bluuebunny/crossref_metadata_2025 huggingface dataset:
Heparan sulfate proteoglycans in the substratum adhesion sites of human neuroblastoma cells: Modulation of affinity binding to fibronectin
Authors: Vallen Elizabeth A. , Eldridge Kevin A. , Culp Lloyd A.
Published: February 2005

Tissue culture substratum adhesion sites from EGTA‐detached Platt human neuroblastoma cells were extracted with a buffer containing octylglucoside, NaCl, guanidine hydrochloride, and a variety of protease inhibitors, an extraction which resulted in quantitative solubilization of the 35SO4=‐radiolabeled proteoglycans and 3H‐leucine‐radiolabeled proteins. Of the sulfate‐radiolabeled material, the vast majority was heparan sulfate proteoglycan (Kav = 0.15 on Sepharose C14B columns) and the remainder was chondroitin sulfate chains (no single chains of heparan sulfate were observed). This extract was then fractionated on DEAE‐Sephadex columns under two different buffer elution conditions. Under DEAE‐I conditions in low ionic strength acetate buffer, two major peaks of 35SO4=‐radiolabeled material (A, B) and a minor peak (C) could be resolved in the NaCl gradient; however, three‐fourths of the material required 4 M guanidine hydrochloride to elute it from the column (peak D). Under DEAE‐II conditions in acetate buffer supplemented with 8 M urea, the vast majority of the proteoglycan material could be eluted in the NaCl gradient as peak AB. Peak D material was shown to contain aggregated proteoglycan, along with nonproteoglycan protein, which high concentrations of urea or guanidine could dissociate, but not nonionic or zwitterionic detergents. Three different affinity chromatography systems were used to further characterize these components. Approximately 60% of peak A heparan sulfate proteoglycan from DEAE‐I binds to the hydrophobic matrix, octyl‐Sepharose, while 80% of the proteoglycan in DEAE‐I peak D binds to this hydrophobic column. A sizable fraction of peak A proteoglycan fails to bind to plasma fibronectin but does bind to platelet factor‐4 affinity columns. In contrast, peak AB proteoglycan from DEAE‐II columns yields a much higher proportion of molecules which do bind to fibronectin. To examine the basis for these differences in affinity binding, nonproteoglycan protein from these adhesion sites was mixed with peak AB proteoglycan prior to affinity chromatography; proteoglycan binding to fibronectin decreased markedly while binding to platelet factor‐4 was unaffected. This modulating activity involves the binding of nonproteoglycan protein in adhesion site extracts to both fibronectin on the column, as well as to heparan sulfate proteoglycan itself, and it could not be mimicked by a number of known proteins in adhesion site extracts or several other proteins. These results demonstrate selectivity and specificity in this modulation and indicate that a previously unidentified protein(s) is responsible. The significance of these findings in the substratum adhesion processes of neuronal cells is discussed.
Stimulation of LDL receptor activity in Hep‐G2 cells by a serum factor(s)
Authors: Ellsworth Jeff L. , Brown Cynthia , Cooper Allen D.
Published: February 2005

The regulation of low‐density lipoprotein (LDL) receptor activity in the human hepatoma cell line Hep‐G2 by serum components was examined. Incubation of dense monolayers of Hep‐G2 cells with fresh medium containing 10% fetal calf serum (FM) produced a time‐dependent increase in LDL receptor activity. Uptake and degradation of 125l‐LDL was stimulated two‐ to four‐fold, as compared with that of Hep‐G2 cells cultured in the same media in which they had been grown to confluence (CM); the maximal 125l‐LDL uptake plus degradation increased from 0.2 μ/mg cell protein/4 to 0.8 μg/mg cell protein/4 h. In addition, a two‐fold increase in cell surface binding of 125l‐LDL to Hep‐G2 cells was observed when binding was measured at 4°C. There was no change in the “apparent” Kd. The stimulation of LDL receptor activity was suppressed in a concentration‐dependent manner by the addition of cholesterol, as LDL, to the cell medium. In contrast to the stimulation of LDL receptor activity, FM did not affect the uptake or degradation of 125l‐asialoorosomucoid. Addition of FM increased the protein content per dish, and DNA synthesis was stimulated approximately five‐fold, as measured by [3H]thymidine incorporation into DNA; however, the cell number did not change. Cellular cholesterol biosynthesis was also stimulated by FM; [14C]acetate incorporation into unesterified and esterified cholesterol was increased approximately five‐fold. Incubation of Hep‐G2 cells with high‐density lipoproteins (200 μg protein/ml) or albumin (8.0 mg/ml) in the absence of the serum factor did not significantly increase the total processed 125l‐LDL. Stimulation of LDL receptor activity was dependent on a heat‐stable, nondialyzable serum component that eluted in the inclusion volume of a Sephadex G‐75 column. Uptake of 125l‐LDL by confluent monolayers of human skin fibroblasts was not changed by incubation with FM or by incubation with Hep‐G2 conditioned medium. Taken together, these data demonstrate that LDL receptor activity in Hep‐G2 cells is stimulated by a serum component. Furthermore, this serum factor shows some specificity for the LDL receptor pathway in liver‐derived Hep‐G2 cells.
Retinoic acid inhibits Ca2+ currents and cell proliferation in a B‐lymphocyte cell line
Authors: Bosma Martha , Sidell Neil
Published: February 2005

Using the whole‐cell variation of the patch‐clamp technique, we have demonstrated that retinoic acid (RA) blocks Ca channels and inhibits cell proliferation in a mouse hybridoma cell line (MHY206) derived from a fusion of murine myeloma and splenic B cells. In 25 mM external Ca, and with an Na internal solution containing aspartate, cAMP, and Mg‐ATP, inward currents were activated in these cells from holding potentials more negative than −70 mV, peaked at voltage steps up to −20 mV, and were voltage‐inactivated within the 125‐msec duration of the pulse. With more positive pulses, outward current carried by Na ions permeating through the Ca channels were seen. Application of RA blocked both inward and outward current through the Ca channels in a dose‐dependent manner, with 50% block at a concentration of around 5 × 10−5 M. Proliferation was blocked by 75% at that concentration, and the same relation between the reduction in current and proliferation was seen throughout the concentration range. A similar reduction of Ca currents and proliferation was demonstrated with octanol, a long‐chain alcohol that has recently been reported to block Ca channels. These results suggest a role for Ca channels in the proliferation of MHY206 cells and implicate blockage of these channels as contributing to the antiproliferative activity of RA.
Involvement of cAMP and calcium in the induction of ornithine decarboxylase activity in an osteoblast cell line
Authors: van Leeuwen J. P. T. M. , Bos M. P. , Herrmann‐Erlee M. P. M.
Published: February 2005

The role of cAMP and calcium in the induction of ornithine decarboxylase (ODC, E.C.4.1.1.17) activity in the osteogenic sarcoma cell line, UMR 106‐01, was studied, with particular interest for parathyroid hormone (PTH). PTH and forskolin dose‐dependently induced the ODC activity and the cAMP production. Protein synthesis is involved in the effect of PTH and forskolin on ODC activity but not on cAMP production. Using quin2 we showed that 20 nM PTH and 10 μM forskolin increased the intracellular ionized calcium concentration ([Ca2+]i), thereby offering the possibility for calcium to play a role as cellular mediator in the action of PTH and forskolin in bone. Data obtained with A23187 showed that solely an increase of the [Ca2+]i is not sufficient to stimulate basal or potentiate PTH‐ and forskolin‐induced ODC activity. However, the effects of calcium channel blockers and EGTA on basal and PTH‐ and forskolin‐induced ODC activity point to a specific role for calcium. Moreover, the effects of calcium channel blockers and EGTA on basal and PTH‐ and forskolin‐induced cAMP production indicate that the involvement of calcium in the induction of ODC activity is primarily located at another site than the adenylate cyclase. These data indicate that calcium is involved in the control of basal ODC activity. Furthermore, these data suggest that both cAMP and calcium are involved in the induction of ODC activity by PTH and forskolin. More precisely, ODC activity in UMR 106‐01 cells can be induced by PTH and forskolin via a calcium‐dependent cAMP messenger system.
Hemopoietic precursor cell defects in nonanemic but stem cell‐deficient W44/W44 mice
Authors: Barker Jane E. , McFarland Eleanor C.
Published: February 2005

Hematopoietic stem cell deficiencies cause a severe macrocytic anemia in W/W″ mice. W44/W44 mice, on the other hand, are not anemic, but, since they accept marrow implants without prior total body irradiation, they have inherited a stem cell lesion. In an attempt to identify the aberrant stem cell(s), we have determined the concentration in W44/W44 marrow of hematopoietic precursors known to be deficient in W/Wv marrow. The in vitro erythroid burst‐forming units (BFU‐E), the in vivo spleen colony‐forming units (CFU‐S), and the cells that repopulate the erythroid compartment of stem cell‐deficient mice were examined. The progenitors of 7‐day bursts are dramatically reduced in W/W″ marrow but are present in normal concentrations in W44/W44 marrow. W44/W44 marrow CFU‐S, unlike W/W″, generate visible spleen colonies 10 days after injection into lethally irradiated recipients. The colonies are, however, smaller and at least 2 times less numerous than those produced from equivalent numbers of +/+ marrow. An additional defect was the inability of W44/W44 stem cells to compete with genetically marked +/+ cells during erythroid repopulation. An estimate of the number of W44/W44 stem cells needed to compete with +/+ cells was provided by enriching W44/W44 progenitors fivefold. Twice as many enriched W44/W44 marrow cells as unfractionated +/+ cells were required to replace competitor cells. This suggests that there are up to 10 times fewer stem cells somewhere in the W44/W44 erythrogenerative pathway. The data support the conclusion that an erythroid progenitor less mature than the BFU‐E is one of the cells most severely affected by expression of the mutant gene.
In vitro studies on adult cardiac myocytes: Attachment and biosynthesis of collagen type IV and laminin
Authors: Lundgren Evy , Gullberg Donald , Rubin Kristofer , Borg Thomas K. , Terracio Marjorie J. , Terracio Louis
Published: February 2005

The interactions between adult rat cardiac myocytes and the basement membrane components collagen type IV and laminin were investigated in attachment experiments and biosynthesis studies and by immunofluorescence staining. Adult myocytes attached equally well to native collagen type IV and laminin but did not attach to collagen type IV solubilized with pepsin (P‐CIV) or to collagen type I. However, when laminin was used to coat P‐CIV, attachment was enhanced. Affinity‐purified antibodies against laminin inhibited the attachment of myocytes to dishes coated with native collagen type IV, indicating that cell surface‐bound laminin mediated attachment of the cells to this substrate. Immunofluorescence staining of freshly isolated myocytes, using antibodies against laminin or collagen type IV, revealed the presence of laminin but not of collagen type IV on the surface of freshly isolated cells, indicating that during the isolation procedure collagen IV was removed from the cell surface. Metabolic labeling followed by immunoprecipitation demonstrated synthesis of both laminin and collagen type IV in cardiac myocytes as they progressed into culture over a 14‐day period. This synthesis was accompanied by the deposition of the collagen type IV and laminin into distinctly different patterns as revealed by immunofluorescence staining. As the cells progressed into culture, newly synthesized laminin formed a network radiating from the center of the reorganizing cell into the pseudopods. The laminin was redistributed and remodeled with time in culture to form a dense layer beneath the cell. Collagen type IV was also synthesized with time in culture, but the pattern was a much finer network as opposed to the denser pattern of laminin staining. These studies demonstrate that adult cardiac myocytes synthesize and remodel the basement membrane as they adapt to the culture environment.
Thrombin and histamine activate phospholipase C in human endothelial cells via a phorbol ester‐sensitive pathway
Authors: Brock Tommy A. , Capasso Elizabeth A.
Published: February 2005

The effects of phorbol esters and synthetic diglycerides on thrombin‐ and histamine‐stimulated increases in inositol trisphosphate (IP3) and cytosolic free calcium ([Ca2+]i) were studied in cultured human umbilical vein endothelial cells (HEC). Thrombin (0.003–3.0 U/ml) and histamine (10−7–10−4 M) induced rapid increases in [Ca2+]i in suspended cells as monitored with the fluorescent calcium indicator fura‐2. In [3H]myoinositol‐labeled cells, both thrombin (3 U/ml)‐ and histamine (10−4 M)‐induced IP3 increases (195% ± 6% and 98% ± 4%, respectively) occurred in less than 15 sec and were temporally correlated with [Ca2+]i increases. Brief incubations (5–60 min) with different protein kinase C activators [4‐β‐phorbol 12‐myristate 13‐acetate (1–100 nM), mezerein (100 nM), and sn‐1,2 dioctanoylglycerol (0.1–10 μM)] attenuated agonist‐induced increases in [Ca2+]i. These compounds also inhibited thrombin‐ and histaminestimulated IP3 formation, thus suggesting a tight coupling between phospholipase C activation and calcium flux in cultured HEC. Overall, these observations suggest that the pathway linking receptors to phospholipase C stimulation in human endothelial cells is sensitive to protein kinase C activation.
Autocrine control of collagenase synthesis by synovial fibroblasts
Authors: Brinckerhoff Constance E. , Mitchell Teresa I.
Published: February 2005

Fibroblasts respond to exogenous stimuli, such as Interleukin 1, phorbol esters, or crystals of monosodium urate monohydrate, by synthesizing and secreting large quantities of collagenase. Here we show that addition of exogenous stimuli results in the production of an autologous protein that is, itself, capable of inducing collagenase. This autocrine has been partially purified. Activity resides in a protein(s) with a pl of 5 or 8 and with Mr of ∼ 15K. Conversely, conditioned medium taken from unstimulated cultures contains an inhibitor of collagenase synthesis. This protein, which has a Mr ∼ 20–25k by HPLC gel filtration antagonizes collagenase synthesis induced by phorbol esters, exogenous ll 1, and the autologous inducer. We conclude that synovial fibroblasts regulate collagenase synthesis via an autocrine mechanism that includes the synthesis of both an inducer and inhibitor. Both proteins are active at nanomolar amounts and may function as polypeptide hormones in regulating collagenase synthesis and, hence, connective tissue remodeling.
Propionic acid‐induced calcium mobilization in human neutrophils
Authors: Naccache Paul H. , Faucher Nicole , Caon Adriana C. , McColl Shaun R.
Published: February 2005

The ability of propionic acid to elicit an increase in the level of cytoplasmic free calcium in human neutrophils was examined in detail. Propionic acid induced a rapid and dose‐dependent mobilization of calcium that relied on both internal and external sources of calcium. The effects of propionic acid on the mobilization of calcium were inhibited by pertussis toxin, but not cholera toxin, implicating a guanine nucleotide binding protein. Furthermore, preincubation of the neutrophils with phorbol 12‐myristate 13‐acetate resulted in a decreased mobilization of calcium. This inhibitory activity of phorbol myristate acetate was antagonized by the protein kinase C inhibitor H‐7. Preincubation of the cells with the synthetic chemotactic factor fMet‐Leu‐Phe caused a reduction in the magnitude of the calcium transient elicited by propionic acid. However, the calcium response to propionic acid was not affected by antagonists of fMet‐Leu‐Phe and platelet‐activating factor binding or by an inhibitor of leukotriene synthesis. Propionic acid did not elicit a mobilization of calcium in monocytes, platelets, lymphocytes, or undifferentiated HL‐60 cells. However, the treatment of the HL‐60 cells with dimethylsulfoxide resulted in the appearance of a calcium response to propionic acid. The potential physiological significance of these findings are discussed.
Bifunctional activity of transforming growth factor type β on the growth of NRK‐49F cells, normal and transformed by kirsten murine sarcoma virus
Authors: Jullien Pierre , Berg Tone M. , de Lannoy Colette , Lawrence David A.
Published: February 2005

Transformation of rat NRK‐49F cells (49F) by Kirsten murine sarcoma virus (Ki‐MSV) renders these cells (Ki‐49F cells) capable of autonomous anchorage independent (Al) growth. As compared to nontransformed 49F cells, the transformation by Ki‐MSV does not modify the cell response to transforming growth factor‐β (TGF‐β) in monolayer conditions, but alters it in A I growth conditions. The growth of nontransformed or Ki‐MSV‐transformed adherent 49F cells is slowed down by porcine TGF‐β, and this effect is reversed by epidermal growth factor (EGF). This decrease in the cell growth rate, induced by TGF‐β, does not affect the cloning efficiency of untransformed and transformed adherent 49F cells. Contrarily, porcine TGF‐β decreases the A I cloning efficiency of Ki‐49F cells in agar‐gelled medium; this effect is only partly reversed by EGF, which does not synergise with TGF‐β to enhance the A I growth as in the case of untransformed 49F cells. Media conditioned by 49F cells, Ki‐49F cells, and chicken embryo fibroblasts contain a latent TGF‐β whose capacity to promote the A I growth of 49F cells and to inhibit that of Ki‐49F cells is unmasked by acidification. The same situation exists concerning TGF‐β from human platelets. Neutral extracts are inefficient in both tests of promotion and inhibition of A I growth and contain an acid‐activable component with an apparent molecular weight of 600 kd. In acid extracts, a 5–9 kd apparent molecular weight component is responsible for the A I growth enhancement of 49F cells and the A I growth inhibition of Ki‐49F cells. Further purification by reverse phase chromatography shows that both activities strictly coelute at the same point (32%) of an acetonitrile gradient. These results indicate that TGF‐β is present in physiological conditions as a latent form which requires activation for inhibiting the A I growth of transformed cells as well as for enhancing that of 49F cells.
Modulation of the mitogenic response of an epidermal growth factor‐dependent keratinocyte cell line by dexamethasone, insulin, and transforming growth factor‐β
Authors: Zendegui Joseph G. , Inman Wendelyn H. , Carpenter Graham
Published: February 2005

The stimulation of DNA synthesis by epidermal growth factor (EGF) has been studied for a cell line having properties useful for investigating the mechanism of action of EGF in epithelial cell populations. These studies employ a mouse keratinocyte cell line (MK), isolated by Weissman and Aaronson (1983), which is stringently dependent on exogenous EGF for growth in serum containing medium. The studies reported here characterize the compliment of EGF receptors present on the surface of MK cells and demonstrate the regulatory influence of other hormones on the capacity of EGF to stimulate DNA synthesis. Up‐regulated MK cells contain approximately 22,000 EGF receptors per cell, but when the cells are grown in the presence of EGF the receptor number is reduced to about 4,000. It is estimated that only a small number of high‐affinity receptors (less than 500) are required for EGF‐dependent cell proliferation. In contrast to its action in fibroblastic cells, dexamethasone is a strong inhibitor of EGF‐stimulated DNA synthesis of MK cells. Insulin at high concentrations, or insulin‐like growth factors I or II (IGF‐I, IGF‐II) at physiological concentrations, synergistically enhance the EGF response. Interestingly, insulin or IGF‐I or II are also able to reverse most of the dexamethasone inhibition of DNA synthesis. Transforming growth factor‐β (TGF‐β) inhibits, in reversible manner, the EGF stimulation of DNA synthesis and this inhibition is not overcome by insulin. TGF‐β receptors have been measured in MK cells and Scatchard analysis indicates approximately 20,000 receptors per cell. None of the modulatory hormones (insulin, dexamethasone, TGF‐β) significantly altered 125I‐EGF binding characteristics in MK cells, suggesting a point of action distal to 125I‐EGF binding.
Differential expression of mRNA coding for heparin‐binding growth factor type 2 in human cells
Authors: Sternfeld Mark D. , Hendrickson Jill E. , Keeble Winifred W. , Rosenbaum James T. , Robertson Joseph E. , Pittelkow Mark R. , Shipley Gary D.
Published: February 2005

The proliferation of normal human fibroblasts, keratinocytes, and melanocytes in vitro can be controlled by purified polypeptide growth factors and serum. We have studied the cellular expression of the heparin‐binding growth factor type 2/basic fibroblast growth factor (HBGF‐2/bFGF) gene to determine whether these cell types synthesize mRNA for this mitogen. Our results indicate that normal human fibroblasts synthesize four distinct mRNAs of 7.0, 3.7, 2.2, and 1.5 kilobases, which hybridize to a specific HBGF‐2/bFGF cDNA probe. In fibroblasts, the level of all four of these transcripts increases dramatically (more than tenfold) within 4 hours of treatment of quiescent cells with fresh fetal bovine serum. Of the purified growth factors tested, transforming growth factor type‐beta also increased HBGF‐2/bFGF mRNA abundance, but not to the levels attained by serum treatment. Treatment of fibroblasts with cycloheximide before and during serum treatment blocked the ability of serum to induce the expression of the HBGF‐2/bFGF gene. The gene is expressed at low levels in human fibroblasts rapidly growing in serum‐free medium and at higher levels in cells rapidly growing in serum‐containing medium. In contrast to fibroblasts, mRNA coding for HBGF‐2/bFGF is undetectable in proliferating normal human keratinocytes, melanocytes, or mammary epithelial cells. Because keratinocytes and melanocytes proliferate in response to purified HBGF‐2/bFGF, our results suggest that HBGF‐2/bFGF may mediate the proliferation of epidermal cells through paracrine mechanisms involving stromal fibroblasts. Moreover, we have shown that a human squamous cell carcinoma cell line (SCC‐25) expresses mRNA coding for HBGF‐2/bFGF, suggesting that the gene may become activated in some carcinomas.
Induction of achromosomal cleavage by hydroxyurea in starfish embryo and the reversal by a combination of deoxyadenosine and deoxycytidine: Possible involvement of salvage pathway for deoxynucleoside triphosphate biosynthesis in the presence of hydroxyurea
Authors: Yamada Hisafumi , Kuraishi Ritsu , Hirai Setsuro , Katoh Yuko , Fusetani Nobuhiro , Amikura Reiko , Okano Keiju , Nagano Hiroshi
Published: February 2005

Effects of hydroxyurea, an inhibitor of ribonucleotide reductase, on cleavage of starfish embryos were studied. In the presence of 1 mM hydroxyurea, fertilized eggs of the starfish, Asterina pectinifera, cleaved up to the 256‐cell stage and decomposed before blastulation. Before the 16‐cell stage, each blastomere contained a normal nucleus or chromosomes with mitotic apparatus. The cleveage after the 16‐cell stage was slow compared to the control embryos, and not all blastomeres contained a nucleus or normal chromosomes. During the fifth cell division (between 16‐cell‐ and 32‐cell‐stage embryos), chromatin mass unassociated with the mitotic apparatus remained near the cleavage furrow. When hydroxyurea was removed before the 16‐cell stage, the embryos developed to normal bipinnalia larvae via normal blastulae. However, the embryos were disintegrated before blastulation when hydroxyurea was removed after the 32‐cell stage. DNA synthesis was normally observed before the 16‐cell stage but not after the 16‐cell stage, but dNTP contents in the embryos remained low throughout development in the presence of hydroxyurea. The achromosomal cleavage observed in the presence of hydroxyurea was reversed by the combination of extracellular dAR and dCR. Therefore, it is assumed that the synthesis of dNTPs required for DNA synthesis in the presence of hydroxyurea occurs via the salvage pathway using deoxynucleosides (dNR) (dNR to dNTP via dNMP and dNDP).
Leukotriene D4‐induced increases in the cytoplasmic pH of human myelocytic leukocytes
Authors: Baud Laurent , Healy Ann , Cragoe Edward J. , Goetzl Edward J. , Koo Catherine H.
Published: February 2005

The effects of leukotriene D4 on the intracellular pH of human myelocytes, derived from cultured HL‐60 cells by dimethylsulfoxide‐induced differentiation, were quantified with the fluorescent indicator 2′,7′‐bis‐(2‐carboxy‐ethyl)‐5,6‐carboxyfluorescein. Leukotriene D4, but not C4 or E4, increased intracellular pH optimally by 3 min with a half‐maximal effect at 1–2 nM. The increases in intracellular pH stimulated by leukotriene D4 were prevented by pretreatment of myelocytes with leukotriene D4 but not peptide chemotactic factors. Analogs of amiloride that inhibit selectively the Na+/H+ antiport also prevented the intracellular alkalinization induced by leukotriene D4. The rate of recovery of intracellular pH after an acid load with 30 mM sodium propionate was approximately 30% higher at each level of intracellular pH for myelocytes exposed to leukotriene D4 than for those challenged in buffer alone. The increase elicited by leukotriene D4 in the adherence of myelocytic leukocytes to surfaces thus is associated with an enhanced sensitivity of the Na+/H+ antiport to intracellular pH, that is, not coupled to an earlier rise in the cytosolic level of Ca+2.
Reassessment of fluid‐phase endocytosis and diacytosis in monolayer cultures of human fibroblasts
Authors: McKinley Dana Noel , Steven Wiley H.
Published: February 2005

We have investigated the kinetics of fluid‐phase endocytosis and diacytosis in confluent monolayers of human fibroblasts by comparing the behavior of three markers that have been previously used to study this process: [14C]sucrose, 125l‐labeled polyvinylpyrrolidone ([125l]PVP), and Lucifer Yellow. Three distinct kinetic compartments were observed with all markers. The first was relatively large (10‐60 fl/cell), reached steady state within 15 min at 37°C, and was rapidly lost from monolayers after removing the markers at 37°C but not at 0°C. These properties indicate that this compartment is the same as that previously proposed to be the major intracellular compartment involved in diacytosis. However, this compartment is probably extracellular fluid trapped between cells since it is rapidly lost into the medium when the cells are either scraped or enzymatically removed from the culture dishes at 0°C. In addition, it very slowly undergoes both filling and emptying at 0°C. However, we did observe a second, much smaller, kinetic compartment (approximately 2 fl/cell) undergoing rapid diacytosis that does seem to be intracellular. A third compartment that we observed accumulates markers at a linear rate (10‐20 fl cell−1 hr−1) and is not lost from cells even after incubation periods greater than 6 hr. The markers [14C]sucrose and [125l]PVP displayed very similar behavior with respect to all three compartments and yielded nearly linear long‐term uptake rates, thus indicating that there is little if any absorbed component in their uptake. However, Lucifer Yellow displayed significantly higher incorporation rates and its uptake rate was strongly nonlinear, indicating its uptake in fibroblasts is predominantly adsorptive. Our observations indicate that the rate of fluid‐phase endocytosis in fibroblasts is significantly less than previously reported and that any compartment involved in diacytosis is very small and turns over very rapidly. Significantly, we estimate that the constitutive internalization of clathrin‐coated pits is sufficient to account for the majority of fluid‐phase endocytosis and thus represents a major mechanism of membrane retrieval in these cells.
Thermal analysis of CHL V79 cells using differential scanning calorimetry: Implications for hyperthermic cell killing and the heat shock response
Authors: Lepock James R. , Frey Harold E. , Rodahl A. Michael , Kruuv Jack
Published: February 2005

Differential scanning calorimetry (DSC) was used to assay thermal transitions that might be responsible for cell death and other responses to hyperthermia or heat shock, such as induction of heat shock proteins (HSP), in whole Chinese hamster lung V79 cells. Seven distinct peaks, six of which are irreversible, with transition temperatures from 49.5°C to 98.9°C are detectable. These primarily represent protein denaturation with minor contributions from DNA and RNA melting. The onset temperature of denaturation, 38.7°C, is shifted to higher temperatures by prior heat shock at 43° and 45°C, indicative of irreversible denaturation occurring at these temperatures. Thus, using DSC it is possible to demonstrate significant denaturation in a mammalian cell line at temperatures and times of exposure sufficient to induce hyperthermic damage and HSP synthesis. A model was developed based on the assumption that the rate limiting step of hyperthermic cell killing is the denaturation of a critical target. A transition temperature of 46.3°C is predicted for the critical target in V79 cells. No distinct transition is detectable by DSC at this temperature, implying that the critical target comprises a small fraction of total denaturable material. The short chain alcohols methanol, ethanol, isopropanol, and t‐butanol are known hyerpthermic sensitizers and ethanol is an inducer of HSP synthesis. These compounds non‐specifically lower the denaturation temperature of cellular protein. Glycerol, a hyperthermic protector, non‐specifically raises the denaturation temperature for proteins denaturing below 60°C. Thus, there is a correlation between the effect of these compounds on protein denaturation in vivo and their effect on cellular sensitivity to hyperthermia.
Maintenance by erythropoietin of viability and maturation of murine erythroid precursor cells
Authors: Koury Mark J. , Bondurant Maurice C.
Published: February 2005

Erythroblasts isolated from the spleens of mice infected with the anemia‐inducing strain of Friend virus (FVA cells) are erythropoietin (EP)‐sensitive cells at the late colony forming unit‐erythroid (CFU‐E) and cluster forming unit stages of differentiation (Koury et al., J. Cell. Physiol. 121:526–532, 1984). We investigate here the EP requirements of FVA cells in vitro for viability, proliferation, and maturation. By delaying the addition of EP to FVA cell cultures or by withdrawing EP at early times of culture, the subsequent viability, cell numbers, and maturation were diminished. The longer the delay in EP addition or the earlier the EP withdrawal, the more diminished these parameters were when compared to cultures which contained EP throughout the 48 h of differentiation. FVA cells had a period of EP requirement in vitro that lasted for only 24 h or less after the initiation of culture. During these crucial first 24 h, EP induced an increase in the synthesis of all size classes of RNA. Protein synthesis was maintained at a stable level in cells cultured with EP, but it declined in cells cultured without it. In contrast, the synthesis rate of DNA and the content of DNA per cell were not affected by the presence of EP in the culture. However, FVA cells cultured without EP had progressive accumulation of small sized DNA due to breakage of higher molecular weight DNA. The rate of DNA breakdown was sufficient to prevent DNA accumulation and thus it probably plays a role in the abortion of cell proliferation. No such breakage was found in cells cultured with EP. Our results indicate that EP exerts an effect on FVA cells in culture which is reflected in their viability, cell number, and maturation. This effect is not mediated by a stimulation of the rate of DNA synthesis, but is accompanied by stimulation of overall RNA synthesis and maintenance of protein synthesis.
Coupling between inositol phosphate formation and DNA synthesis in smooth muscle cells stimulated with neurokinin A
Authors: Hultgårdh‐Nilsson Anna , Nilsson Jan , Jonzon Bror , Dalsgaard Cari‐Johan
Published: February 2005

The two mammalian neuropeptides substance P (SP) and neurokinin A (NKA) have been demonstrated to stimulate DNA synthesis in connective tissue cells, suggesting that peripheral neurons may play a role in development and tissue regeneration. In this study we have tried to identify intracellular messengers required for SP‐ and NKA‐induced DNA synthesis. SP and NKA, as well as platelet‐derived growth factor (PDGF) stimulated formation of inositol phosphates in smooth muscle cells (SMC), whereas no effect on inositol phosphates formation occurred in response to nonmitogenic neuropeptides. Pretreatment of the cells with pertussis toxin markedly decreased DNA synthesis induced by NKA. This toxin inhibits formation of inositol phosphates by acting on a regulatory G‐protein. Calcium and calmodulin antagonists also inhibited NKA‐induced DNA synthesis. These results imply that the mitogenic signal(s) produced by activated neuropeptide receptors involves formation of inositol phosphate and activation of a calcium/calmodulin dependent process. We further report that other neuropeptides occurring in peripheral neurons, i.e., vasoactive intestinal polypeptide, calcitonin gene‐related peptide, neuropeptide Y, somatostatin, or cholecystokinin, are without growth‐stimulatory effect on cultured SMC.
Basic fibroblast growth factor and insulin‐like growth factor I are strong mitogens for cultured mouse keratinocytes
Authors: Ristow Hans‐Jürgen , Messmer Trudy O.
Published: February 2005

Basic fibroblast growth factor (bFGF), but not acidic fibroblast growth factor (aFGF), was found to be mitogenic for cultured mouse keratinocytes. A six‐to‐nine fold increase in 3H‐thymidine (3H‐dT) incorporation into the acid insoluble pool and a similar increase of the labeling index can be measured when bFGF, at a concentration between 1 and 10 ng/ml, is added to keratinocytes arrested in serum‐free and growth factor‐free medium with a Ca++‐concentration below 0.1 mM. The half‐maximal response is observed between 0.2 and 0.7 ng/ml. In the same culture system, insulin‐like growth factor I/somatomedin C (IGF‐I) and insulin with 100–500 ng/ml. Basic FGF, IGF‐I and insulin can be classified as strong stimulators of DNA synthesis in mouse keratinocytes. In this regard they are comparable to epidermal growth factor, which shows a half‐maximal stimulation at a concentration between 1.5–2 ng/ml. These results show that in addition to mesenchymal cells, FGF is a growth factor not only for neuroectodermal cells, but ectodermal cells in general. They further support the idea that the growth promoting effect of insulin on keratinocytes may be mediated by the IGF‐I receptor.
Induction of competence and progression signals in human T lymphocytes by phorbol esters and calcium ionophores
Authors: Kumagai Naoki , Benedict Stephen H. , Mills Gordon B. , Gelfand Erwin W.
Published: February 2005

We have investigated the induction of competence (IL‐2 responsiveness) and progression in human T lymphocyte proliferation triggered by phorbol ester and calcium ionophore. The degree of proliferation induced with the phorbol ester, phorbol 12,13‐dibutyrate (PDB) and the calcium ionophore ionomycin was dependent on the duration of exposure to these agents, with more than 6 h required for obtaining maximum proliferation. Following brief exposure to both agents for 30 min, which did not cause significant proliferation, T cells became competent to proliferate in response to exogenous interleukin 2 (IL‐2). These competent T cells also progressed to DNA synthesis following incubation with PDB in the absence of ionomycin. Induction of competence to proliferate in response to either PDB or IL‐2 was blocked by EGTA, suggesting that transmembrane Ca2+ flux was obligatory at this stage. Since other phorbol esters and synthetic diacylglycerols also stimulated DNA synthesis in competent cells, it is likely that progression was triggered by activation of protein kinase C. Following a brief exposure to PDB and ionomycin, subsequent incubation with PDB induced gene expression and secretion of IL‐2 and augmented the expression of IL‐2 receptors in the competent cells. Thus, we have demonstrated that Ca2+ mobilization is required for rendering T cells competent to express functional IL‐2 receptors, to produce IL‐2 in response to subsequent incubation with PDB, and that sustained activation of protein kinase C seems necessary for IL‐2 production and subsequent progression of competent T cells to DNA synthesis.
Tumor necrosis factor inhibits the terminal events in mesenchymal stem cell differentiation
Authors: Filipak Michiko , Sparks Rodney L. , Tzen Chin‐Yuan , Scott Robert E.
Published: February 2005

Control of the terminal event in cellular differentiation is an important normal regulatory process, and the expression of defects in the control of this process has been implicated in the pathogenesis of cancer. To determine if tumor necrosis factor (TNF), which is an important biological response modifier, can inhibit terminal differentiation, we have studied 3T3 T mesenchymal stem cells. This experimental cell system was employed because a well‐defined series of steps in differentiation has been defined and cells at each stage of differentiation can be isolated. For example, nonterminal differentiated cells can be isolated, and their transition to a terminal differentiation state can be evaluated. The most interesting results in the current studies show that TNF blocks the terminal event in mesenchymal stem cell differentiation. Inhibition of the terminal event of differentiation by TNF is reversible and is not associated with inhibition of selective or general protein synthesis. Evidence is also presented that cell clones that are defective in their ability to undergo the terminal event in differentiation secrete factor(s) that inhibit the terminal event in differentiation. These observations suggest that the inhibition of the terminal event in differentiation may be mediated via autocrine or paracrine regulatory molecules such as tumor necrosis factor.
DNA topoisomerase inhibitors block erythropoiesis and delay hemoglobinization in vitro
Authors: Dainiak Nicholas , Kreczko Sandra , Hanspal Manjit , Strauss Phyllis R.
Published: February 2005

To examine the importance of topological constraints on DNA during erythroid development, we measured the effects of camptothecin and teniposide, two tumoricidal agents which are also specific inhibitors of type I and type II topoisomerases respectively, on the formation of hematopoietic colonies by cultured human bone marrow cells. When added to bone marrow culture, each inhibitor alone impairs the formation of early BFU‐E‐derived colonies, late CFU‐E‐derived colonies and mixed hematopoietic (CFU‐GEMM‐derived) colonies by up to 100%. Inhibition of colony formation is directly related to the time of inhibitor addition and the inhibitor concentration tested. Although either inhibitor alone reduces colony formation by 90%, when added together at a submaximal concentration, camptothecin and teniposide exert a synergistic suppressive effect. Furthermore, addition of topoisomerase inhibitors to culture impairs hemoglobinization of colony erythroblasts in a time‐dependent fashion. In contrast to the effects of topoisomerase inhibitors, the antiproliferative agent aphidicolin reduces erythroid colony number and size without altering hemoglobinization of colony erythroblasts. Since neither topoisomerase inhibitor alters the morphology of cultured cells, the capacity of cells to exclude trypan blue or the potential to form erythroid colonies through the interval required for the first progenitor cell division, it is unlikely that camptothecin or teniposide are cytotoxic to hematopoietic cells. Human mononuclear cells enriched in bone marrow lymphocytes and nucleated erythroblasts from both human and mouse sources release DNA into the detergent soluble fraction. Release requires functional topoisomerases and is altered by acute exposure to topoisomerase inhibitors. Our results suggest that topoisomerases are critical not only to proliferation but also to differentiation of human marrow erythroid progenitor cells and stem cells in culture.
Enhancement of plasminogen activator activity in cultured endothelial cells by granulocyte colony‐stimulating factor
Authors: Kojima Soichi , Tadenuma Hirohiko , Inada Yuji , Saito Yuji
Published: February 2005

A hitherto unknown function of granulocyte colony‐stimulating factor (G‐CSF) was found using cultured endothelial cells. G‐CSF stimulated activity of plasminogen activator (PA) in both extracellular and intracellular milieus of endothelial cells obtained from bovine carotid and aortic artery. This effect was dependent on the concentration of G‐CSF added to the culture medium and on the treatment time. The extracellular activity was enhanced approximately 5‐fold at a concentration of 5,000 colony‐forming unit (CFU)/ml (2.6 nM) and in about a 15‐hr treatment period. Analyses by fibrin and reverse fibrin autography revealed that activity of PA was much more increased than that of PA inhibitor in endothelial cells treated with G‐CSF.
Heparin potentiates the action of acidic fibroblast growth factor by prolonging its biological half‐life
Authors: Damon Deborah H. , Lobb Roy R. , D'Amore Patricia A. , Wagner John A.
Published: February 2005

The mechanism(s) by which heparin influences the biological activities of acidc and basic fibroblast growth factors (aFGF and bFGF) is not completely understood. One mechanism by which heparin could alter the biological activities of aFGF and bFGF is by altering their biological half‐lives. We investigated the possibility that heparin potentiates aFGF‐induced neurite outgrowth from PC12 cells by prolonging its biological half‐life. Under conditions where heparin potentiated aFGF‐induced neurite outgrowth, we observed that heparin increased the biological half‐life of aFGF from 7 to 39 hr. We determined that >25 hr of exposure to active aFGF was required for induction of neurite outgrowth. If aFGF activity was maintained for greater than 25 hr by periodic readdition of factor, heparin no longer potentiated aFGF‐induced neurite outgrowth. These observations strongly suggest that heparin potentiates the activity of aFGF by prolonging its biological half‐life. The protease inhibitors hirudin, leupeptin, and pepstatin A did not potentiate aFGF‐induced neurite outgrowth, indicating that proteases inhibited by these inhibitors are not responsible for the loss of aFGF activity that we observed. However, aprotinin potentiated aFGF neurite‐promoting activity approximately sevenfold, indicating that proteases that are inhibited by aprotinin are at least partially responsible for aFGF inactivation. These observations suggest that heparin regulates the activity of aFGF by regulating its proteolytic degradation, thereby regulating its biological half‐life.
Kinetic characteristics and regulation of hexose transport in a galactokinase‐negative Chinese hamster fibroblast cell line: A good model for studies on sugar transport in cultured mammalian cells
Authors: Germinario Ralph J. , Lakshmi Tannirkulam M. , Thirion Jean‐Paul
Published: February 2005

We report the kinetic characteristics for D‐galactose, 2‐deoxy‐D‐glucose and 3‐0‐methyl‐D‐glucose transport in a galactokinase null‐allele mutant of a Chinese hamster V79 cell line. GalKl cells exhibited a Km and Vmax for D‐galactose, 2‐deoxy‐D‐glucose, and 3‐0‐methyl‐D‐glucose transport of 8.6 ± 2.6 mM and 26.1 ± 7.2nmol/mg p/min, 4.1 ± 1.2 mM and 40.3 ± 9.5 nmol/mg p/min, and 7.01 ±.85 mM and 11.6 ± 4.8 nmol/mg p/30 s, respectively. Nonsaturable hexose uptake was determined using cytochalasin B inhibition of galactose uptake (89.6 ± 3.7% of the galactose uptake was cytochalasin B inhibitable) and L‐glucose uptake (7.5% of the galactose uptake). D‐Galactose was not metabolized and effluxed rapidly from preloaded cells. The K1s for the inhibition of D‐galactose transport were 4.5 ± 2.5 mM for D‐glucose, 7.0 ± 2.0 mM for 2‐deoxy‐D‐glucose, 6 mM for 2‐deoxy‐D‐galactose and 6.0 ± 0.6 mM for 3‐0‐methyl‐D‐glucose. This indicates the operation of a single common carrier. The hexose transport rate decreased 50‐60% after 24 h serum deprivation. Addition of insulin was shown to increase hexose transport (more than twofold) in serum‐deprived cells. Hexose transport rates increased substantially in glucose‐deprived, D‐fructose‐ or D‐galactose‐fed cells as compared to glucose‐fed cells. Since GalKl does not metabolize galactose, the hexose transport increases induced by feeding cells galactose suggest that carrier interaction with ligand is not a significant factor in transport regulation in GalKI. The kinetic and regulatory characteristics of D‐galactose transport in the GalKI cell line indicate that this system is a good model to study sugar transport from a mechanistic and regulatory point of view.
Effect of retinoic acid on the phosphorylation of endogenous proteins in HL‐60 cells
Authors: Spearman Terry N. , Fontana Joseph A. , Butcher Fred R. , Durham John P.
Published: February 2005

HL‐60 cells were incubated with [32P]‐Pi in order to label endogenous phosphoproteins in situ. These were then resolved via two‐dimensional electrophoresis and autoradiograms were made from the gels. A comparison of autoradiograms made from retinoic‐acid‐differentiated cells with those made from control cells revealed a small number of phosphoproteins whose labeling was enhanced in differentiated cells. Incubating HL‐60 cells with [35S]‐methionine revealed that RA induced the synthesis of one of these proteins, (53 kDa, pl 4.8) although not to a degree sufficient to account for the increased phosphate labeling observed in differentiated cells. Difluoromethylornithine (DFMO), which arrests HL‐60 cell proliferation without inducing differentiation, had no effect on endogenous protein phosphorylation. Treatment of DFMO‐arrested cells with retinoic acid, however, increased the phosphorylation of the proteins and resulted in differentiation of the cells. Densitometric analysis of autoradiograms made from two‐dimensional gels revealed that the phosphorylation of the 53‐kDa, pl 4.8 protein was significantly elevated in cells exposed to RA for as little as 12 hours, i.e., before the cells were committed to differentiate and before a significant increase in the number of phenotypically mature cells was observed. It therefore appears that this protein may be an intermediate in the retinoic‐acid‐induced differentiation process.