bluuebunny/crossref_metadata_2025 huggingface dataset:
Isoprenylated proteins in cultured cells: Subcellular distribution and changes related to altered morphology and growth arrest induced by mevalonate deprivation
Authors: Maltese William A. , Sheridan Kathleen M.
Published: February 2005

In the presence of lovastatin (mevinolin), an inhibitor of endogenous mevalonate synthesis, C1300 murine neuroblastoma cells incorporated (2‐14C)mevalonate into several discrete polypeptides that were separable by SDS‐PAGE. The electrophoretic pattern of the labeled proteins did not vary substantially when cells were homogenized with Ca++, Mg++, high concentrations of NaCl or phosphatase inhibitor, or when cells were lysed immediately in trichloroacetic acid. When cells that had been prelabeled with (14C)mevalonate were incubated with lovastatin and simultaneously deprived of exogenous mevalonate, there was a 50–60% decline in the concentration of protein‐bound isoprenoid label within 17 h. In contrast, there was little change in the radioactivity in the sterol, dolichol, or ubiquinone fractions. The time course of the decline in mevalonate‐derived label in cellular polypeptides paralleled the onset of neurite outgrowth and preceded the decline of DNA synthesis, suggesting that a decreased intracellular concentration of protein‐bound isoprenoid groups may contribute to the well‐documented effects of mevalonate deprivation on cell morphology and cell cycling. Fractionation of neuroblastoma cells by differential centrifugation and sucrose density‐gradient centrifugation revealed that mevalonate‐labeled proteins of 53 kDA, 22–26 kDa, and 17 kDa were concentrated in the cytosol. Proteins migrating at 45 kDa were found in both the soluble and particulate fractions, including those enriched in mitochondria and plasma membrane. The isoprenylated proteins migrating at approximately 66 kDa were localized exclusively in the nuclear fraction. When chromatin was removed from the nuclei by extraction with 2 M NaCl, the 66 kDa isoprenylated proteins remained associated with the residual components of the nuclear matrix and lamina. Isoprenylated proteins with electrophoretic mobilities similar to those observed in neuroblastoma cells were detected in a variety of established cell lines. However, there was considerable variation among cell lines in the overall efficiency of protein labeling with (14C)mevalonate and in the prominence and mobilities of specific labeled proteins in the 45–70 kDa range. Comparisons of paired transformed vs. nontransformed fibroblast cell lines suggested that the profile of mevalonate‐labeled proteins in a given cell line is not altered by malignant transformation. The finding that electrophoretically distinct isoprenylated proteins are localized in discrete subcellular compartments indicates that they do not represent dissociated subunits of a single multimeric protein complex, and provides a possible explanation for the diverse effects of mevalonate deprivation. The presence of these proteins in a wide variety of mammalian cells suggests that they play a fundamental role in cell structure and/or regulation.
Alpha tocopheryl succinate inhibits melanocyte‐stimulating hormone (MSH)‐sensitive adenylate cyclase activity in melanoma cells
Authors: Sahu Satya N. , Edwards‐Prasad Judith , Prasad Kedar N.
Published: February 2005

D‐alpha tocopheryl succinate (vitamin E succinate), which is known to induce differentiation and growth inhibition in murine B‐16 melanoma cells, reduced basal and melanocyte‐stimulating hormone (MSH)‐stimulated adenylate cyclase (AC) activity in vitro. Vitamin E succinate treatment also reduced sodium fluoride‐ and forskoline‐stimulated AC activity of melanoma cells in vitro. Treatment of cells with vitamin E succinate (6 μg/ml) for a period of 24 hours was sufficient to reduce MSH‐stimulated AC activity. Other forms of vitamin E, such as d1‐alpha tocopheryl nicotinate, d1‐alpha tocopheryl acetate, and d1‐alpha tocopherol, which did not affect growth or morphology of melanoma cells, were relatively less effective in altering basal and MSH‐stimulated AC activity. Retinoic acid, which inhibited the growth of B‐16 melanoma cells, also reduced basal and MSH‐, NaF‐, and forskolin‐stimulated AC activity in vitro. Prostaglandin A2, which inhibited growth and altered morphology, did not change basal or MSH‐stimulated AC activity. These results show that one of the mechanisms of action of vitamin E succinate and retinoic acid on melanoma cells may involve reduction of basal and MSH‐sensitive AC activity, and this vitamin effect is not necessarily related to growth inhibition.
Fibroblast‐dependent growth of mouse mast cells in vitro: Duplication of mast cell depletion in mutant mice of w/wv genotype
Authors: Fujita Jun , Nakayama Hiroki , Onoue Hitoshi , Kanakura Yuzuru , Nakano Toru , Asai Hidekazu , Takeda Shun‐Ichi , Honjo Tasuku , Kitamura Yukihiko
Published: February 2005

In spite of the apparent depletion of mast cells in tissues of mutant mice of W/Wv genotype, cells with many features of mast cells do develop when bone marrow cells of W/Wv mice are cultured in the presence of pokeweed mitogen‐stimulated spleen cell‐conditioned medium (PWM‐SCM). In order to resolve this discrepancy and facilitate the analysis of the W mutation, we attempted to establish an in vitro system in which the in vivo defect of W/Wv mice can be reproduced. Cultured mast cells (CMC) were developed from bone marrow cells of either W/Wv or congenic +/+ mice, and then co‐cultured with NIH/3T3 mouse fibroblasts in media supplemented only with fetal calf serum (i.e., in the absence of PWM‐SCM). Under this condition, CMC from +/+ mice continued to divide and were maintained for more than 4 weeks. The supportive effect of NIH/3T3 cells required close‐range interactions with CMC and was not due to synthesis of the known mast cell growth factors, interleukins 3 and 4. By contrast, CMC from W/Wv mice were not maintained, and the number of mast cells remaining after 4 weeks of co‐culture was only 1% of the normal +/+ counterparts. Thus, the humoral factor‐independent and cell contact‐dependent system presented here revealed the intrinsic defects in growth and differentiation of CMC derived from W/Wv mice and might be useful for biochemical and molecular analysis of the gene product(s) encoded at the W locus.
Characterization of functional histamine H1 receptors on a cultured smooth muscle cell line
Authors: Mitsuhashi Masato , Payan Donald G.
Published: February 2005

Cultured cells of the smooth muscle line DDT1MF‐2, which was derived from a hamster vas deferens tumor, expressed histamine H1‐type receptors and responded biochemically and functionally to H1‐specific stimulation. The H1‐receptor antagonist [3H]‐pyrilamine bound specifically to 9.7 × 106 sites/DDT1MF‐2 cell with a dissociation constant (Kd) of 219 nM. The addition of histamine to suspensions of fura‐2‐loaded DDT1MF‐2 cells elicited a rapid, transient, and stimulus concentration‐dependent increase in the intracellular concentration of Ca2+ with an EC50 of 3 × 10−5 M, which demonstrated H1 receptor specificity. Moreover, in order to evaluate in vitro contractile response of individual DDT1MF‐2 cells, the degree of intracellular actin polymerization was quantified by a DNase inhibition assay. The percentage of nonpolymerized or G‐actin in DDT1MF‐2 cells was reduced in a histamine concentration‐dependent manner with an EC50 of 1 × 10−5 M and H1 receptor specificity. Histamine‐induced actin polymerization was accompanied by changes in cell shape that were consistent with cellular contraction, as assessed by flow cytometry. The H1‐type receptors of cultured DDT1MF‐2 cells thus couple histamine stimulation to a variety of functional responses of smooth muscle cells.
Recombinant interferon‐γ inhibits the mitogenic effect of platelet‐derived growth factor at a level distal to the growth factor receptor
Authors: Hosang M.
Published: February 2005

Highly purified preparations of recombinant human interferons (rIFNs)‐αA, ‐β, and ‐γ all inhibited platelet‐derived growth factor (PDGF)‐induced DNA synthesis in normal human dermal fibroblasts, as monitored by incorporation of [3H]‐thymidine into trichloroacetic acid (TCA)‐insoluble material. rIFN‐γ was the most potent, since it blocked the PDGF response by 50% at about 10 U/ml or 0.3 ng/ml, whereas with rIFN‐αA and rIFN‐β 4000 U/ml and 600 U/ml, respectively (10 ng/ml in both cases), were required to achieve the same effect. There was a close parallelism between the ability of these rIFNs to inhibit PDGF mitogenic activity and their capacity to inhibit cell proliferation in serum‐containing medium. None of the rIFNs inhibited specific binding of 125I‐PDGF to fibroblasts, and none interfered with receptor internalization. The mechanism of action of rIFN‐γ was analyzed further. rIFN‐γ did not inhibit uptake of [3H]‐thymidine into these cells. However, it shifted if the time point of initiation of DNA synthesis from about 14 h after stimulation with PDGF to about 18 to 21 h and decreased significantly the rate of the DNA synthesis. rIFN‐γ could be added up to 6 h following stimulation with PDGF with no loss of its inhibitory effect. rIFN‐γ also blocked the mitogenic activity of epidermal growth factor and basic fibroblast growth factor. Taken together these results implicate that rIFN‐γ exerts its antimitogenic effect by inhibiting a process that occurs late in the PDGF signaling pathway and onto which the activity pathways of other mitogens converge. In view of the important role PDGF may play in wound‐healing and in the pathogenesis of the proliferative lesions of arteriosclerosis, these data point to a possible role IFN‐γ may play as a regulator of these processes in vivo.
Mitogenic action of tumor necrosis factor in human fibroblasts: Interaction with epidermal growth factor and platelet‐derived growth factor
Authors: Palombella Vito J. , Mendelsohn John , Vilček Jan
Published: February 2005

We have previously shown that tumor necrosis factor (TNF) can increase the number of epidermal growth factor (EGF) receptors on human FS‐4 fibroblasts and that this increase may be related to the mitogenic action of TNF in these cells. Here we show that TNF stimulated the growth of FS‐4 fibroblasts in a chemically defined, serum‐free medium in the absence of EGF. Anti‐EGF receptor antibody, which blocked the mitogenic effects of EGF in FS‐4 cells, did not inhibit the mitogenic action of TNF in serum‐free or serum‐containing medium, indicating that EGF or an EGF‐like molecule was not responsible for the mitogenic effects of TNF. However, the simultaneous addition of TNF and EGF to cells grown in serum‐free medium resulted in a synergistic stimulation of DNA synthesis and cell growth. The actions of TNF and EGF were also examined in growth‐arrested FS‐4 cells and were compared with the action of platelet‐derived growth factor (PDGF). In the absence of other growth factors, TNF was a relatively weak mitogen in growth‐arrested cells, compared with EGF or PDGF. Nevertheless, TNF synergized with EGF or high doses of PDGF in stimulating DNA synthesis. Furthermore, antibodies specific for TNF or the EGF receptor were used to selectively inhibit the actions of these two factors, after specific incubation periods, in growth‐arrested cells treated concurrently with EGF and TNF. To produce an optimal stimulation of DNA synthesis, EGF had to be present for a longer period of time than TNF. We conclude that in their synergistic action on growth‐arrested FS‐4 cells, EGF was responsible for driving the majority of the cells into S phase, while TNF appeared to make the cells more responsive to the mitogenic action of EGF. The findings indicate that TNF can cooperate with, and enhance the actions of, EGF in promoting DNA synthesis and cell division.
Osmotic forces are not critical for Ca2+‐induced secretion from permeabilized human neutrophils
Authors: Stoehr Sally Jo , Smolen James E.
Published: February 2005

In order to examine the role of osmotic forces in degranulation, the effects of solutes and osmolality on granule secretion were explored using both FMLP‐stimulated, intact neutrophils and Ca2+‐stimulated, permeabilized cells. We employed a HEPES‐based buffer system which was supplemented with: (a) permeant (KCl or NaCI) or impermeant (Na‐isethionate or choline‐CI) ions, or (b) permeant (urea) or impermeant (sucrose) uncharged solutes. Intact and permeabilized cells had significantly different solute requirements for degranulation. FMLP‐stimulated release from intact cells was supported by NaCI or Na‐isethionate > KCl > choline‐Cl or sucrose > urea. In contrast, the rank order of Ca2+‐stimulated release from permeabilized cells was choline‐C > Na‐isethionate, KCl, or NaCl > sucrose > urea. Hypo‐osmotic conditions caused increased levels of background granule release from both intact and permeabilized neutrophils. However, hypo‐osmolality inhibited both FMLP‐stimulated degranulation from intact cells and Ca2+‐induced release from permeabilized neutrophils. While hyperosmotic conditions inhibited stimulated release from intact cells, this inhibition was much less pronounced in permeabilized cells when the granules were directly exposed to these solutions. In fact, hyperosmotic sucrose greatly enhanced Ca2+‐induced secretion. Although isolated specific and azurophil granules showed some lytic tendencies in hypo‐osmotic buffers, the overall stability of the isolated granules did not indicate that swelling alone could effect degranulation. These results suggest that degranulation in permeabilized cells is neither due to nor driven by simple osmotic forces (under resting or stimulated conditions) and emphasize differences obtained by bathing both the granules and plasma membrane (as opposed to membranes alone) in various solutes.
Demonstration of 1α,25‐dihydroxyvitamin D3 receptor‐like molecule in ST 13 and 3T3 L1 preadipocytes and its inhibitory effects on preadipocyte differentiation
Authors: Sato Mayumi , Hiragun Akiyoshi
Published: February 2005

The active metabolite of vitamin D3, 1α,25‐dihydroxyvitamin D3 (1,25(OH)2D3), inhibited morphologic and enzymatic expression during differentiation of preadipocyte to adipocyte. In the presence of ∼6.4–20 × 10−10 M 1,25(OH)2D3, the triacylglycerol accumulation was only 50% of that of fully differentiated control cells. High‐affinity binding sites for 1,25(OH)2D3 binding component sediments at 3.3 S in 4–24% (w/v) sucrose gradients prepared in hypertonic buffer. Binding assay revealed that Nmax was 70 fmol/mg protein and 90 fmol/mg protein, and Kd value was 170 pM and 37 pM in cell lines ST 13 and 3T3 L1, respectively. We also found that differentiated adipocytes did not contain specific receptors for 1,25(OH)2D3. 1,25(OH)2D3, 1(OH)D3, 24,25(OH)2D3, and 24(OH)D3 all suppressed differentiation of preadipocytes to adipocytes, and the dose required closely reflected the affinities of the various metabolites and the synthetic derivative for 1,25(OH)2D3 receptor. It is suggested that the action of vitamin D3 on preadipocyte differentiation may result from a receptor‐mediated event.
Incubation of endothelial cells in a superoxide‐generating system: Impaired low‐density lipoprotein receptor‐mediated endocytosis
Authors: Poumay Y. , Ronveaux‐Dupal M.‐F.
Published: February 2005

Endothelial cells (EC) of blood vessels are submitted to oxidative stress under various circumstances. These conditions may modify EC functions; therefore, in the present work we have studied the receptor‐mediated endocytosis of low‐density lipoproteins (LDL) and malondialdehyde‐modified LDL by the LDL receptor and the “scavenger” receptor, respectively, in cultured human umbilical vein EC after short (0‐120 minutes) incubations in a superoxide anion (O2−) generating system. In both receptor‐mediated processes, the oxidative stress produces a significant decrease at four different LDL concentrations (5‐50 μg/ml) after 120 minutes of oxidation. On the other hand, the fluid‐phase endocytosis of sucrose by EC seems to be stimulated by these conditions. Furthermore, incorporation of antioxidant enzymes in the O2−−producing system shows that H2O2 is an obligatory intermediate in order to produce the effect on the receptor‐mediated processes. Hypotheses concerning the mechanisms involved in the modifications of endocytotic processes and their implications in vivo are discussed.
Release of arachidonic acid from vascular endothelial cells: Fatty acyl specificity is observed with receptor‐mediated agonists and with the calcium ionophore A23187 but not with melittin
Authors: Rosenthal Miriam D. , Jones Janet E.
Published: February 2005

Vascular endothelial cells respond to a variety of physiological and pharmacological stimuli by releasing free arachidonic acid from membrane phospholipids, thus initiating synthesis of prostacyclin. Previous work in our laboratory has demonstrated that the thrombin‐stimulated deacylation is specific for arachidonate and structurally similar polyunsaturated fatty acids that contain a delta‐5 double bond. We now report that histamine, bradykinin, and the calcium ionophore A23187 exhibit the same fatty acid specificity as does thrombin. Experiments with both human umbilical vein and calf pulmonary artery endothelial cells indicate that these agonists stimulate the release of previously incorporated [14C]arachidonate but not 8,11,14‐[14C]eicosatrienoate or [14C]docosatetraenoate. By contrast, melittin stimulates the release of 8,11,14‐eicosatrienoate, docosatetraenoate, and oleate as well as arachidonate. These results suggest that histamine, bradykinin, and A23187 activate a common calcium‐dependent phospholipase A2. Melittin appears either to alter the substrate specificity of the receptor‐linked phospholipase A2 activity or to activate additional enzymes as well.
Specific binding, internalization, and degradation of human recombinant interleukin‐3 by cells of the acute myelogenous, leukemia line, KG‐1
Authors: Gesner Thomas G. , Allan Mufson R. , Norton Christine R. , Turner Katherine J. , Yang Yu Chung , Clark Steven C.
Published: February 2005

We have studied the interaction of 35S‐labeled recombinant IL‐3 with the acute myelogenous leukemia cell line, KG‐1. 35S‐IL‐3 bound to these cells in a time dependent, saturable, and specific manner at 4°C. Scatchard transformation of binding isotherms demonstrated the existence of a small number (200) of binding sites, with an apparent dissociation constant of 70‐105 pM. After a temperature shift from 4°C to 37°C, surface‐bound 35S‐IL‐3 was rapidly internalized and processed into a trichloroacetic acid soluble form that was released into the medium. Experiments to address the specificity of the IL‐3 binding site revealed that neither human IL‐2, M‐CSF, erythropoietin, transferrin, bovine insulin, nor murine nerve growth factor compete with IL‐3 for binding to KG‐1 cells. Both human and gibbon recombinant IL‐3 and, surprisingly, human recombinant GM‐CSF effectively competed the binding of the labeled IL‐3 to these cells at 4°C. The competition by GM‐CSF was found to be concentration dependent, but much higher concentrations were required to achieve the levels obtained with IL‐3. These results suggest that GM‐CSF may also interact with the high‐affinity IL‐3 binding site on KG‐1 cells or, alternatively, that GM‐CSF binding to its own receptor may decrease the affinity of the IL‐3 receptor for its ligand.
Identification of the fibroblast growth factor receptor in human vascular endothelial cells
Authors: Neufeld Gera , Gospodarowicz Denis
Published: February 2005

The fibroblast growth factor (FGF) receptor of human umbilical vein‐derived endothelial (HUE) cells has been identified by affinity labeling. It has an apparent molecular weight of 130,000. It binds both basic and acidic FGF, but not with epidermal growth factor, insulin, or transferrin. The lectin concanavalin‐A does not inhibit the binding of 125l‐bFGF to HUE cell‐surface receptors, whereas it inhibits bFGF binding to BHK‐21 cell‐surface FGF receptor. This suggests that both types of receptors may differ in their degree of glycosylation. In contrast to other cell types, heparin only slightly inhibits the binding of basic FGF to its receptor. Protamine sulfate, which is anti‐angiogenic in vivo, and suramin, a drug used in the therapy of trypanosomiasis and onchocerciasis, also inhibit the binding of basic FGF to the receptor.
Amphiphile‐induced heart muscle‐cell (Myocyte) injury: Effects of intracellular fatty acid overload
Authors: Janero David R. , Burghardt Charles , Feldman Dorothy
Published: February 2005

Lipid amphiphile toxicity may be an important contributor to myocardial injury, especially during ischemia/reperfusion. In order to investigate directly the potential biochemical and metabolic effects of amphiphile overload on the functioning heart muscle cell (myocyte), a novel model of nonesterified fatty acid (NEFA)‐induced myocyte damage has been defined. The model uses intact, beating neonatal rat myocytes in primary monolayer culture as a study object and 5‐(tetradecyloxy)‐2‐furoic acid (TOFA) as a nonmetabolizable fatty acid. Myocytes incubated with TOFA accumulated it as NEFA, and the consequent NEFA amphiphile overload elicited a variety of cellular defects (including decreased beating rate, depletion of high‐energy stores and glycogen pools, and breakdown of myocyte membrane phospholipid) and culminated in cell death. The amphiphile‐induced cellular pathology could be reversed by removing TOFA from the culture medium, which resulted in intracellular TOFA “wash‐out.” Although the development and severity of amphiphile‐induced myocyte injury could be correlated with both the intracellular TOFA/NEFA content (i.e., the level of TOFA to which the cells were exposed) and the duration of this exposure, removal of amphiphile overload did not inevitably lead to myocyte recovery. TOFA had adverse effects on myocyte mitochondrial function in situ (decoupling of oxidative phosphorylation, impairing respiratory control) and on myocyte oxidative catabolism (transiently increasing fatty acid β oxidation, citric acid cycle flux, and glucose oxidation). The amphiphile‐induced bioenergetic abnormalities appeared to constitute a state of “metabolic anoxia” underlying the progression of myocyte injury to cell death. This anoxic state could be ameliorated to some extent, but not prevented, by carbohydrate catabolism.
Human macrophage colony‐stimulating factor inhibits bone resorption by osteoclasts disaggregated from rat bone
Authors: Hattersley G. , Dorey E. , Horton M. A. , Chambers T. J.
Published: February 2005

Colony stimulating factors (CSFs) regulate the survival, proliferation and differentiation of haemopoietic progenitor cells, as well as the functional activity of mature cells. Because the osteoclast is derived from haemopoietic tissue, and because osteoblastic cells produce CSFs, we tested the effects of several CSFs on bone resorption by osteoclasts disaggregated from neonatal rat long bone. We found that recombinant macrophage (M)‐CSF was a potent inhibitor of bone resorption, causing significant inhibition at concentrations similar to those required to support the growth of macrophage colonies in agar. Unlike other inhibitors of osteoclastic resorption, M‐CSF did not alter cytoplasmic motility in time‐lapse recordings, suggesting that M‐CSF may inhibit osteoclasts through a different transduction mechanism. None of the remaining cytokines tested (granulocyte‐macrophage CSF, interleukin 3, interleukin 6, or interferon γ) influenced bone resorption. M‐CSF production may be a mechanism by which osteoblastic cells, which produce M‐CSF, may regulate osteoclastic function. Alternatively, inhibition of osteoclastic resorption by a CSF that is responsible for amplification of the macrophage compartment may reflect a close lineage relationship between mononuclear phagocytes, in which M‐CSF induces a diversion of lineage resources away from osteoclastic function.
Transfer of functional EGF receptors to an IL3‐dependent cell line
Authors: Collins Mary K. L. , Downward Julian , Miyajima Atsushi , Maruyama Kazuo , Arai Ken‐Ichi , Mulligan Richard C.
Published: February 2005

Epidermal growth factor (EGF) is a small protein that acts as a mitogen for various epidermal, epithelial, and fibroblastic cells that bear specific EGF receptors. The molecule that binds EGF is a 175‐kD transmembrane protein, with an extracellular ligand binding domain and an intracellular domain that possesses tyrosine kinase activity, thought to be involved in the mitogenic signalling process. Here we have constructed a recombinant murine retrovirus that transduces a human cDNA encoding the 175‐kD protein and used this retrovirus to infect BAF3, a murine, bone marrow‐derived cell line, which is dependent on the haematopoietic factor interleukin‐3 (IL3) for its growth in culture. The EGF receptors expressed on the infected cells exhibit two affinity states, as well as EGF‐stimulated autophosphorylation. Furthermore, EGF can replace IL3 in supporting short‐term proliferation of these cells. These data identify functional properties of the EGF receptor upon expression of the 175‐kD EGF binding protein in a haemotopoietic cell that does not express endogenous receptors. They also suggest that gene transfer of growth factor receptors to heterologous cells may allow novel growth stimuli to be exploited.
Quantitation of specific binding of erythropoietin to human erythroid colony‐forming cells
Authors: Sawada K. , Krantz S. B. , Sawyer S. T. , Civin C. I.
Published: February 2005

Highly purified human erythroid colony‐forming cells (ECFC), which consist predominately of colony‐forming units‐erythroid (CFU‐F), were prepared from human blood and used to study the binding and processing of erythropoietin (Ep). When radioiodinated human recombinant Ep (125I‐rEp) was incubated with these cells, binding was specific and saturable. Specific binding was directly proportional to cell concentration and did not occur with other human cells. Saturation of specific binding at 3°C occurred at 1 nM (3.9/U/ml), and Scatchard analysis revealed two classes of binding sites on the cell surface. Of a total of 1,050 binding sites per ECFC, one‐fifth had a Kd of 0.10 nM, while the remainder had a Kd of 0.57 nM. Specific binding was twofold greater at 37°C than at 3°C, and removal of surface‐bound Ep with acid indicated that 125I‐rEp was internalized into the cells after incubation at 37°C. Further incubation at this temperature showed a decline of cellular radioactivity, with a release of small molecular weight degradation fragments into the medium. These studies demonstrate two classes of receptors for Ep on normal human ECFC. Internalization and degradation of EP occur, and the biologic effect of the hormone is produced by a small number of Ep molecules, as demonstrated in murine erythroid progenitor cells.
Disturbance of cell proliferation by two model compounds of lipid peroxidation contradicts causative role in proliferative senescence
Authors: Poot Martin , Esterbauer Hermann , Rabinovitch Peter S. , Hoehn Holger
Published: February 2005

Cumene hydroperoxide (Chp), a lipophilic peroxide, and hydroxy‐nonenal (HNE), a breakdown product of lipid peroxides, were used as model compounds to assess the effects of lipid peroxidation upon cell proliferation. Amniotic fluid fibroblastlike (AFFL) cells and human diploid skin‐derived (HDFL) cells were cultured with the two model compounds and cell proliferation was assayed via bromodeoxyuridine‐Hoechst flow cytometry. At low doses Chp elicited an accumulation of cells in the S and G2 phase, while at higher doses the fraction of nonproliferating cells increased as well. Low doses of HNE caused an accumulation of cells in the G1 and G2 phase, whereas an additional increase of cells in S phase and in the nonproliferating fraction was found at an elevated concentration. A delay of onset of proliferation was obtained with both Chp and HNE. Permanent arrests in the S, G2, and G1 compartment are provoked by Chp only when Chp was applied together with serum. HNE, to the contrary, elicited a permanent arrest in the G2 and the G1 compartment even if added to quiescent cell cultures. Additionally, HNE caused a combination of a prolongation of the G1 phase of the cell cycle and an arrest in this compartment, which is reminiscent of cell differentiation. HDFL cells were much more sensitive toward Chp than were AFFL cells, but both cell types showed similar sensitivities toward HNE. We conclude that lipophilic peroxides exert toxic effects upon cell proliferation distinct from the pattern elicited by aldehydic breakdown products of lipid peroxides. The pattern of cell cycle arrest induced by Chp and HNE makes it unlikely that Chp and HNE, or related products of lipid peroxidation, are responsible for the limitation of the proliferative life span of human fibroblasts in culture.
Reciprocal regulation of adult rat hepatocyte growth and functional activities in culture by dimethyl sulfoxide
Authors: McGowan Joan A.
Published: February 2005

Hepatocyte DNA synthesis, initiated by epidermal growth factor (EGF), is reversibly inhibited by 2% dimethyl sulfoxide (DMSO). At that concentration, both the survival of the cells in culture and the expression of differentiated functions are prolonged. DMSO does not affect thymidine uptake or EGF receptor binding. Moreover, EGF receptor binding is maintained at 84% of initial 12 hr binding when cells are cultured for several days in the presence of DMSO, whereas specific receptor binding declines to 49% of initial binding under standard culture conditions without DMSO. Studies of hepatocyte functional activity indicate that, during early culture, total cellular export protein synthesis, specific albumin synthesis, and glycogen synthesis are enhanced in the presence of DMSO. Dexamethasone is required for the effect of DMSO on survival, and although dexamethasone alone enhances hepatocyte DNA synthesis in the presence of EGF, it does not reverse the inhibitory effect of 2% DMSO on DNA replication. The correlation of prolonged survival with growth inhibition supports the hypothesis that hepatic growth and differentiated functional activity may be reciprocally regulated.
Insulin‐like growth factor binding protein secretion by muscle cells: Effect of cellular differentiation and proliferation
Authors: McCusker Robert H. , Clemmons David R.
Published: February 2005

Several cell types have been shown to secrete insulin‐like growth factor binding proteins (IGF‐BP) in vitro. Since IGF‐BP influences cell responsiveness to IGF, three muscle cell types were investigated to determine if they produced IGF‐BP and to identify factors that regulate IGF‐BP secretion. Porcine smooth muscle cells (pSMC), rat L6 skeletal muscle cells, and mouse BC3H‐1 myocytes were used. IGF‐BP activity in serum‐free conditioned media was quantitated with a polyethylene glycol precipitation method. All three cell types secreted IGF‐BP activity into the medium. Insulin was a potent stimulant of IGF‐BP secretion for each cell type. Specifically, 1 ug/ml insulin increased the IGF‐BP concentration in conditioned media from 10.5 ± 1.3 to 15.0 ± 1.5 ng/ml in confluent L6 myotubes, from 42.5 ± 11.1 to 90.5 ± 9.8 ng/ml in confluent BC3H‐1 cells, and from 2.1 ± 0.1 to 3.8 ± 0.1 ng/ml in confluent pSMC. L6 myotubes required more insulin (8 ug/ml) to achieve a half‐maximal stimulation of IGF‐BP secretion than confluent pSMC, differentiation deficient L6.DD cells or BC3H‐1 cells, where half‐maximal stimulation occurred between 125 and 300 ng/ml. L6 myoblasts were 40‐fold more sensitive to insulin stimulation of IGF‐BP secretion than L6 myotubes. IGF‐I, although it interferes with the assay and thereby lowers the amount of detectable IGF‐BP, stimulated the secretion of IGF‐BP from all three cell types. Dexamethasone, (10−7 M) decreased IGF‐BP secretion into the media by approximately 50% for all three cell types. Affinity cross‐linking and ligand blotting of 125I‐IGF‐I to conditioned media from each cell type showed (IGF‐BP)‐(IGF‐I) complexes with molecular weights ranging 32–40 kDa (24–32 kDa for IGF‐BP and 7.5 kDa for IGF‐I). Insulin stimulated cell proliferation for both L6 myoblasts and BC3H‐1 myocytes. This cell proliferative response was associated with an increase in IGF‐BP secretion/cell in response to insulin. In contrast dexamethasone decreased L6 myoblast proliferation and decreased IGF‐BP secretion/cell. We conclude that IGF‐BP is secreted by each muscle cell type and that the state of cellular differentiation or quiescence influences its basal and insulin‐stimulated secretion. Insulin and IGF‐I are stimulators of IGF‐BP secretion, whereas dexamethasone inhibits IGF‐BP secretion. Because these hormones control muscle cell growth and differentiation, the IGF‐BP may play an important regulatory role in these processes.
Properties of binding sites for chloroquine in liver lysosomal membranes
Authors: Colombo Maria Isabel , Bertini Francisco
Published: February 2005

Chloroquine (CQ) is an antimalarial and antirheumatic drug that accumulates in lysosomes. We purified liver lysosomal membranes of tritosomes from albino mice injected with Triton WR 1339. The membranes were used for the binding assay with CQ in 0.01 M Tris‐HCl buffer (pH 7.4). This binding was saturable, with a KD value of 6.2 μM. To understand the nature of CQ affinity, the binding was done under conditions that alter membrane structure and composition. Changes in pH, high ionic strength, and bivalent cations reversibly decreased the binding, while the effect of non‐ionic detergents was partially reversed. The cationic detergent Hyamine strongly decreased the binding, and its effect was irreversible. Binding increased after treatment with phospholipase C or D, while trypsin and neuraminidase had no effect. The results indicate the existence of binding sites for CQ in liver lysosomal membranes, which were strongly affected by changes of charge in the molecules involved in the binding. The treatment with the enzymes suggests that loss of polar groups of phospholipids increases the affinity of CQ by exposing protein sites located deep in the membrane, or by permiting a closer interaction between the drug and membrane lipids. CQ lysosomotropism and other effects of CQ on the lysosomal apparatus studied by other authors may be due not only to its accumulation inside the acid milieu of the lysosomes, in the same manner as other weak bases, but also to the affinity of CQ for binding sites in the lysosomal membrane.
Induction of cyclic AMP synthesis by forskolin is followed by a reduction in the expression of c‐myc messenger RNA and inhibition of 3H‐thymidine incorporation in human fibroblasts
Authors: Heldin Nils‐Erik , Paulsson Ylva , Forsberg Karin , Heldin Carl‐Henrik , Westermark Bengt
Published: February 2005

We have studied the effect of increased intracellular levels of cyclic AMP on the growth response to platelet‐derived growth factor (PDGF) of human foreskin fibroblasts in culture. It was found that forskolin, a potent stimulator of adenylate cyclase activity, inhibits the stimulatory effect of PDGF on 3H‐thymidine incorporation with a dose dependence similar to that observed with regard to cyclic AMP formation. A time‐course study indicated that forskolin has no effect on ongoing DNA synthesis but affects events in the prereplicative phase. The cell‐cycle block induced by forskolin was found to be reversible; after removal of the drug, DMA synthesis was initiated after a lag period, similar to that of the prereplicative phase of control cells. Forskolin had no effect on PDGF binding, receptor autophosphorylation, or c‐fos mRNA expression. However, a reduction in PDGF‐induced c‐myc mRNA expression was observed in cultures given forskolin. Forskolin was also found to have a marked stimulatory effect on the expression of interferon‐β2 mRNA expression. However, we were unable to demonstrate that the growth‐inhibitory effect of forskolin is mediated by interferon‐β. In conclusion, an increase in cAMP levels leads to a reversible inhibition of PDGF‐induced DNA synthesis in human fibroblasts, which may be related to an inhibition of c‐myc mRNA expression.
Localization of basic fibroblast growth factor to the developing capillaries of the bovine retina
Authors: Hanneken Anne , Lutty Gerard A. , McLeod D. Scott , Robey Frank , Harvey Anita K. , Hjelmeland Leonard M.
Published: February 2005

The basic fibroblast growth factor (FGF) is a potent mitogen that has vascular endothelium as one of its principle target cells. Recent work has provided both the complete amino acid sequence of basic FGF and the nucleotide sequence of the genes for both human and bovine basic FGF. Although capillary endothelial cells have been shown to produce basic FGF in vitro and to deposit basic FGF in their extracellular matrix in vitro as well, no direct evidence yet exists for the distribution of basic FGF in vivo. Antipeptide antibodies were prepared against a 15‐amino‐acid sequence from the amino terminus of basic FGF in order to avoid cross‐reactivity with acidic FGF, a protein with 55% overall homology to basic FGF. After affinity purification, these antisera were used to localize the basic fibroblast growth factor in the fetal and adult bovine retina. Immunoreactive material was found in capillaries of the inner nuclear layer, a capillary network undergoing development during the third trimester in the fetal bovine eye. Although the resolution of the technique does not permit a unique assignment of cellular localization, the presence of stain immediately adjacent to the lumen of capillaries suggests that capillary endothelial cells may produce the basic fibroblast growth factor in vivo during vascular development.
Butyric acid causes morphological changes in cultured chondrocytes through alterations in the extracellular matrix
Authors: Bretton Randolph H. , Pennypacker John P.
Published: February 2005

Butyric acid induces characteristic changes in the morphology of chick embryo chondrocytes. Chick embryo chondrocytes when cultured in the absence of butyrate exhibit a spherical morphology and synthesize cartilage‐specific chondroitin sulfate proteoglycan (CSPG). When these cultures are initiated and maintained in the presence of butyric acid, chondrocytes exhibit a mesenchymal morphology, a 90% reduction in the synthesis of CSPG, and a 75% reduction in DNA synthesis. The reduced synthesis of CSPG and DNA was shown not to be dependent on the morphological change. Chondrocytes require CSPG in order to express a spherical morphology, since including chondroitinase ABC in the culture media caused the cells to spread. In addition, the treatment of chondrocytes with purified CSPG prior to culture in media containing butyric acid resulted in spherical cells. The butyrate‐induced spreading was shown to require either serum or fibronectin and could be prevented with antiserum against chick cell‐surface fibronectin (cFn). Cell‐surface fibronectin, which was present on both spherical and flattened chondrocytes, organized into fibrils beneath cells which spread. Increased fibronectin synthesis was not responsible for the butyrate‐induced morphological change. From this evidence, it is concluded that the mechanism by which butyrate alters the morphology of these cells in culture involves inhibiting CSPG synthesis, thus preventing CSPG accumulation in the extracellular matrix (ECM). The absence of CSPG in the ECM allows fibronectin to mediate spreading of chondrocytes in culture.
Interaction of heparin with human basic fibroblast growth factor: Protection of the angiogenic protein from proteolytic degradation by a glycosaminoglycan
Authors: Sommer Andreas , Rifkin Daniel B.
Published: February 2005

Fibroblast growth factors (FGF) are a family of heparin‐binding angiogenic polypeptide mitogens. In the presence of heparin, recombinant human basic fibroblast growth factor (bFGF) is fully protected from tryptic digestion and partially protected from chymotryptic digestion. Complete protection of bFGF by heparin is achieved at ratios of growth factor:heparin of approximately 10 or less (w/w). The protection requires bioactive bFGF because inactivated bFGF is rapidly degraded by trypsin or chymotrypsin in the presence of heparin. The bFGF‐heparin interaction forms hydrophobic complexes which become insoluble in aqueous buffers at bFGF:heparin ratios of 8 to 10 (w/w). The heparin was found to bind up to a tenfold excess of bFGF on a weight basis. bFGF in the presence of heparin is as active as bFGF alone in inducing 3H‐thymidine incorporation into Swiss 3T3 fibroblast DNA.
Bicarbonate abolishes intracellular alkalinization in mitogen‐stimulated 3T3 cells
Authors: Szwergold Benjamin S. , Brown Truman R. , Freed Jerome J.
Published: February 2005

An increase in intracellular pH (pHi) following mitogenic stimulation has been reported in a variety of mammalian cells (W. Moolenaar, Annu. Rev. Physiol., 48:363–376, 1986; E. Rozengurt, Science, 234:161–166, 1986). This increase is currently believed to constitute a “permissive” signal in the process of cell activation (A.E. Lagarde and J.M. Pouyssegur, Cancer Biochem. Biophys. 9: 1–14, 1986). Since the majority of studies of this phenomenon have been conducted in the nonphysiological milieu of bicarbonate‐free solutions, we have undertaken a study of the effects of bicarbonate and CO2 on mitogen‐induced intracellular alkalinization in NIH 3T3 cells.

Using nuclear magnetic resonance (NMR) spectroscopy and novel 31P NMR pH indicators (2‐amino‐phosphono‐carboxylic acids) we found that mitogen induces an increase in pHi of 0.16 units only in cells bathed in medium containing low concentrations of bicarbonate (less than 1 mM) and not in cells bathed in medium containing physiological levels of bicarbonate (10–30 mM). In addition to abolishing the mitogen‐induced alkalinization, bicarbonate stabilizes pHi at 7.25 units as the external pH (pHe) is varied from 7.0 to 7.6. In contrast, in a bicarbonate‐free medium pHi increases from 6.9 to 7.3 over the same range of external pHs.

At a constant external pH, increasing the bicarbonate/CO2 concentration results in an increase in pHi from 6.9 in bicarbonate‐free solution to 7.25 in a bicarbonate‐buffered medium. This relationship is hyperbolic with half‐maximal effect occurring at a concentration of 0.4 mM bicarbonate at pH 7.05 and 37°C.

Our results suggest that the observations of mitogen‐induced alkalinization may be due to the use of nonphysiological bicarbonate‐free media. Since this increase in pHi is not observed in physiological media where bicarbonate concentrations are usually greater than 20 mM, we conclude that an increase in pHi is not an obligatory or usual part of the cellular response to growth factors in vivo.
FGF and EGF act synergistically to induce proliferation in BC3H1 myoblasts
Authors: Kelvin David J. , Simard Gilles , Connolly Joe A.
Published: February 2005

BC3H1 muscle cells proliferate when grown in high concentrations of FBS (20%). Lowering the FBS concentration to 0.5% causes the cells to stop proliferating and is permissive for the morphological and biochemical differentiation of BC3H1 cells. Exposure of differentiated BC3H1 myocytes to high concentrations of serum or to the purified growth factors FGF or TGF‐b induced a shutdown of this differentiation program but did not induce cell proliferation (Olson et al.,J. Cell Biol.,103: 1799‐1805, 1986; Lathrop et al., Cell Biol.,100:1540–1547, 1985, and Cell Biol., 101:2194–2198,1985). We explored the possibility that BC3H1 cells require factors to act synergistically to induce proliferation. We found that EGF and FGF function in a synergistic fashion to stimulate BC3H1 proliferation. Moreover, the temporal requirement for these growth factors suggest that they are functioning as competence and progression factors for BC3H1 cell proliferation.
Stimulation of cartilage‐matrix proteoglycan synthesis by morphologically transformed chondrocytes grown in the presence of fibroblast growth factor and transforming growth factor‐beta
Authors: Inoue Hiroyuki , Kato Yukio , Iwamoto Masahiro , Hiraki Yuji , Sakuda Mamoru , Suzuki Fujio
Published: February 2005

The effects of transforming growth factor‐beta (TGF‐beta) on the synthesis of cartilage‐matrix proteoglycan by cultured rabbit chondrocytes were examined. Rabbit chondrocytes were seeded at low density and exposed to a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F‐12 medium supplemented with 0.5% fetal bovine serum, 1% bovine serum albumin, 50μg/ml ascorbic acid, and 2 × 10−7 M hydrocortisone (Medium A). Various combinations of TGF‐beta, insulin‐like growth factor–l (IGF–I), and fibroblast growth factor (FGF) were also added to Medium A, and the chondrocytes were grown to confluency. Chondrocytes grown with TGF‐beta or FGF alone became flat or fibroblastic, those grown with FGF and TGF‐beta became very elongated and formed distinct foci, and those grown with FGF and IGF‐I showed the spherical configuration characteristic of overtly differentiated chondrocytes. Nevertheless, the incorporation of 3H With glucosamine into the large, chondroitin sulfate proteoglycan synthesized by cultures with FGF and TGF‐beta was similar to that in cells grown with FGF and IGF‐I and five times that in cells cultured with FGF alone. The increases in incorporation of 3H reflected real increases in proteoglycan synthesis, because chemical analyses showed an increase in the accumulation of macromolecules containing uronic acid in cultures with FGF and TGF‐beta or with FGF and IGF‐I. However, FGF in combination with either TGF‐beta or IGF‐I had little effect on the incorporation of 3H into small proteoglycans or hyaluronic acid. These results indicate that chondrocytes morphologically transformed with TGF‐beta and FGF fully express the differentiated proteoglycan phenotype rather than the transformed glycosaminoglycan phenotype.
Differences of Ca2+ regulation in skin fibroblasts from blacks and whites
Authors: Nakamura Akitoshi , Gardner Jeffrey , Hatori Norio , Nakamura Makiko , Fine Burton P. , Aviv Abraham
Published: February 2005

Black people have a higher propensity than caucasions toward essential hypertension. To explore the possibility that this racial difference relates to cellular Ca2+ metabolism, we measured 45Ca2+ washout and uptake and cytosolic free concentration of Ca2+ [Ca2+], in serially passed skin fibroblasts from normotensive black and white males. Depending on the experimental conditions, 45Ca2+ washout in these cells was described by either two or three exponential functions, whereas 45Ca2+ uptake was described only by a two‐exponent function. There were no racial differences in 45Ca2+ uptake and washout of unstimulated fibroblasts. However, stimulation by human serum resulted in an increase in the 45Ca2+ washout that was higher in fibroblasts from blacks than from whites. The racial differences were expressed primarily by higher values of the apparent washout rate constant (k1) of 45Ca2+ from the largest and most rapidly exchangeable cellular pool. The effect of human serum was not related to its origin (blacks vs. whites). In 2 mM Ca2+ medium and 10% serum from blacks, the respective k1 (mean ± SEM; × 10−2/min) values for fibroblasts from blacks and whites were 89.68 ± 5.23 and 73.29 ± 4.0; in the presence of 10% serum from whites, the k1 values for cells from blacks and whites were 84.14 ± 2.80 and 76.36 ± 3.23 (overall significance of P .01). In Ca2+‐deficient medium in the presence of 10% human serum, the k1 for fibroblasts from blacks and whites were 115.57 ± 3.76 and 102.15 ± 3.30 (P < .05). Serum substantially increased the 45Ca2+ uptake in fibroblasts from both blacks and whites; however, racial differences were not observed. Basal levels of [Ca2+], were not different in fibroblasts of blacks vs. whites (46.8 ± 6.8 and 43.2 ± 7.1 nM for blacks and whites, respectively). However, the peak response of Ca2+ transients for cell stimulated by 5% human serum was significantly higher in blacks than whites (blacks = 963 ± 213, whites = 481 ± 162 nM; P = .0286). We conclude that Ca2+ regulation is different in serum‐stimulated fibroblasts from blacks and whites and that, at least in part, this difference may relate to a greater agonist‐induced mobilization of Ca2+ in fibroblasts from blacks.