bluuebunny/crossref_metadata_2025_split huggingface dataset:
Insulin‐like growth factor II binding and action in human fetal fibroblasts
Authors: Conover Cheryl A. , Rosenfeld Ron G. , Hintz Raymond L. 
Published: February 2005

AbstractTo investigate the role of insulin‐like growth factor II (IGF‐II) in human prenatal growth, IGF‐II binding and biological action were studied in four lines of fetal and three lines of postnatal human fibroblasts. Specific biniding of IGF‐II was similar in both groups: 15.7% and 14.9% for fetal and postnatal fibroblasts, respectively. This was 5–10 times the amount of IGF‐I binding found in these cells. IGF‐I and IGF‐II caused dose‐dependent increases in [14C]aminoisobutyric acid (AIB) uptake. IGF‐II was sevenfold less potent than IGF‐I in stimulating this metabolic response in both fetal and postnatal fibroblasts. The maximal effect of IGF‐II in stimulating [14C]AIB uptake approached that of IGF‐I. Similar results were obtained when IGF‐I and IGF‐II stimulation of [3H]thymidine incorporation was compared in fetal and postnatal fibroblasts. Incubation in the presence of alphalR‐3, a monoclonal antibody to the type I IGF receptor, inhibited the ability of both IGF‐I and IGF‐II to stimulate [14C]AIB uptake and [3H]thymidine incorporation in fetal and postnatal cells. A monoclonal antibody to the insulin receptor did not affect IGF action. These data indicate that IGF‐II is a potent metabolic and mitogenic stimulus for human fetal firoblasts. However, despite the presence of abundant type II IGF receptors on both fetal and postnatal human fibroblasts, IGF‐II stimulation of amino acid transport and DNA synthesis appears to be mediated through the type I rather than through its own type II IGF receptor.
Effect of osmolar and ionic composition of the extracellular fluid on transferrin endocytosis and exocytosis and iron uptake by reticulocytes
Authors: Bowen Barbara J. , Morgan Evan H. 
Published: February 2005

AbstractThe effects of osmolar and ionic factors on endocytosis and exocytosis were investigated using rabbit reticulocytes and 125I‐59Fe labelled transferrin. Endocytosis and exocytosis of transferrin and the uptake of iron were inhibited by increasing the osmolality or decreasing the ionic strength or pH of the cell incubation medium. However, elevation of the pH above 8.0 inhibited endocytosis but not exocytosis. Replacement of the NaCl in the incubation medium by Nal, NaF, NaSCN, NaCIO4, Na2SO4, Na phosphate, or Na Hepes inhibited endocytosis and iron uptake but only Nal, NaF, and NaSCN inhibited exocytosis. Transferrin exocytosis was insensitive to inhibitors of anion or cation transport, but endocytosis and iron uptake were inhibited by several anion transport inhibitors. Overall, transferrin endocytosis was more sensitive than exocytosis to most of the factors which were investigated, and the effects on the rates of endocytosis and iron uptake were quantitatively very similar. The results provide strong support for the concept that transferrin endocytosis is a necessary step in iron uptake by reticulocytes. They do not support the chemiosmotic models of exocytosis in their present formulations, out do not rule out the possible role of an osmotic event in exocytosis.
Activation and proliferation signals in murine macrophages: Stimulation of Na+, K+‐ATPase activity by hemopoietic growth factors and other agents
Authors: Vairo Gino , Hamilton John A. 
Published: February 2005

AbstractPurified colony stimulating factor (CSF‐1) stimulates the Na+, K+‐ATPase activity of murine bone marrow‐derived macrophages (BMM) and resident peritoneal macrophages (RPM) measured as ouabain‐sensitive 86Rb+ uptake. Similar concentrations of CSF‐1 stimulate the Na+, K+‐ATPase activity and DNA synthesis in BMM whilst ouabain, a specific inhibitor of the Na+, K+‐ATPase, also inhibits this CSF‐1‐mediated DNA synthesis. Other purified hemopoietic growth factors, granulocyte‐macrophage CSF (GM‐CSF) and interleukin‐3 (IL‐3), and the tumor promoter, 12‐0‐tetradecanoyl‐phorbol‐13‐acetate (TPA), even though differing in their mitogenic capabilities, are also stimulators of the Na+,K+‐ATPase activity in BMM and RPM. The non‐mitogenic agents, lipopolysaccharide (LPS) and Concanavalin A (Con A), are also active. CSF‐1 stimulation of the Na+,K+‐ATPase was shown to be dependent on elevation of intracellular Na+ via an amiloride sensitive Na+‐channel, most likely representing Na+/H+ exchange activity. Such stimulation of Na+,K+‐ATPase activity via activation of the Na+/H+ exchange appears to be a necessary but insufficient early macrophage response for subsequent DNA synthesis.
Time dependence of transmembrane potential changes and intracellular calcium flux in stimulated human monocytes
Authors: Bernardo John , Brink Holly F. , Simons Elizabeth R. 
Published: February 2005

AbstractAn important characteristic of the functional differentiation of the blood monocyte is the development of its capacity to recognize and respond to stimuli. This ability is mediated to a large extent by specific receptor glyco‐proteins located on the cell surface. Stimulation of mononuclear phagocytes via these receptors results in a rapid rise in intracellular Ca+ + concentration, accompanied or followed by a change in membrane potential, generation of oxidative products, degranulation, and effector functions such as phagocytosis, aggregation, or locomotion. While the development of these characteristics is difficult to characterize in vivo, several investigators have demonstrated in vitro changes in these cells that correlate with the development of effector function. To examine the mechanisms of specific membrane‐stimulus interactions of monocytes as they differentiate into macrophage‐like cells, we studied the responses of human monocytes and of monocytes incubated in serum‐containing medium for up to 96 hr to the chemotactic peptide formyl‐methionyl‐leucyl‐phenylalanine (fMLP). Freshly isolated monocytes exhibited little change in transmembrane potential following stimulation with an optimal concentration of peptide and underwent a significant increase only after 48 hr in culture. While constant resting intracellular Ca+ + concentrations were maintained during the culture period, intracellular Ca+ + levels following fMLP stimulation increased with with incubation in serum, for up to 96 hr. In contrast, fMLP‐induced respiratory burst activity increased from 0 to 24 hr in culture; it remained elevated at 48 hr but declined again by 96 hr. Incubation of the cells for 24 hr increased their random (unstimulated) motility in modified Boyden chambers but did not alter the cells' directed (chemotactic) response to fMLP in comparison to the response of freshly isolated monocytes. Peptide binding to the cells did not increase during the incubation period, indicating that an increase in receptor number or in affinity for fMLP was not responsible for the enhanced responsiveness to fMLP as incubation time increased. These studies indicate that incubation of monocytes in serum‐containing medium leads to a complex, altered series of responses to fMLP that correlate with the differentiation of the original monocytes in vitro and may relate to the in vivo differentiation of monocytes to macrophages.
Roles of protein kinases in neurotransmitter responses in Xenopus oocytes injected with rat brain mRNA
Authors: Ito I. , Hirono C. , Yamagishi S. , Nomura Y. , Kaneko S. , Sugiyama H. 
Published: February 2005

AbstractMicroinjection of rat brain mRNA in Xenopus oocytes induced acetylcholine, neuroteisin, serotonin, and glutamate receptors in the cells. These receptors stimulate an intracellular reaction pathway, including G‐protein activation, inositol trisphosphate (IP3) formation, and Ca2+‐dependent CI− channels. In the present study, we examined the roles of several protein kinases in these responses by means of inhibitors and activators of these kinases. Isoquinolinesulfonamides, inhibitors of protein kinases, caused no current responses and affected no receptor‐mediated responses when injected into the oocytes at low doses (30–50 pmol), which inhibit cyclic nucleotide‐dependent kinases or kinase C specifically, but abolished the receptor‐mediated responses at a higher dose (300 pmol), which inhibit most protein kinases nonspecifically. Calmodulin inhibitors blocked the receptor‐mediated responses strongly. Activation of cyclic nucleotide‐dependent kinases or kinase C by injection of cAMP (or cGMP) or perfusion with phorbol esters caused no direct current responses but suppressed receptor‐mediated responses. Current responses triggered by IP3 injection were not suppressed by these treatments. These results suggest that cAMP‐ (or cGMP‐)dependent kinases or kinase C may not be involved in the pathway directly but may modulate it by inhibiting the initial part of the pathway (receptors, G‐proteins, and/or phospholipase C), and they suggest that calmodulin may most likely be involved in the activation of Ca2+‐dependent CI− channels.
Myogenic stem cell commitment probability remains constant as a function of organismal and mitotic age
Authors: Quinn Lebris Smith , Norwood Thomas H. , Nameroff Mark 
Published: February 2005

AbstractChicken myogenic stem cells can undergo symmetric and asymmetric cell divisions. Symmetric divisions produce two stem cells or two cells committed to terminal muscle differentiation. Asymmetric divisions produce one stem cell and one committed cell. Committed cells undergo four divisions, and their progeny differentiate into postmitotic, biochemically distinct muscle cells, which can be identified immunocytochemically. The control of stem cell commitment was investigated in vitro by means of cell cloning and subcloning experiments, and computer modeling. We found that stem cell commitment is a process which can be modeled as a stochastic event, with a central tendency or probability of 0.2 ± 0.1. This value is independent of organismal or mitotic age of the stem cells, cell density, or growth in a mitogen‐poor environment. Myogenic stem cells stop dividing after approximately 30 divisions in vitro. Since the probability of commitment to terminal differentiation remains below 0.5, clonal senescence and terminal differentiation are separate processes in this system.
Serial propagation of human ovarian surface epithelium in tissue culture
Authors: Siemens Craig H. , Auersperg Nelly 
Published: February 2005

AbstractMost human ovarian cancers are thought to arise in the ovarian surface epithelium (OSE). The precise role of OSE in carcinogenesis has not been defined because no appropriate animal models for the study of this tissue exist and culture of human OSE has been limited to primary outgrowths. In this report, we describe conditions for serial cultivation of normal human OSE. Premenopausal ovarian tissue was obtained at surgery. OSE growth was compared in media MCDB 202, 199 and Waymouth's 752/1 (WM) supplemented with 5, 15, or 25% fetal bovine serum (FBS), with/without 20 ng/ml epidermal growth factor (EGF) and 0.4 γg/ml hydrocortisone (HC). The rate and extent of OSE outgrowths from explants in primary culture were greatest in either WM or 199/202 (1:1), supplemented with 15% FBS/EGF/HC. In early passage cultures, cell proliferation was most rapid and extensive in 199/202 with 15% FBS, EGF, and HC. In this medium, OSE cells were subcultured up to 10 times and underwent 20–25 population doublings over 5 weeks. The population doubling time during rapid growth was approximately 48 h. Seeding efficiencies of up to 53% and cloning efficiencies of up to 13% were obtained. Early passage OSE cells reversibly modulated from a slow growing, epithelial, intensely keratin‐positive form in 199/202 medium lacking EGF/HC, to a rapidly proliferating, elongate, less keratin‐positive form in medium with EGF/HC. OSE cells grown in WM/5–15% FBS were epithelial and near‐stationary. Thus, culture conditions have been defined for ovarian carcinogen assays requiring either proliferating or stationary cell populations, and for further studies of the role of OSE in ovarian biology.
Effects of diacylglycerols on LLC‐PK1 renal epithelia: Similarity to phorbol ester tumor promoters
Authors: Mullin James M. , McGinn Mary T. 
Published: February 2005

AbstractAs previously shown using phorbol ester tumor promoters (see Mullin and O'Brien: Am. J. Physiol., 251:C597–C602, 1986), diacylglycerols induce leakiness in LLC‐PK1 renal epithelial tight junctions. The similarity between phorbol ester and diacylglycerol action includes effects on (1) cell morphology, (2) dome formation, (3) transepithelial resistance and potential difference, (4) transepithelial flux of D‐mannitol, and (5) mitogenesis. Four diacylglycerols have been tested: 1,2‐dioctanoylglycerol; 1,2‐dicaprylglycerol; 1,2‐dioleoylglycerol; and 1‐oleoyl‐2‐acetoyl‐sn‐3‐glycerol. Their relative effectiveness depended upon the phenomenon being observed. Unlike phorbol esters, diacylglycerol effects were reversible within hours at 37°C in the continued presence of diacylglycerol, and effects were more pronounced when cell sheets were exposed to diacylglycerols from the basolateral cell surface. Overall, these findings indicate that previous results with phorbol esters may be attributed to the protein kinase C signal transduction system, and this system may therefore exert a role in transepithelial permeability.
Direct visualization and quantitative analysis of epidermal growth factor‐induced receptor clustering
Authors: van Belzen Nico , Rijken Philip J. , Hage Willem J. , de Laat Siegfried W. , Verkleij Arie J. , Boonstra Johannes 
Published: February 2005

AbstractSeveral observations have indicated that clustering of growth factor receptors plays an important role in the action of growth factors. In this investigation, we have used the label fracture method to study the effects of epidermal growth factor (EGF) on the lateral distribution of its receptors in A431 epidermoid carcinoma cells. This method allows a direct visualization of immunogold‐labeled plasma membrane receptors on ultrastructural level and in addition permits an quantitative analysis of their lateral distribution. EGF receptors were immunogold‐labeled according to standard procedures with the monoclonal anti‐EGF receptor antibody 2E9 (lgG1), which binds to the EGF receptor in a 1:1 ratio. In the absence of EGF, EGF receptors located on the surface of A431 cells were found to be clustered, as deduced from Poisson variance analysis (p < 0.001). Following treatment of A431 cells with EGF, receptor clustering increased rapidly, reaching the maximum within 10 min. Maximal clustering was maintained for 1 h, after which the lateral distribution of receptors returned to the control situation within another hour.
Role of the cytoskeleton in laminin induced mammary gene expression
Authors: Blum Joanne L. , Wicha Max S. 
Published: February 2005

AbstractThe differentiation of rat mammary epithelial cells is characterized both by morphologic changes and by the expression of a group of milk protein genes. We have previously shown that by culturing these cells on the basement membrane glycoprotein laminin, the synthesis of the milk proteins, transferrin, α‐casein, and α‐lactalbumin is induced. In order to determine if this effect is mediated through the cytoskeleton, we have treated these cells with cytochalasin D and colchicine. Treatment with cytochalasin D or colchicine for 24 h inhibits the accumulation of α‐casein, transferrin, and α‐lactalbumin without significant effect on general protein synthesis. Pulse chase studies show that cytochalasin D does not alter the intracellular turnover of α‐casein or transferrin. Additionally, treatment with cytochalasin D causes an early (within 1 h) increase in secretion of α‐casein and transferrin suggesting that the actin cytoskeleton provides a meshwork for secretory vesicles. The disruption of this network enhances the secretion of preformed proteins. However, long term (24 h) treatment with cytochalasin D inhibits synthesis of these milk proteins. Northern blot analysis indicates that treatment with cytochalasin D or colchicine inhibits the laminin induced increase in α‐casein, α‐lactalbumin, and transferrin mRNAs. These studies indicate that the major effect of the cytoskeleton on laminin induced milk protein gene expression occurs at the level of accumulation of mRNAs for these proteins. We conclude that the expression of laminin induced milk protein gene expression in primary rat mammary cultures depends on the integrity of the actin and microtubule cytoskeleton.
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

AbstractTissue 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

AbstractThe 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

AbstractUsing 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.