protein synthesis, unlike the second step, which was dependent on de novo protein synthesis.
Ecto‐nucleotide triphosphatase activity of human lymphocytes: Studies of normal and CLL lymphocytes
Authors: Segel George B. , Ryan Daniel H. , Lichtman Marshall A.
Published: February 2005

We have studied the apparent kinetic parameters of the ecto‐nucleotide triphosphatase from CLL B lymphocytes and compared them to blood and tonsillar B and T cells. The Vmax of the ecto‐ATPase activity in CLL B lymphocytes, was 65 ± 10 fmol Pi/cell per 30 min compared to 37 ± 2.1 in blood B lymphocytes, and 8.5 ± 1.7 in blood T lymphocytes. The ATPase of membranes prepared from CLL, tonsillar B and T, and blood T lymphocytes had a relationship among the cell types similar to that seen in intact cells. However, no difference in the km for ATP, .17 mM, or the km for magnesium, .15 mM was found in the ecto‐ATPase of CLL lymphocytes as compared to blood or tonsillar B cells. The ectoenzyme of CLL cells hydrolyzed GTP, ITP, CTP, and UTP as well as ATP. Further, ATP added to an enzyme assay containing an alternative nucleotide did not result in increased phosphate release. Nucleotide acceptance of blood B and T lymphocytes was very similar to that of CLL B cells. ATP inhibited phosphate release when present in excess of magnesium in both CLL and blood B lymphocytes. These data indicate that there is greater ectonucleotide triphosphatase activity in tonsillar and blood B lymphocytes, including CLL, as compared either to blood or tonsillar T lymphocytes. However, CLL cells showed no qualitative difference from blood or tonsillar B cells in ectonucleotidase activity. Thus, the higher activity in CLL cells is “B cell‐like” and might reflect, also, their maturation stage or monoclonal origin.
Late G1 amino acid restriction point in human dermal fibroblasts
Authors: Cooper Janet L. , Wharton Walker
Published: February 2005

Human dermal fibroblasts arrested in G0 by maintenance in medium supplemented with 0.1% serum were not restimulated to divide when fresh medium containing 10% dialyzed serum but lacking group B amino acids (cystine, isoleucine, lysine, phenylalanine and tyrosine) was added. Unlike rodent cells, the addition of fresh serum‐supplemented medium lacking only isoleucine did not cause a growth arrest. The amino acid sensitive growth arrest in human fibroblasts was dependent both on presynchronization in G0 as well as a prestarvation for amino acids prior to stimulation with high serum.

When cells were restimulated in the absnece of amino acids, they arrested predominantly in G1, although a small percentage of cells entered early S phase. When medium containing a complete complement of amino acids was then added, cells initiated DNA synthesis following a minimum lag of 2–3 hr. Growth arrested cells initiated DNA synthesis even when complete unsupplemented medium was added, although the addition of high concentrations of insulin or 10% serum increased the rate of entry.
Identification of novel, stage‐specific polypeptides associated with the differentiation of mammary epithelial stem cells to alveolar‐like cells in culture
Authors: Paterson Frieda C. , Rudland Philip S.
Published: February 2005

A linear pathway of morphologically intermediate cells has been identified between the cuboidal epithelial stem cells and the doming alveolar‐like cultures of the cell line Rat Mammary (Rama) 25 in the order: cuboidal → grey → dark → dark droplet cell → doming cultures. The overall process can be accelerated by dimethyl sulfoxide (DMSO) or retinoic acid (RA) in the presence of mammotrophic hormones. From 400–450 [35S]methionine‐labeled polypeptides that are routinely separated by two‐dimensional gel electrophoresis approximately only 3% change during this process. As the Rama 25 cultures become confluent, three polypeptides of molecular weights (MW) 35 kD (pl = 7.7), 45 kD (pl = 7.5) and 33 kD (pl = 7.7) increase dramatically in radioactive abundance. These increases correspond to increases in numbers of grey cells for the 35 kD polypeptide, to increases in numbers of dark cells together with increases in peanut lectin‐binding‐ability for the 45 kD polypeptide, and to increases in the numbers of dark cells and in the numbers of droplet cells for the 33 kD polypeptide. After treatment with DMSO, RA or in spontaneously doming cultures, a second set of four polypeptides of MW 26 kD (pl = 5.9), 27 kD (pl = 6.2), 30 kD (pl = 7.2), and the same 33 kD polypeptide as above increase with the increase in numbers of droplet cells, domes, and increase in casein secretion. A variant of Rama 25, Rama 259, which fails to produce droplet cells, domes, or to secrete casein with DMSO and hormones also shows the same changes in the first set but not in the second set of polypeptides. The elongated, myoepithelial‐like cell line derived from Rama 25, Rama 29, which cannot undergo any of the above intercellular conversions, fails to show changes in any of these polypeptides. Major changes in radioactive polypeptides have been confirmed for nonradioactive polypeptides and for polypeptides labeled for 4 hr with [35S]methionine. The synthesis of these novel polypeptides thus marks specific morphological stages of the differentiation of mammary epithelial stem to alveolar‐like cells in culture, and as such may mark similar differentiation stages in vivo.
The activation of specific gene transcription in the adipose conversion of 3T3 cells
Authors: Djian Philippe , Phillips Marjorie , Green Howard
Published: February 2005

During the adipose conversion of 3T3 cells, there occur sequential changes in cellular protein and mRNA composition. To determine if there are also changes in transcription, we have studied the transcripts of specific genes in isolated nuclei prepared before and after adipose conversion. Transcription of three genes encoding adipocyte‐specific proteins was detectable only in adipocytes, whereas transcription of actin and collagen type I genes occurred in both adipocytes and preadipocytes. The activation of transcription of adipocyte‐specific genes was not synchronous. Thus temporal differences in the appearance of different adipocyte mRNAs probably result from differences in the times of activation of transcription.
Fibrin‐enhanced endothelial cell organization
Authors: Olander J. V. , Bremer M. E. , Marasa J. C. , Feder J.
Published: February 2005

The formation of cloned bovine endothelial cells into capillary‐like tubes is accelerated from 3–7 days to 2–18 h in the presence of fibrin. Indirect immu‐nofluorescence showed the presence of both fibrin and fibronectin in the strands along which the cells organized. Electronmicroscopy revealed the same type of cell structures as form in the absence of fibrin; it also revealed a gradual decrease with time of the fibrin within the putative lumen. Fibrin and fibronectin are commonly present during angiogenesis in vivo, thus these in vitro observations may well have relevance to the in vivo process.
Cell growth and differentiation in vitro in mouse macrophages transformed by a tsA mutant of simian virus 40. III. Large T antigen level and cell proliferation and survival in an SV40 tsA640‐transformed macrophage line
Authors: Tanigawa Takahiko , Shimura Hideo , Yamada Koji , Okuda Atsuyuki , Takayama Hisao , Takagi Atsushi , Tanaka Yoshinori , Kimura Genki
Published: February 2005

The levels of simian virus 40 (SV40) large T antigen in a tsA‐transformed mouse macrophage line at the permissive (33°C) and the nonpermissive (30°C) temperature were examined by immunofluorescence, sodium dodecylsulfate‐polyacrylamide gel electrophoresis, complement fixation, and enzyme‐linked immunosorbent assay. When the cells were confluent and rested at 33°C, and then were shifted to 39°C, the amount of large T antigen per cell decreased, and most cells survived and remained phagocytic. When the cells were proliferating at 33°C, and then were shifted to 39°C, the cells died with only a small reduction in the amount of large T antigen. Therefore, the physiological state of the cells may determine the survival of cells by affecting the level of large T antigen after exposure to 39°C. The confluent cells may be rested with a concomitant decrease of large T antigen. The proliferating cells may not survive in the presence of a relatively high level of functionally defective large T antigen at 39°C.
Calcium‐mediated regulation of the low density lipoprotein receptor and intracellular cholesterol synthesis in human epidermal keratinocytes
Authors: Ponec Maria , Havekes Louis , Kempenaar Johanna , Lavrijsen Sjan , Wijsman Marian , Boonstra Johannes , Vermeer Bert Jan
Published: February 2005

Unlike cells cultured under physiological Ca2+ concentrations (1–2 mM), keratinocytes cultured in media containing Ca2+ in low concentrations (less than 0.1 mM) do not stratify. The latter cells also differ with respect to several features of the regulation of cholesterol synthesis. In keratinocytes cultured in medium containing high Ca2+ concentrations (1.6 mM) and fetal calf serum, the rate of cholesterol synthesis was 20–30 times higher than in keratinocytes exposed to a low Ca2+ concentration. The rate of cholesterol synthesis did not change when high‐calcium cells were deprived of extracellular sources of cholesterol but increased (8–10 fold) in deprived low‐calcium cells. Furthermore, the addition of low density lipoprotein (LDL) reduced cholesterol synthesis markedly in low‐calcium cells but had no effect on high‐calcium cells. Finally, in keratinocytes cultured at low calcium concentrations the association and degradation of 125I‐LDL was 20–30 times higher than in keratinocytes cultured under high‐calcium conditions. Switching of the cells from the low‐calcium to the high‐calcium medium resulted in the induction of terminal differentiation within 15 hours and was accompanied by increased cholesterol and protein synthesis, increased competence of cells to form cornified envelopes, and reduced association of 125I‐LDL. A gradual increase of the extracellular Ca2+ concentration was accompanied by a corresponding increase of cholesterol and protein synthesis and a decrease of the response of intracellular cholesterol synthesis to changes in the extracellular concentrations of lipoprotein. Various morphological techniques showed virtually no binding and internalization of LDL by keratinocytes cultured at the high‐calcium level, whereas both were observed at the low‐calcium level. Once internalized, the LDL was delivered to dense bodies representing lysosomes. It is concluded that in human epidermal keratinocytes, the expression of the LDL receptor and the endogenous synthesis of cholesterol are regulated by the conditions determined by the differentiation stage of the cells.
Effects of bicarbonate on resting potentials in mammalian skeletal muscle
Authors: Grossie J.
Published: February 2005

In the absence of external HCO3, resting membrane potentials (Vm) in extensor digitorum longus muscle were depolarized as compared to the normal Vm in the presence of HCO3. Removal of Na or Cl from the HCO3‐free media induced repolarization. In muscle in HCO3 buffer at 20°C, internal K, Na, and Cl activities were analyzed with liquid ion selective microelectrodes. The averages were respectively, 119.7 ± 2.1, 6.69 ± 0.3, and 3.41 ± 0.06 mM. In a high proportion of cells analyzed, the equilibrium potential for Cl was negative to Vm. Removing external HCO3, decreased internal K while internal Na and Cl increased. An increase in temperature and the application of HCO3 significantly lowered internal activities of both Na and Cl. Removal of HCO3 with temperature held constant caused a rapid depolarization, an increase in internal Na and Cl, and a decrease in internal K. Furosemide (10 μM) induced a repolarization of cells that were previously depolarized in the HCO3‐free state, but the drug does not decrease internal Na.
Reinitiation of cellular DNA synthesis in BrdU‐selected nondividing senescent WI‐38 cells by simian virus 40 infection
Authors: Gorman Scott D. , Cristofalo Vincent J.
Published: February 2005

Bromodeoxyuridine‐selected nondividing senescent WI‐38 cells were stimulated to synthesize DNA, as evidenced by incorporation of [3H]thymidine into nuclei of senescent cells, after infection with simian virus 40 (SV40). Cellular DNA synthesis was confirmed by DNA‐DNA hybridization experiments and the use of temperature‐sensitive A gene mutants. The DNA synthesis was, at least in part, semiconservative, as microdensitometry of Feulgen‐stained nuclei revealed increased DNA content in a large fraction of the cells in the infected population. Thus, senescent cells retain the capacity to replicate their DNA, despite their intrinsic inability to initiate DNA synthesis.
Microtubule‐disrupting drugs increase the frequency of conversion of a rat mammary epithelial stem cell line to elongated, myoepithelial‐like cells in culture
Authors: Paterson Frieda C. , Rudland Philip S.
Published: February 2005

Rat mammary (Rama) 25 cuboidal epithelial stem cells convert at a low frequency to elongated, Thy‐1‐positive, myoepithelial‐like cells in culture; one such cell line is termed Rama 29. Addition of increasing concentrations of the microtubule‐disrupting drug colchicine to sparse cultures of Rama 25 dramatically increases the percentage of colonies containing elongated cells and the percentage of Thy‐1‐positive cells when the drug is removed. Similar results on the formation of elongated cell colonies are obtained with other microtubule disruptors, such as vinblastine, vincristine, demecolcine, and nocodazole. The inactive analogues of colchicine β‐ and δ‐lumicolchicine and the microfilamental‐disruptors cytochalasin B and D are without effect on the formation of elongated cell colonies and Thy‐1‐positive cells. For a given concentration of colchicine the percentage of elongated cell colonies and Thy‐1‐positive cells increases the longer the cells are exposed to the drug (range 8–96 hr) and the longer the drug‐treated cultures are subsequently grown in drug‐free medium. Colchicine fails to display this morphological change on Rama 29 elongated cells and on Rama 600 epithelial cells from a rat mammary metastasizing tumor. Immunofluorescent localization of antisera to tubulin confirms that colchicine disrupts the microtubules in all three cell lines at similar concentrations (0.1 to 1 μM) to those required to increase the percentage of elongated cell colonies in Rama 25. The DNA synthesis inhibitor cytosine arabinoside fails to inhibit this conversion process, and time‐lapse cinematographic studies confirm that the conversion of a cuboidal to an elongated cell can take place without cell division. However, cell division may sometimes be required for subsequent stabilization events. Treatment of Rama 25 cells with colchicine under the same conditions also increases the abundance of elongated cell (Rama 29)‐associated polypeptides, and elongated cell clones isolated after such treatment show an overall pattern of protein synthesis very similar to that of Rama 29.
Diacylglycerols and mezerein activate neutrophils by a phorbol myristate acetate‐like mechanism
Authors: O'Flaherty Joseph T. , Redman Jimmy F. , McCall Charles E. , Wykle Robert L. , Schmitt Jeffery D.
Published: February 2005

rac‐1‐O‐Myristoyl‐2‐O‐acetylglycerol, rac‐1‐O‐palmitoyl‐2‐O‐acetylglycerol, and rac‐1‐O‐oleoyl‐2‐O‐acetylglycerol acted like phorbol myristate acetate and mezerein in stimulating human neutrophil aggregation. Responses to these agents were equally influenced by cytochalasin B, extracellular calcium and magnesium, arachidonate antimetabolites, and procedures that rendered the cells desensitized to other agonists. The compounds also inhibited the binding of [3H]‐phorobol myristate acetate to its receptor on neutrophils. Thus, these agents are biologically homologous. They act by binding to a common receptor. This receptor may function physiologically as a transducer for endogenous glycerides that form in cells challenged by other stimuli.
Interactions between cyclic AMP‐ and phorbol ester‐dependent phosphorylation systems in S49 mouse lymphoma cells
Authors: Kiss Zoltá;n , Steinberg Robert A.
Published: February 2005

High‐resolution two‐dimensional gel electrophoresis of proteins labeled with either 32Pi or [35S]methionine was used to study interactions between cyclic AMP and tetradecanoyl phorbol acetate (TPA) at the level of intracellular protein phosphorylation. Cultured S49 mouse lymphoma cells were used as a model system, and mutant sublines lacking either the catalytic subunit of cyclic AMP‐dependent protein kinase or the guanyl nucleotide‐binding “Ns” factor of adenylate cyclase provided tools to probe mechanisms underlying the interactions observed. Three sets of phosphoproteins responded differently to TPA treatment of wild‐type and mutant cells: Phosphorylations shown previously ot be responsive to activation of intracellular cyclic AMP‐dependent protein kinase were stimulated by TPA in wild‐type cells but not in mutant cells, a subset of phosphorylations stimulated strongly by TPA in mutant cells was inhibited in wild‐type cells, and two novel phosphoprotein species appeared in response to TPA only in wild‐type cells. The latter two classes of TPA‐mediated responses specific to wild‐type cells could be evoked in adenylate cyclase‐deficient cells by treating concomitantly with TPA and either forskolin or an analog of cyclic AMP. Three conclusions are drawn from our results: (1) TPA stimulates adenylate cyclase in wild‐type cells causing increased phosphorylation of endogenous substrates by cyclic AMP‐dependent protein kinase, (2) activated cyclic AMP‐dependent protein kinase inhibits phosphorylation (or enhances dephosphorylation) of a specific subset of TPA‐dependent phosphoproteins, and (3) cyclic AMP‐dependent events facilitate TPA‐dependent phosphorylation of some substrate proteins.
Chloroquine allows the secretion of internalized 125I‐epidermal growth factor from fibroblasts
Authors: Wakshull E. , Cooper Janet L. , Wharton Walker
Published: February 2005

Incubation of cells with labelled hormone in the presence of the lysosomotropic agent chloroquine produces an enhanced intracellular accumulation of hormone and receptor. Using a pulse‐chase paradigm in which cell surface receptors were labelled with 125I‐EGF at 4°C, it was found that when 100 μM chloroquine was present in the 37°C chase medium intact hormone was accumulated in the medium. Without chloroquine, low molecular weight (mw) degradation products were found in the medium. The processes of receptor‐mediated endocytosis and subcellular distribution of 125I‐EGF‐receptor complexes were unchanged by chloroquine. The source of the intact hormone accumulating in the medium was therefore an intracellular compartment(s). The 125I‐EGF released from the cells could rebind to surface receptors and be re‐internalized; rebinding was inhibited by unlabelled EGF or Concanavalin A in the incubation medium. The concentration of unlabelled EGF required to inhibit rebinding was more than three orders of magnitude greater than the amount of 125I‐EGF whose rebinding was inbibited. Thus, the 125I‐EGF whose rebinding was inhibited. Thus, the 125I‐EGF released from intracellular sites was rebound preferentially over exogenous EGF. The possible pathways for secretion of intact 125I‐EGF and mechanisms of its preferential rebinding are discussed.