bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation of bovine aortic endothelial cell plasma membranes: Identification of membrane‐associated cytoskeletal proteins
Authors: Ketis Nika V. , Hoover Richard L. , Karnovsky Morris J.
Published: February 2005

The plasma membrane of bovine aortic endothelium was isolated, characterized, and found to contain at least four membrane‐associated cytoskeletal proteins. Exposure of the plasma membranes to salt media (up to 1M KCl) resulted in the release of 30% of the total plasma membrane‐associated proteins and extraction with 1% Triton X‐100, 60%. At least four heavily glycosylated bands (185‐, 165‐, 150‐, and 130,000 mol‐wt) were evident. The Triton‐insoluble pellet fraction contained several major polypeptides (30‐, 43‐, 58‐, and 240,000 mol‐wt), two of which were identified by immunoblotting as cytoplasmic actin (43,000 mol‐wt) and vimentin (58,000 mol‐wt). Strikingly, vimentin and a 240,000 mol‐wt polypeptide were routinely present in approximately a mole ratio of 4:1 in more than 60% of the plasma membrane preparations. We also report the presence of a 2.1‐like and a 4.1‐like protein associated with plasma membranes. The 2.1‐like protein demonstrated similar solubilities and apparent molecular weight (210,000) as erythroid protein 2.1. Likewise, the endothelial 4.1‐like protein exhibited similar solubilities and apparent molecular weight as erythroid protein 4.1. Immunofluorescence staining of fixed and permeabilized cultures with anti‐2.1 antibodies showed a fibrillar pattern. In contrast, cells stained with anti‐protein 4.1 were brightly fluorescent, bearing both a diffuse and punctate pattern.

This paper presents severalnovel observations pertaining to the composition of bovine aortic endothelial cell plasma membranes, namely: (1) the presence of two erythroid‐like cytoskeletal polypeptides; (2) the presence of vimentin and a 240,000 mol‐wt polypeptide in a 4:1 mole ratio in more than 60% of the plasma membrane preparations and the co‐elutionin a 4:1 mol ratio with a protein perturbant; and (3) the inability to release actin from the plasma membrane preparations, suggesting the association of actin with other molecules in the plasma membrane preparation.
Serotonin, norepinephrine, and histamine mediation of endothelial cell barrier function in vitro
Authors: Bottaro Donald , Shepro David , Peterson Scott , Hechtman Herbert B.
Published: February 2005

The effects of serotonin (5‐hydroxytryptamine, 5‐HT), norepinephrine (NE), and histamine on endothelial cell barrier function were examined in vitro. Bovine aortic endothelial (BAE) cells grown to confluence on microcarriers formed a measurable barrier to the passage of a trypan blue dye‐bovine serum albumin conjugate (TB‐BSA) from the culture medium into the microcarrier matrix. Vascular smooth muscle (VSM) cells or Swiss 3T3 fibroblasts impeded TB‐BSA diffusion only 42% and 56%, respectively, relative to BAE cells. These results suggest that barrier formation may be an endothelial cell‐specific phenomenon. Treatment of BAE cells with histamine was associated with 2‐to 3‐fold increases in the rate of TB‐BSA diffusion. In contrast, treatment with 5‐HT or NE at concentrations ranging from normal to pathophysiological circulating plasma levels significantly impeded TB‐BSA diffusion by up to 43% and 33%, respectively, relative to untreated controls. The barrier‐modulating effects of the vasoactive amines were dose‐dependent, cell‐specific, and in some cases appear to be receptor‐mediated. These results are consistent with previous reports that histamine increases vascular permeability in part by affecting diffusion between endothelial cells; they support the hypothesis that 5‐HT and NE contribute to the maintenance of the endothelial barrier in vivo.
Sensitivity of preadipose 3T3 cells to growth hormone
Authors: Morikawa Minoru
Published: February 2005

Cultured preadispose 3T3 cells are able to undergo a process of differentiation through which they are converted into adipose cells. Growth hormone induces this conversion in resting cultures but not in growing cultures. It was of interest to determine the period of cell sensitivity to the hormone and the timing of the induction of glycerophosphate dehydrogenase, a key enzyme in lipogenesis. It was found that 3T3‐F442A cells became highly sensitive to rat growth hormone at confluence but that high sensitivity remained for only 3 days; thereafter, the responsiveness to the rat growth hormone declined rapidly. Refeeding of the cells with fresh medium did not lead to the recovery of the hormone sensitivity, indicating that the decrease in sensitivity was not due to exhaustion of medium components but that it seemed to be a specific property of F442A cells. As glycerophosphate dehydrogenase activity was detected at nearly the same time as its mRNA was measurable, it is likely that the mRNA is translated immediately after its synthesis.
Localization of human histone gene transcripts predominantly in the nonmatrix nuclear fraction
Authors: Bandyopadhyay Ram , Stein Gary , Stein Janet
Published: February 2005

Using S1 nuclease protection assays, we have examined the representation of cell cycle‐dependent H4 histone RNAs in the nuclear matrix and nonmatrix nuclear fractions of human cells. Cytoplasmic and nuclear fractions were prepared from exponentially growing HeLa S3 cells by double detergent (sodium deoxycholate and NP40) lysis. The nuclear matrix and nonmatrix nuclear fractions were then prepared by digestion of nuclei with RNase‐free DNase I and subsequent high‐salt [0.4 M (NH4)2SO4] extraction. Subcellular fractions were characterized by (1) DNA, RNA, and protein composition; (2) electrophoretic analysis of the proteins in each fraction; (3) the representation of 45S ribosomal RNA precursors and processed 18S and 28S ribosomal RNAs; and (4) the presence of mitochondrial RNAs. In contrast to ribosomal and messenger RNA precursors, which are largely associated with the nuclear matrix, the human H4 histone RNAs in the nucleus were found predominantly in the nonmatrix nuclear fraction. The presence of H4 histone RNA in the nonmatrix nuclear fraction appeared to be coupled to DNA replication, since inhibition of DNA synthesis by hydroxyurea resulted in a loss of histone RNA from the nucleus. Our results suggest either that the association of histone RNAs with the nuclear matrix is very transient or that posttranscriptional modifications of the rapidly processed histone gene transcripts do not involve the nuclear matrix.
In vitro actions on hemopoietic cells of recombinant murine GM‐CSF purified after production in Escherichia coli: Comparison with purified native GM‐CSF
Authors: Metcalf D. , Burgess A. W. , Johnson G. R. , Nicola N. A. , Nice E. C. , Delamarter J. , Thatcher D. R. , Mermod J.‐J.
Published: February 2005

Recombinant murine GM‐CSF produced in Escherichia coli was purified to homogeneity and tested in parallel with purified native GM‐CSF. Both recombinant and native GM‐CSF stimulated granulocyte and/or macrophage colony formation by adult and fetalmouse progenitor cells, and with adult marrow cells the specific activity of the recombinant GM‐CSF (25 × 108 U/mg) was similar to that of the native form (15 × 108 U/mg). At high concentrations (> 200 U/ml), both forms of GM‐CSF also stimulated eosinophil colony formation by adult marrow cells and, at very high concentrations (> 800 U/ml), megakaryocyte and some erythroid and mixed‐erythroid colony formation. Recombinant GM‐CSF was as effective in stimulating the proliferation of the GM‐CSF‐dependent cell line FD as the native molecule. Both recombinant and native GM‐CSF were able to induce partial differentiation in colonies of WEHI‐3B myeloid leukemic cells. Recombinant GM‐CSF competed effectively for the binding of 125l‐labeled native GM‐CSF to hemopoietic cells, and anti‐serum to recombinant GM‐CSF also neutralized the biological activity of native GM‐CSF. The bacterially synthesized GM‐CSF was a slightly more effective stimulus for megakayocyte colony formation than then native molecule. The demonstration that purified bacterially synthesized GM‐CSF is biologically active in vitro now permits studies to be undertaken on the in vivo effects of this material.
Effects of 25‐hydroxycholesterol, cholesterol, and isoprenoid derivatives on the G1 progression in Swiss 3T3 cells
Authors: Larsson Olle , Zetterberg Anders
Published: February 2005

The effect of inhibition of 3‐Hydroxy‐3‐methylglutaryl Coenzyme A reductase (HMG CoA reductase) on cell cycle progression in proliferating 3T3 cells was studied. It was found that short transient exposures to the HMG CoA reductase inhibitor 25‐hydroxycholesterol temporarily blocked the cell cycle traverse in the postmitotic half of G1 (G1 pm), whereas cells in the subsequent cell cycle phases were unaffected. The kinetics of the cell cycle delay, induced by 25‐hydroxycholesterol, resembled the kinetics of the delay induced by serum depletion, which also inhibited the activity of HMG CoA reductase. In contrast to the case of serum depletion, platelet derived growth factor (PDGF), which efficiently prevented the decrease of HMG CoA reductase in serum‐free medium, was not capable of preventing the growth inhibitory effect following treatment by 25‐hydroxycholesterol. However, cholesterol and two isoprenoids, dolichol and coenzyme Q, were effective in this respect. In addition, dolichol counteracted the cell cycle delay following short periods of serum starvation.
Long‐term maintenance of hepatocyte functional activity in co‐culture: Requirements for sinusoidal endothelial cells and dexamethasone
Authors: Morin Odette , Normand Claire
Published: February 2005

Sinusoidal cells isolated from adult rat liver have been established in primary culture and in cell line. The presence of factor VIII R:Ag and peroxidatic/phagocytosis activities were the criteria used to distinguish in freshly isolated cells the endothelial cells from the Kupffer cells and suggested the endothelial origin of the cell line. Using a co‐culture system, the effect of sinusoidal liver cells on hèpatocyte functional activity was characterized. A plateau in which the state of differentiation was stabilized could be generated for co‐cultured hepatocytes isolated from adult rat and a disappearance of the initial expression of α1‐fetoprotein (AFP) and the increase and/or maintenance of albumin secretion were measured with co‐cultured hepatocytes isolated from suckling rat. The presence of dexamethasone was required for such beneficial effect. The hepatocyte‐stabilizing activity was also produced by a pulmonary endothelial cell line.
Vasopressin rapidly stimulates protein kinase C in quiescent Swiss 3T3 cells
Authors: Rodriguez‐Pena Angeles , Rozengurt Enrique
Published: February 2005

Addition of vasopressin to quiescent cultures of Swiss 3T3 cells caused a rapid increase in the phosphorylation of an acidic molecular weight 80,000 cellular protein (termed 80K). The effect was concentration‐ and time‐dependent; enhancement in 80K phosphorylation could be detected as early as 30 sec after the addition of the hormone. Recently, a rapid increase in the phosphorylation of an 80K cellular protein following treatment with phorbol esters or diacylglycerol has been shown to reflect the activation of protein kinase C in intact Swiss 3T3 cells. Here we show that the 80K phosphoproteins generated in response to vasopressin and phorbol 12,13‐dibutyrate (PBt2) were identical as judged by one‐ and two‐dimensional polyacrylamide gel electrophoresis (PAGE) and peptide mapping following partial proteolysis with Staphylococcus aureus V8 protease. In addition, prolonged pretreatment of 3T3 cells with PBt2 which leads to the disappearance of protein kinase C activity blocked the ability of vasopressin to stimulate the phosphorylation of 80K. The effect of vasopressin on 80K phosphorylation and mitogenesis was selectively blocked by the vasopressin antagonist (Pmp1‐O‐Me‐Tyr2‐Arg8) vasopressin suggesting that these responses are mediated by its specific receptor in these cells. The removal of vasopressin leads to dephosphorylation (within minutes) of the 80K phosphoprotein. We conclude that vasopressin rapidly stimulates protein kinase C activity in intact 3T3 cells.
Rat cerebral microvascular smooth muscle cells in culture
Authors: Diglio Clement A. , Grammas Paula , Giacomelli Filiberto , Wiener Joseph
Published: February 2005

This report describes the development and establishment of long‐term serial cultures of adult rat vascular smooth muscle cells (SMC) derived from cerebrocortical resistance vessels (small arteries and arterioles). Electron microscopic examination of microvessels isolated off a 150 μm nylon mesh sieve clearly demonstrated the predominance of these vessel types. Initial outgrowth from collagenase‐elastase‐lreated microvessel fragments yielded both endothelium and smooth muscle cells. However, at confluency (2–3 weeks) these cultures consisted of a homogeneous population of broad, polygonal cells that grew in a multilayered “hill and valley” pattern typical of SMC in vitro. For comparative morphological and functional studies, SMC cultures were also initiated from rat thoracic aortas utilizing ring segments as explants. The smooth muscle origin of cultures derived from both resistance vessel (RV) and aorta (RA) was further demonstrated by positive immunofluorescent staining by the specific smooth muscle α‐actin and myosin antibodies. Ultrastructural examination of these SMC cultures revealed similar morphologic features consisting of typical cytoplasmic myofilament bundles with associated dense bodies and numerous pinocytotic vesicles. Cell growth studies on early ( < P15)‐ and late ( > P15)‐passage RV‐ and RA‐SMC populations revealed markedly different cell growth responses. Representative growth curves of early‐ and late‐passage RA‐SMC showed a significantly higher growth rate (two‐ to fourfold) than RV‐SMC cultures. Both cultures, however, exhibited a marked increase in growth potential at higher passage levels. Heparin, at a concentration of 100 μg/ml inhibited the growth of RV‐SMC during the first 3 days after addition in both exponential and growth‐arrested culture states, whereas RA‐SMC cultures showed no inhibitory response. These studies indicate that long‐term RV‐SMC cultures can serve as a useful model system to study functional and metabolic properties of this cell type and provide the means to explore further the heterogeneity of SMC derived from different vasculatures in normal as well as various disease states.
Relation between the regulation of DNA synthesis and the production of two secreted glycoproteins by 12‐O‐tetradecanoylphorbol‐13‐acetate in 3T3 cells and in phorbol ester nonresponsive 3T3 variants
Authors: Fienup Virginia K. , Jeng Meei‐Huey , Hamilton Richard T. , Nilsen‐Hamilton Marit
Published: February 2005

12‐O‐tetradecanolyphorbol‐13‐acetate (TPA), a potent tumor promoter, acts similarly to growth factors by selectively increasing the rate of production of the secreted proteins, mitogen regulated proteins (MRP) and major excreted protein (MEP) by murine 3T3 cells. MRP, a 34 kilodalton (kDa) glycoprotein, is a member of the prolactin‐growth hormone family of proteins. MEP, a 39 kDa glycoprotein, is a lysosomal thiol protease that is also secreted. The aim of our investigation was to determine the relation between increases in MRP and MEP production and the initiation of DNA synthesis in response to mitogens. The TNR‐9 cell lines is a variant of 3T3 cells in which growth factors, but not TPA and teleocidin, stimulate DNA synthesis and cell division. Usibng [35S]methionine to metabolically label proteins and SDS polyacrylamide gel electrophoresis to resolve the proteins, we found that growing cultures of 3T3 and TNR‐9 cells responded equally well to TPA and teleocidin with increased rates of production of MRP and MEP. by conrast, the responses of quiescent TNR‐9 cells to these tumor promoters in the increased production of MRP and MEP was greatly diminished compared with quiescent 3T3 cells. The changes in production of MRP in response to tumor promopters in quiescent and growing cells paralleled similar changes in the level of MRP mRNA. In summary, the ability to TPA and teleocidin to increase the rate of production of MRP and MEP correlated with the ability of these tumor promoters to stimulate DNA synthesis in quiescent 3T3 and TNR‐9 cells. Evidently the biochemical condition that distinguishes TNR‐9 from 3T3 cells and that limits the ability of tumor promoters to stimulate the production of MEP and MRP, and perhaps also DNA synthesis in TNR‐9 cells occurs only when the cells are quiescent.
Ion‐sensitive microelectrode measurements of free intracellular chloride and potassium concentrations in hyperthermia‐treated neuroblastoma cells
Authors: Borrelli M. J. , Carlini W. G. , Ransom B. R. , Dewey W. C.
Published: February 2005

Murine NG108‐15 neuroblastoma cells were heated for times of 5–40 min at 45.5°C, and survival ranged from 0.7–0.0015, respectively. Ion‐sensitive micro‐electrodes (ISM) were used to measure the free intracellular concentrations of Cl− and K+ immediately after heating and up to 30 hr later. The free intracellular Cl− and K+ concentrations, [Cl−]i and [K+]i respectively, of the heated cells remained identical to those of the controls for the first 10 hr after heating. At later times, some cells had increased [Cl−]i values and decreased [K+]i values identical to those of the extracellular medium. These cells had a mottled morphology, no longer excluded the vital stain trypan blue, and had no membrane potential. The number of these dye‐including, physiologically dead cells increased with time, and was always greater following longer heating times. No changes in mean cellular volume were observed until 25 hr after heating. All trypan‐blue‐excluding, physiologically live cells had the same [Cl−]i and [K+]i as the control cells, even when the majority of them were destined for clonogenic death.
Spontaneous differentiation of murine teratocarcinoma stem cells at low temperature
Authors: Cremisi Chantal , Duprey Philippe
Published: February 2005

Embryonal carcinoma cells differentiate in vitro after treatment with different drugs or under certain culture conditions. We show here that at 31°C PCC‐aza‐1 cells differentiated spontaneously into cells with characteristics resembling those of epithelial cells. With this temperature, we observed morphological changes; reorganization of the cytoskeleton with polymerization of actin cables, tight junctions, and cytokeratin endo A messenger RNA and protein expression The cells also became totally permissive to Py virus but remained resistant to SV40 infection. This new pathway of differentiation has the advantage of being reversible at 37°C.
Stimulation of beta‐adrenoceptors inhibits calcium‐dependent potassium‐channels in mouse macrophages
Authors: Rosati Clelia , Hannaert Patrick , Dausse Jean‐Pierre , Braquet Pierre , Garay Ricardo
Published: February 2005

K+ efflux in mouse macrophages exhibited a rate constant (kK) of 0.67 ± 0.04 (h)−1 (mean ± SEM of 16 experiments). This was strongly stimulated by increasing concentrations of the Ca2+ ionophore A23187 up to a maximal value of 4.01 ± 0.25 (h)−1 with an IC50 of 7.6 ± 1.9 μM (mean ± SEM of 6 experiments). Similar results were obtained with the Ca2+ ionophore ionomycin. Binding experiments with 3H‐dihydroalprenolol revealed a high density of beta‐adrenergic receptors (97.5 ± 5.2 fmol/mg protein) with apparent dissociation constant of 2.03 ± 0.06 nM. Isoproterenol at a concentration of 10−6‐10−5 M induced a two‐ to threefold stimulation of endogenous levels of cyclic AMP (cAMP). A23187‐stimulated K+ efflux was partially inhibited by (i) stimulation of adenylate cyclase with isoproterenol, forskolin or, PG1; (ii) exogenous cAMP; and (iii) inhibition of phosphodiesterase with MIX (1‐methyl‐3‐isobutylxanthine). Maximal inhibition of K+ efflux was obtained by simultaneous addition of isoproterenol and MIX. In dose‐response curves, the isoproterenol‐sensitive K+ efflux was half‐maximally inhibited (IC50) with 2–5 × 10−10 M of isoproterenol concentration. Propranolol was able to completely block the effect of isoproterenol, with an IC50 of about 1–2 × 10−7 M. Isoproterenol and MIX were also able to partially inhibit ionomycin‐stimulated K+ efflux. Isoproterenol and MIX did not inhibit A23187‐stimulated K+ efflux in an incubation medium where NaCI was replaced by sucrose (or choline), suggesting the involvement of an Na + : Ca2+ exchange mechanism. Our results show that stimulation of beta‐adrenoceptors in mouse macrophages counterbalances the opening of K+ channels induced by the calcium ionophore A23187. This likely reflects a decrease in cytosolic free calcium content via a cAMP‐mediated stimulation of Na + :Ca2+ exchange.
Phorbol diesters and transferrin modulate lymphoblastoid cell transferrin receptor expression by two different mechanisms
Authors: Alcantara O. , Phillips J. L. , Boldt D. H.
Published: February 2005

Expression of transferrin receptors (TfR) by activated lymphocytes is necessary for lymphocyte DNA synthesis and proliferation. Regulation of TfR expression, therefore, is a mechanism by which the lymphocyte's proliferative potential may be directed and controlled. We studied mechanisms by which lymphoblastoid cells modulate TfR expression during treatment with phorbol diesters or iron transferrin (FeTf), agents which cause downregulation of cell surface TfR. Phorbol diester‐induced TfR downregulation occurred rapidly, being detectable at 2 min and reaching maximal decreases of 50% by 15 min. It was inhibited by cold but not by agents that destabilize cytoskeletal elements. Furthermore, this downregulation was reversed rapidly by washing or by treatment with the membrane interactive agent, chlorpromazine. In contrast, FeTf‐induced TfR downregulation occurred slowly. Decreased expression of TfR was detectable only after 15 min and maximal downregulation was achieved after 60 min. Although FeTf‐induced downregulation also was inhibited by cold, it was inhibited in addition by a group of microtubule destabilizing agents (colchicine, vinblastine, podophyllotoxin) or cytochalasin B, a microfilament inhibitor. Furthermore, FeTf‐induced downregulation was not reversed readily by washing or by treatment with chlorpromazine. The inactive colchicine analogues, β‐ and γ‐lumicolchicine, did not inhibit FeTf‐induced TfR downregulation. Similarly, when cells were pretreated with taxol to stabilize microtubules, colchicine no longer inhibited FeTf‐induced downregulation. Therefore, FeTf causes TfR downregulation in lymphoblastoid cells by a cytoskeleton‐dependent mechanism. Phorbol diesters cause TfR downregulation by a cytoskeleton‐independent mechanism. In other experiments, treatment of cells with both a phorbol diester and FeTf, either simultaneously or sequentially, produced additive effects on TfR expression. These data indicate that TfR expression is regulated by two independent mechanisms in lymphoblastoid cells, and they provide the possibility that downregulation of TfR by different mechanisms may result in different effects in these cells.
Glycosphingolipid patterns in primary mouse kidney cultures
Authors: Lyerla Timothy A. , Gross Sonja K. , McCluer Robert H.
Published: February 2005

Primary kidney cultures from C57BL/6J mice, 6 weeks of age or older, were produced using D‐valine medium to select for epithelial cell growth. After allowing the cells to attach and proliferate for 1 week following plating, medium was changed once per week. Cells formed nearly confluent monolayers during the second week of culture.

The cultured cells contained all of the glycosphingolipids seen in the adult kidney, analyzed by high performance liquid chromatography as their perbenzoyl derivatives. Glucosylceramide, however, was highly predominant in the cultured cells, whereas dihexosyl‐ and trihexosylceramides predominate in the intact kidney. Sex differences in glycolipid contents found in the intact kidney were also apparent in these cultured cells: The concentration of neutral glycolipids, in general, was higher in male cells than in those derived from females, and the male‐specific glycolipid nonhydroxy fatty acid digalactosylceramide was high in male cells but very low in female cells.

Neutral glycosphingolipids were labeled in 2‐week‐old cultures using [3H]palmitate. The [3H]palmitate was incorporated into all of the glycolipids within 2 hr of labeling. Hence, adult mouse kidney cells in D‐valine medium retain their differentiated characteristics for a sufficient period of time to allow investigation of glycolipid syntheses in monolayer cultures of epithelial cells derived from this organ.
Mouse Balb/c3T3 cell mutant with low epidermal growth factor receptor activity: Induction of stable anchorage‐independent growth by transforming growth factor β
Authors: Kuratomi Yuichiro , Ono Mayumi , Yasutake Chie , Mawatari Masasumi , Kuwano Michihiko
Published: February 2005

A mutant clone (Mo‐5) was originally isolated as a clone resistant to Na+/K+ ionophoric antibotic monensin from mouse Balb/c3T3 cells. MO‐5 was found to show low receptor‐endocytosis activity for epidermal growth factor (EGF): binding activity for EGF in MO‐5 was less than one tenth of that in Balb/c3T3. Anchorage‐independent growth of MO‐5 was compared to that of Balb/c3T3 when assayed by colony formation capacity in soft agar. Coadministration of EGF and TGF‐β efficiently enhanced anchorage‐independent growth of normal rat kidney (NRK) cells, but neither factor alone was competent to promote the anchorage‐independent growth. The frequency of colonies appearing in soft agar of MO‐5 or Balb/c3T3 was significantly enhance by TGF‐β while EGF did not further enchance tha tof MO‐5 or Balb/c3T3. Colonies of Balb/c3T3 formed in soft agar in the presence of TGF‐β showed low colony formation capacity in soft agar in the absence of TGF‐β. Colonies of MO‐5 formed by TGF‐β in soft agar, however, shoed high colony formation capacity in soft agar in the absence of TGF‐β. Pretreatment of MO‐5 with TGF‐β induced secretion of TGF‐β‐like activity from the cells, while the treatment of Balb/c3T3 did not induce the secretion of a significant amount of TGF‐β‐like activity. The loss of EGF‐receptor activity in the stable expression and maintenance of the “transformed” phenotype in MO‐5 is discussed.
Characterisation of stroma‐dependent blast colony‐forming cells in human marrow
Authors: Gordon M. Y. , Dowding C. R. , Riley G. P. , Greaves M. F.
Published: February 2005

Human bone marrow contains a population of haemopoietic progenitor cells that can be distinguished by their ability to adhere to preformed stromal layers cultured in the presence of methylprednisolon [MP+] and form blast cell colonies. The stromal layers function in the colony assay after they have been heavily irradiated but not after they have been passaged. The binding of the progenitor cells to the stromal cells is complete after 2 hours of coincubation, and stromal layers of 9.6 cm2 can provide adhesion sites for at least 2,000 blast colony‐forming cells. The blast colony‐forming cells were shown by micromanipulation to self‐renew as well as to give rise to multipotential and lineage‐committed colony‐forming progenitor cells.
Toxicity of oxidized low‐density lipoprotein to cultured fibroblasts is selective for S phase of the cell cycle
Authors: Kosugi Keisuke , Morel Diane W. , DiCorleto Paul E. , Chisolm Guy M.
Published: February 2005

Oxidized LDL (o‐LDL) is toxic to a variety of cultured cells. Preliminary results suggested that susceptibility is enhanced by cell proliferation. As a step toward determining the mechanism of cytotoxicity, we chose to identify the cell cycle phase(s) during which exposure of cultured human fibroblasts to o‐LDL leads to death. Cytochalasin B, which blocks cell migration and proliferation, and irradiation, which prevents mitosis but not migration, both blocked cytotoxicity. Colchicine, which arrests cells in mitosis but does not inhibit DNA synthesis, did not block cytotoxicity. Treatment of cells with hydroxyurea, which blocks cells prior to S phase, prevented cell death. Addition of o‐LDL to cells immediately after S phase allowed mitosis without death. The above results coupled with results using cells synchronized by three different means indicate that cell death is selective for proliferating cells and occurs after exposure to o‐LDL during S phase. Understanding the mechanism of o‐LDL‐induced death may have implications for tissue damage in vivo in the numerous instances of pathology in which oxidized lipoproteins or lipids are present.
Further analysis of spontaneous membrane potential activity and the hyperpolarizing response to parathyroid hormone in osteoblastlike cells
Authors: Ferrier Jack , Ward‐Kesthely Angela , Homble Fabrice , Ross Stephen
Published: February 2005

Whole cell voltage clamp measurements using the patch technique on well‐attached and well‐spread cells of an osteoblastlike line (ROS 17/2.8) show the same spontaneous membrane potential activity as measurements with inserted microelectrodes. Furthermore, membrane potential measurements during the first 80 milliseconds (ms) following microelectrode penetration of the cell membrane usually show no decay. There is also good agreement between values of cell membrane resistance obtained by the microelectrode technique, the whole cell patch clamp technique, and the single channel patch clamp technique. These results indicate that our microelectrode measurements are not dominated by leak‐induced artifacts, and that the spontaneous membrane potential activity is not induced by Ca2+ leakage around the microelectrode. The spontaneous membrane potential activity is eliminated in the presence of the Ca2+ ionophore A23187, also in serum‐free medium, and by K+ and Ca2+ channel blockers, but it is not affected by the hyperpolarizing responses to parathyroid hormone (PTH) and dibutyryl cAMP, which persist under all of these conditions. These results support the hypothesis that the spontaneous membrane potential activity is related to repeated fluctuations of internal [Ca2+] and that such fluctuations result from a feedback loop involving Ca2+ channels or Ca2+ pumps in the cell membrane.
Heat stress stimulates inositol trisphosphate release and phosphorylation of phosphoinositides in CHO and Balb C 3T3 cells
Authors: Calderwood Stuart K. , Stevenson Mary Ann , Hahn George M.
Published: February 2005

Exposure of eukaryotic cells to elevated temperature leads to profound switches in cell metabolism and gene expression which may be involved in cellular homeostatic mechanisms. We have investigated the effect of heat shock (45°C) on the metabolism of the phosphoinositides, a class of phospholipids involved in the function of Ca2+‐linked membrane receptors. Heat shock led to stimulation of phosphoinositide turnover in HA1‐CHO and Balb C 3T3 cells, resulting in the rapid accumulation of inositol trisphosphate (lP3). Mitogenic and α1 adrenergic stimulation, with serum or phenylephrine, led to similar increases in lP3. Heat shock also caused rapid increase in phosphorylation of polyphosphoinositides (PPl). Prolonged exposure to heat >15 min at 45°C led to progressive cellular toxicity which was associated with depletion of PPl. This decline in PPl concentration appeared to result from inhibition of PPl resynthesis. In this respect, heat may resemble some other types of cellular stresses in stimulating membrane phospholipases to deplete classes of membrane phospholipids. The induction of PPl turnover may, therefore, be involved in both plelotropic responses to brief heat shock and toxicity resulting from prolonged thermal stress.
Isolation and characterization of a cloned growth factor dependent macrophage cell line, BAC1.2F5
Authors: Morgan Claudia , Pollard Jeffrey W. , Stanley E. Richard
Published: February 2005

The SV40 transformed murine macrophage cell line, BAC1, proliferates in response to the colony stimulating factor, CSF‐1 (Schwarzbaum et al., J. Immunol., 132:1158, 1984). In order to obtain a cell line suitable for biochemical and genetic studies of CSF‐1 signal transduction, clones of BAC1 were established. Clones ranged from being completely autonomous to being completely dependent on CSF‐1 for growth. Cells of one clone (2F5), which proliferated in response to either CSF‐1 or granulocyte‐macrophage CSF (GM‐CSF) were characterized in detail. The kinetics of receptor‐mediated internalization and intracellular destruction of CSF‐1 were comparable to the kinetics observed with peritoneal exudate macrophages. CSF‐1 was shown to regulate cell spreading, cell survival, protein degradation, and the duration of the G1 and S phases of the cell cycle. The 2F5 clone therefore exhibits a number of CSF‐1 stimulated responses and is being used for genetic and biochemical studies of CSF‐1 action.
Ionic responses and growth stimulation in rat astroglial cells: Differential mechanisms of gangliosides and serum
Authors: Skaper Stephen D. , Varon Silvio
Published: February 2005

Rat astroglial cells respond to fetal calf serum (FCS) and gangliosides, including GM1, by undergoing proliferation. Here, we show that addition of FCS but not GM1 causes an increase in Na+, K+‐pump activity, as measured by ouabain‐sensitive 86Rb+ influx. The increase of Na+, K+‐pump activity by FCS was due to increased Na+ influx (measured with 22Na+). This increased Na+ influx was sensitive to amiloride, an inhibitor of Na+/H+ exchange. Amiloride also blocked the FCS‐stimulated incorporation of [3H]thymidine into DNA. Two defined polypeptide growth factors, epidermal growth factor and fibroblast growth factor were also able to elicit an amiloride‐sensitive Na+ influx and an ouabain‐sensitive K+ uptake in these astroglial cells, in the presence of FCS or insulin. Thus, GM1 differs from serum and growth factors in the mechanisms by which these agents stimulate the proliferation of the astroglial cells used here.
Relationship of thrombin‐stimulated arachidonic acid release and metabolism to mitogenesis and phosphatidylinositol synthesis
Authors: Raben Daniel M. , Yasuda Kathleen M. , Cunningham Dennis D.
Published: February 2005

Thrombin and certain prostaglandins are both capable of stimulating the proliferation of cultured cells. Since thrombin stimulates the release and metabolism of arachidonic acid, the precursor of prostaglandins, we examined the relationship between this release and metabolism and the stimulation of cell division in cultured fibroblasts. We also examined the role of prostaglandin synthesis in thrombin‐stimulated phosphatidylinositol synthesis.

The data in this report demonstrate that the release and metabolism of arachidonic acid are not necessary for thrombin‐stimulated cell division. The presence of a low concentration of chymotrypsin prevented thrombin‐stimulated arachidonic acid release and metabolism without affecting the stimulation of cell division. Furthermore, thrombin‐stimulated cell division occurred in the presence of indomethacin concentrations that prevented cyclooxygenase‐mediated metabolism of arachidonic acid.

The following experiments showed that thrombin‐stimulated phosphati‐dylinositol synthesis was brought about by a cyclooxygenase‐mediated metabolite(s) of arachidonic acid. Indomethacin inhibited the cyclooxygenase‐mediated metabolism of arachidonic acid without affecting the thrombin‐stimulated release of arachidonic acid. Indomethacin also inhibited thrombin‐stimulated phosphatidylinositol synthesis. The dose dependence of this inhibition paralleled the inhibition by indomethacin of cyclooxygenase‐mediated metabolism of arachidonic acid. In addition, prostaglandin F2, stimulated phosphatidylinositol synthesis in the presence of indomethacin concentrations which prevented thrombin‐stimulated phosphatidylinositol synthesis.
Specific gene expression during compensatory renal hypertrophy in the rat
Authors: Beer David G. , Zweifel Kathleen A. , Simpson David P. , Pitot Henry C.
Published: February 2005

The compensatory growth of the kidney which is induced by unilateral ne‐phrectomy is a highly regulated process resulting principally in hypertrophy of the remaining kidney. The events which regulate this process are unknown. We have examined the levels of transcripts for the proto‐oncogenes, myc, H‐ras, K‐ras, and fos, and the cellular genes, H4 histone, ornithine aminotrans‐ferase, and gamma‐glutamyl transpeptidase, following unilateral nephrec‐tomy in the rat. The pattern of expression of c‐myc, c‐H‐ras, and c‐K‐ras during compensatory growth of the kidney differs from the pattern of expression of these proto‐oncogenes during liver regeneration, in which, unlike the kidney, hyperplasia rather than hypertrophy predominates. The lack of change in the abundance of these proto‐oncogene transcripts following unilateral nephrec‐tomy suggests a primary relationship between the expression of these proto‐oncogenes and DNA synthesis and indicates there may be separate signals for cell growth, one to double cell size and one to replicate DNA. Increased mRNA transcripts for the enzymes ornithine aminotransferase and gamma‐glutamyl transpeptidase were induced in the contralateral kidney after ne‐phrectomy. The time course of expression for these two enzymes differs. The early expression of the gamma‐glutamyl transpeptidase gene may indicate an involvement of this glutathione‐metabolizing enzyme during renal compensatory growth, while the function of the delayed increase in ornithine amino‐transferase transcripts in the remaining kidney is not apparent.
Homologous and heterologous desensitization of proto‐oncogene cfos expression in murine peritoneal macrophages
Authors: Introna Martino , Bast Robert C. , Johnston Paul A. , Adams Dolph O. , Hamilton Thomas A.
Published: February 2005

Treatment of murine peritoneal macrophages for 30 min with lipopolysaccharide (LPS) resulted in a transient increase in c‐fos proto‐oncogene mRNA levels (Introna et al., 1986). After 2 h from the initial treatment, c‐fos mRNA could no longer be detected and its expression could not be restimulated either by LPS or by other signals including colony stimulating factor‐1 (CSF‐1) and phorbol myristate acetate (PMA), both of which are able to induce expression of the c‐fos gene in unstimulated macrophages. When LPS was removed after an initial 30 min incubation, responsiveness to a second exposure to LPS began to reappear after 3 h and was completely restored by 20 h. The same pattern of desensitization of c‐fos induction was observed when CSF‐1 stimulated macrophages were subsequently exposed to LPS. The loss of sensitivity to PMA following pretreatment with LPS was selective for c‐fos expression as LPS treated macrophages remained responsive to PMA with respect to the ability to stimulate secretion of H2O2. The mechanism of desensitization was localized, at least in part, at the level of transcription as demonstrated by analysis of c‐fos transcripts in nuclei isolated from macrophages pretreated and restimulated with LPS.
Magnesium deprivation inhibits the expression of differentiation‐related phenotypes in human promyelocytic leukemia HL‐60 cells
Authors: Okazaki Toshiro , Mochizuki Toshihiro , Tashima Masaro , Sawada Hiroyoshi , Uchino Haruto
Published: February 2005

The role of magnesium ions in the differentiation of human promyelocytic leukemia HL‐60 cells was investigated. When HL‐60 extracellular magnesium was deficient (less than 0.01 mM), the total intracellular magnesium content and [3H] leucine incorporation rates decreased to 61 and 28%, respectively, on day 3. When the cells were treated with various inducers (100 nM 1α, 25 dihydroxyitamine D3 (1,25(OH)2D3), 100 nM β‐all‐trans retinoic acid (RA), 20 nM 12‐o‐tetradecanoyl phorbol‐13‐acetate (TPA), 1.25% dimethylsulfoxide (DMSO) and 30 nM aclacinomycin (AcM)) in magnesium‐deficient medium, the expression of differentiation‐related phenotypes (nitroblue tetrazolium (NBT) reducing ability, nonspecific esterase (NSE) activity and monoclonal antibody, OKM1 binding activity) was almost completely inhibited. After a 2‐day treatment with 100 nM 1,25(OH)2D3 in magnesium‐deficient medium, the expression of differentiation‐related phenotypes was restored by further incubation in the absence of inducer in standard magnesium medium (0.4 mM). These results suggested that magnesium deprivation inhibited the expression of HL‐60 differentiation‐related phenotypes but not their commitment to differentiation. These phenotypes were expressed without inducer in standard magnesium medium after a 2‐day simultaneous treatment with 1,25(OH)2D3 and cyclohexamide (protein synthesis inhibitor) in magnesium‐deficient medium, but not after simultaneous pretreatment with 1,25(OH)2D3 and α ‐amanitin (RNA synthesis inhibitor). Thus, it was suggested that the magnesium‐requiring step in HL‐60 cell differentiation is in protein but not mRNA synthesis. This conclusion is supported by the findings that changes in c‐myc and c‐fms mRNA levels in HL‐60 cells treated with 100 nM 1,25(OH)2D3 in magnesium‐deficient medium and those in standard magnesium medium were the same. In addition, dibutyryl cyclic adenosine monophosphate (dbc AMP) could restore expression of differentiation‐related phenotypes inhibited by magnesium deprivation but not those inhibited by cyclohexamide, even though magnesium deprivation inhibited protein synthesis as much as did cyclohexamide. This suggests that magnesium‐requiring step in HL‐60 cell differentiation is different from that inhibited by cyclohexamide.
Exposure to caffeine and suppression of DNA replication combine to stabilize the proteins and RNA required for premature mitotic events
Authors: Schlegel Robert , Croy Robert G. , Pardee Arthur B.
Published: February 2005

Caffeine had been shown to induce mitotic events in Syrian hamster fibroblast (BHK) cells that were arrested during DNA replication (Schlegel and Pardee, Science 232:1264–1266, 1986). Inhibition of protein synthesis blocked these caffeine‐induced events, while inhibition of RNA synthesis showed little effect. We now report that the protein(s) that are required for inducing mitosis in these cells were synthesized shortly after caffeine addition, the activity was very labile in the absence of caffeine, and the activity was lost through an ATP‐dependent mechanism. Caffeine dramatically increased the stability of these putative proteins while having no effect on overall protein degradation. Experiments with an inhibitor of RNA synthesis indicated that mitosis‐related RNA had accumulated during the suppression of DNA replication, and this RNA was unstable when replication was allowed to resume. These results suggest that the stability of RNA needed for mitosis is regulated by the DNA replicative state of the cell and that caffeine selectively stabilizes the protein product(s) of this RNA. Conditions can therefore be selected that permit mitotic factors to accumulate in cells at inappropriate times in the cell cycle. Two‐dimensional gel electrophoresis has demonstrated several protein changes resulting from caffeine treatment; their relevance to mitosis‐inducing activity remains to be determined.
Heparin modulation of the neurotropic effects of acidic and basic fibroblast growth factors and nerve growth factor on PC12 cells
Authors: Neufeld Gera , Gospodarowicz Denis , Dodge Linda , Fujii Dennis K.
Published: February 2005

Nerve growth factor (NGF) and acidic or basic fibroblast growth factor (aFGF and bFGF, respectively) induce neurite outgrowth from the rat pheochromocytoma cell line, PC12. The neurites induced by these three factors are stable for up to a month in cell culture in the continued presence of any of the above growth factors. bFGF (ED50 = 30 pg/ml) is 800 fold more potent in stimulating neurite outgrowth than aFGF (ED50 = 25 ng/ml) and 260 fold more potent than NGF (ED50 = 8 ng/ml). While the neurotropic activities of aFGF and NGF are potentiated by heparin, that of bFGF is both partially inhibited or stimulated, depending upon the concentration of bFGF. Radioreceptor binding experiments show that aFGF and bFGF bind to a common binding site on the PC12 cell surface. Affinity labeling studies demonstrate a single receptor with an apparent molecular weight of 145,000 daltons, which corresponds to the high molecular weight receptor identified in BHK‐21 cells. NGF does not appear to compete with aFGF or bFGF for binding to the receptor. Heparin blocked the binding of bFGF to the receptor but had only a small inhibitory effect on the binding of aFGF to the receptor. Thus, it appears that heparin inhibition of the neurotropic effects of bFGF occurs, at least in part, by impairing the interaction of bFGF with the receptor, while having little effect on that of aFGF. The stimulatory effects of heparin on the neurotropic activity of aFGF, bFGF, and NGF may occur through a site not associated with the respective cellular receptor for the growth factors.
Temporal requirement for epidermal growth factor and insulin in the stimulation of hepatocyte DNA synthesis
Authors: Sand Tor‐Erik , Christoffersen Thoralf
Published: February 2005

Primary monolayer cultures of adult rat hepatocytes were used to study the temporal interaction of epidermal growth factor (EGF) and insulin in their stimulation of DNA synthesis. The hepatocytes were cultured both under defined conditions and with serum. EGF and insulin interacted synergistically. The entry into S phase (G1 exit) followed first‐order kinetics both in untreated and hormone‐stimulated cells. Addition of EGF and insulin at the time of plating did not alter the lag period before the DNA synthesis started (25–26 h), but the rate constant for the S phase entry increased five‐ to sixfold. Experiments where the time of hormone addition was varied indicated that insulin exerted its strongest effect at the time of plating, whereas the cells became more responsive to EGF after being cultured for up to 40–50 h. The responsiveness to EGF at these later stages required an early exposure of the hepatocytes to insulin. When the administration of EGF to insulin‐pretreated hepatocytes was postponed for 44 h after plating in serum‐free medium, the cellular sensitivity was increased as compared to EGF treatment at 0 h (a one‐log shift of the dose‐effect curve), the rate of S phase entry was more rapid, and the lag period for the onset of the EGF effect (i.e., shift of rate constant) was shortened (6–7 h vs. 26 h).
Resolution of multiple endosomal compartments associated with the internalization of epidermal growth factor and transferrin
Authors: Gorman Regina M. , Poretz R. D.
Published: February 2005

Morphological studies have indicated divergent pathways for the endocytosis of epidermal growth factor (EGF) and transferrin (Tf). In order to obtain biochemical evidence for the pathways associated with the endocytosis of EGF and Tf, a series of Percoll density gradients were employed to separate individual cellular components. Subcellular fractionation of murine fibro‐blasts exposed to a 2‐min pulse of either 125l‐Tf or 125I‐EGF results in the detection of a total of six cellular compartments related to the internalization process of these ligands. The results of kinetic analysis of the entry of EGF into five membranous fractions is consistent with a model in which ligand is transferred sequentially from the plasma membrane through three distinct prelysosomal environments prior to reaching secondary lysosomes. Each prelysosomal compartment exhibits distinct density and temporal properties in a Percoll density gradient and may represent preexisting endocytic vesicles and/or specific domains of a continuous tubular structure, vesicularized during the process of cell disruption. In addition, the observed differential migration on Percoll density gradients of Tf and EGF containing compartments indicates that the majority of cell bound Tf segregates from EGF and enters a compartment lacking EGF within 5 min of internalization.