bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of cycloheximide or puromycin on induction of thermotolerance by sodium arsenite in Chinese hamster ovary cells: Involvement of heat shock proteins
Authors: Lee Yong J. , Dewey William C. 
Published: February 2005

AbstractAfter sodium arsenite (100 μM) treatment, the synthesis of three major heat shock protein families (HSPs; Mr = 110,000, 87,000, and 70,000), as studied with one‐dimensional gels, was enhanced twofold relative to that of unheated cells. The increase of unique HSPs, if studied with two‐dimensional gels, would probably be much greater. In parallel, thermotolerance was observed as a 100,000‐fold increase in survival from 10−6 to 10−1 after 4 hr at 43°C, and as a thermotolerance ratio (TTR) of 2–3 at 10−3 isosurvival for heating at 45.5°C. Cycloheximide (CHM: 10 μg/ml) or puromycin (PUR: 100 μg/ml), which inhibited total protein synthesis and HSP synthesis by 95%, completely suppressed the development of thermotolerance when either drug was added after sodium arsenite treatment and removed prior to the subsequent heat treatment. Therefore, thermotolerance induced by arsenite treatment correlated with an increase in newly synthesized HSPs. However, with or without arsenite treatment, CHM or PUR added 2–6 hr before heating and left on during heating caused a 10,000–100,000‐fold enhancement of survival when cells were heated at 43°C for 4 hr, even though very little synthesis of heat shock proteins occurred. Moreover, these cells manifesting resistance to heating at 43°C after CHM treatment were much different than those manifesting resistance to 43°C after arsenite treatment. Arsenite‐treated cells showed a great deal of thermotolerance (TTR of about 10) when they were heated at 45°C after 5 hr of heating at 43°C, compared with less thermotolerance (TTR of about 2) for the CHM‐treated cells heated at 45°C after 5 hr of heating at 43°C. Therefore, there are two different phenomena. The first is thermotolerance after arsenite treatment (observed at 43°C or 45.5°C) that apparently requires synthesis of HSPs. The second is resistance to heat after CHM or PUR treatment before and during heating (observed at 43°C with little resistance at 45.5°C) that apparently does not require synthesis of HSPs. This phenomenon not requiring the synthesis of HSPs also was observed by the large increase in thermotolerance to 45°C caused by heating at 43°C, with or without CHM, after cells were incubated for 6 hr following arsenite pretreatment. For both phenomena, a model based on synthesis and redistribution of HSPs is presented.
Na+ for H+ exchange in rabbit erythrocytes
Authors: Escobales Nelson , Rivera Alicia 
Published: February 2005

AbstractThe effect of a transmembrane pH gradient on the ouabain, bumetanide, and phloretin resistant H+ efflux was studied in rabbit erythrocytes. Proton equilibration was reduced by the use of DIDS (125 μM) and acetazolamide (1 mM). H+ efflux from acid loaded erythrocytes (pHi = 6.1) was measured in a K+ (145 mM) medium, pHO = 8.0, in the presence and absence of 60 μM 5,N,N‐dimethyl‐amiloride (DMA). The H+ efflux rate in a K+‐containing medium was 116.38 ± 4.5 mmol/l cell × hr. Substitution of NaO+ for KO+ strongly stimulated H+ efflux to 177.89 ± 7.9 mmol/l cell × hr. The transtimulation of H+ efflux by NaO+ was completely abolished by DMA falling to values not different from controls with an ID50 of about 8.6 × 10−7 M. The sequence of substrate selectivities for the external transport site were Na ⋙ Li > choline, Cs, K, and Glucamine. The transport system has no specific anion requirement, but is inhibited by NO3−. The DMA sensitive H+ efflux was a saturable function of [Na+]O, with an apparent Km and Vmax of about 14.75 ± 1.99 mM and 85.37 ± 7.68 mmol/l cell × hr, respectively. However, the NaO+‐dependent and DMA‐sensitive H+ efflux was sigmoidally activated by [H+]i, suggesting that Hi+ interacts at both transport and modifier sites. An outwardly directed H+ gradient (pHi 6.1, pH = 8.0) also promoted DMA sensitive Na+ entry (61.2 ± 3.0 mmol/l cell × hr) which was abolished when pHO was reduced to 6.0. The data is therefore consistent with the presence of a Na+/H+ exchange system in rabbit erythrocytes.
Endogenous thiol levels in heterogeneous murine tumor cells as a function of the physiological state and the response to X‐irradiation
Authors: Dethlefsen Lyle A. , Biaglow John E. , Peck Vickie M. , Ridinger David N. 
Published: February 2005

AbstractThe endogenous thiols (PSH, protein sulfhydryls; NPSH, nonprotein sulfhydryls; and GSH, glutathione) were measured in the 66 and 67 murine carcinoma cells growing under different physiological conditions in vitro (e.g., proliferation, P; nutrient‐deprived quiescence Ql; and Ql cells stimulated by refeeding the monolayer in situ and assayed 4 (St4) and 14 (St14) h later). The aerobic radiation response was also studied as a function of the physiological state and thiol concentration. The changes in PSH levels suggest that the proportion of thiol‐containing proteins changed whenever the cells were in transition between different physiological states (e.g., when Ql cells were stimulated by refeeding, the proportion of PSH was elevated dramatically over either Ql or P cells). The NPSH and GSH levels were both down significantly in the Ql vs. P cells as was the total thiol level (PSH plus NPSH). Fourteen h but not 4 h after stimulation, the NPSH and GSH levels had returned to or exceeded the P‐cell levels. Also, the proportion of GSH in the NPSH fraction varied as a function of the physiological state. The 66 and 67 Ql cells were both more radiosensitive than the respective P cells. Also, the 66 cell radiation‐induced cytotoxicity had returned to the P response by about 4 h after refeeding but the stimulatec 67 cells had not. However, no overall correlation was apparent between the various aerobic radiation responses and the pool sizes of either the total thiols or of the various subsets of thiols. The depressed total thiol level and the increased radiosensitivity of the Ql cells could represent a cause‐and‐effect relationship or these parameters could be independent phenomena only related indirectly through the reduced metabolic activity of the quiescent cells.
Induction and decay of thermotolerance in rainbow trout fibroblasts
Authors: Mosser D. D. , van Oostrom J. , Bols N. C. 
Published: February 2005

AbstractThermotolerance was studied in the rainbow trout fibroblast cell line RTG‐2. RTG‐2 cultures that had been incubated at 28°C for 24 h were better able to withstand ultimately lethal temperatures above 28°C than RTG‐2 cultures that had been maintained at the routine growth temperature of 22°C. This thermotolerance developed rapidly between 3 and 6 h and was fully developed by 24 h at 28°C. After development for 24 hr at 28°C, thermotolerance showed little change over 72 h at 0 and 5°C but approximately a 40 and 60% reduction at 10 and 22°C, respectively. This is the first demonstration of heat‐induced thermal resistance in the cells of a poikilothermic vertebrate.
The glioma cell‐derived neurite promoting activity protein is functionally and immunologically related to human protease nexin‐I
Authors: Knauer Daniel J. , Orlando Robert A. , Rosenblatt Dorrie 
Published: February 2005

AbstractProtease nexin‐I (PN‐I, Mr ∼ 43,000) is representative of a newly described class of cell‐secreted protease inhibitors. PN‐I has been purified to apparent homogeneity, partially sequenced, and monospecific antibodies have been raised against it. PN‐I is a potent inhibitor of urokinase, thombin, plasmin, and trypsin. In addition, cells have specific receptors that mediate the uptake of covalently linked complexes formed between PN‐I and its protease substrates. In the present studies, we have investigated the relationship between human PN‐I and a protease inhibitor derived from C6 glioma cells in culture that has neurite‐promoting activity. On the basis of co‐purification on heparin‐Sepharose, identical molecular weight, antibody cross‐reactivity, and receptor cross‐reactivity, we conclude that PN‐I and the glioma‐cell‐derived inhibitor are equivalent molecules.
Elevation of a potassium current in differentiating human leukemic (HL‐60) cells
Authors: Wieland Steven J. , Chou Robin H. , Chen Thelma A. 
Published: February 2005

AbstractHuman promyelocytic leukemia (HL‐60) cells display a novel voltage‐dependent outward current under voltage clamp. This current is present at low levels in the proliferative state and in granulocytes derived from HL‐60 cells which were induced to differentiate with retinoic acid. It is elevated in macrophages derived from HL‐60 cells after exposure to phorbol‐12‐myristate‐13‐acetate (PMA). The current is carried primarily by K+, is blocked by Cs+ and by increased intracellular concentrations of Cl−. From a holding potential of −80 mV, significant activation required depolarization to +20 mV membrane potential. Activation was not influenced by intracellular Ca2+ (1–2 × 10−6M). These properties appear to differ significantly from the Ca2+‐activated K+ channel and the delayed rectifier. The increase of this voltage‐activated current in differentiation toward the macrophage, but not the granulocyte, suggests that this current is correlated specifically with macrophage differentiation.
Phorbol myristate acetate inhibits growth in S49 cells: Isolation of resistant variants
Authors: Leach Karen L. , Powers Elaine A. , Mayo Judy K. , Abraham Irene , Burnett Bonnie‐Ann , Groppi Vincent E. 
Published: February 2005

AbstractWe have used S49 mouse lymphoma cells to study phorbol ester effects on growth. Treatment of wild‐type (wt) cells with phorbol 12‐myristate 13‐acetate (PMA) results in growth arrest within 72 hr. We have selected variants that are resistant to PMA‐induced growth arrest, based on a selection in the presence of 10 nM PMA. We have characterized one of these variants, termed 21.1, in detail. The 21.1 and wt cells contain similar levels of protein kinase C (PKC) as determined by [3H]phorbol 12,13‐dibutyrate ([3H]PDBu) binding. Treatment of both wt and 21.1 cells with PMA results in translocation of PKC to the membrane, suggesting that the coupling between PKC and an immediate biological response is intact. PMA treatment leads to the phosphorylation of many similar proteins in wild‐type and 21.1 cells. However, in the 21.1 cells there is a prominent substrate of approximately 70 kilodaltons (kD) which is no longer phosphorylated after PMA treatment. In wild‐type cells ornithine decarboxylase (ODC) activity and mRNA levels are decreased within 1 hr of PMA treatment. Likewise, ODC levels are decreased in the 21.1 cells after exposure to PMA even though PMA only slightly modulates the growth of these cells. The 21.1 cells represent a unique line with a dominant phenotype in which ODC expression is uncoupled from the growth state of the cell. These cells may represent a good model system in which to examine the steps involved in phorbol ester growth regulation in S49 cells.
Erythropoiesis in murine long‐term bone‐marrow cell cultures: Dependence on erythropoietin and endogenous production of an erythropoietic stimulating activity
Authors: Oddos Thierry , Nacol‐Lizard Sarab , Blanchet Jean Paul 
Published: February 2005

AbstractErythropoiesis was obtained in murine long‐term bone‐marrow cell cultures (LTBMCs) in the presence of erythropoietin (Epo) when the medium was frequently renewed. The level of the erythropoietic differentiation was shown to be a function of the erythropoietin concentration. In response to Epo addition, an activity which stimulates CFU‐E proliferation in semisolid cultures of fresh bone marrow cells was detected in the LTBMC supernatants. These results suggest that another factor, whose synthesis may be under Epo control, participates in the stimulation of erythropoiesis in vitro.
Role of biosynthesis of cholesterol and isoprenoid derivatives in regulation of G1 progression and cell proliferation of 3T6 cells
Authors: Larsson Olle 
Published: February 2005

AbstractThe growth of 3T6‐cells was rapidly decreased, although not completely stopped, as a consequence of treatment of the cell cultures with serum‐free medium. By analyzing the cell cycle traverse it was found that the decrease in growth rate was attributable to a 6–8‐h delay in progression through the proximal part of G1. These effects of serum depletion on cell‐cycle traverse and cell proliferation were compared with simulataneous effects on the activity of 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG CoA) reductase and the biosynthesis of cholesterol and the two isoprenoid derivatives coenzyme Q and dolichol. The activity of HMG CoA reductase was unaffected during the first 8 h of serum depletion and depressed by approximately 50% during the next 16 h. In contrast, the rate of coenzyme Q and dolichol synthesis, as related to cholesterol synthesis, was substantially decreased, and within 4 h after shift to serum‐free medium both were reduced by 70–80%. As distinguished from dolichol synthesis, the synthesis of coenzyme Q was even decreased following exposures of the cell cultures to cholesterol‐poor serum. This indicates that the rate of coenzyme Q synthesis is dependent on the concentration of serum cholesterol, whereas rate of dolichol synthesis is stimulated by some other serum factor(s). In addition, supplementation with dolichol to serum‐depleted cells partially normalized G1 traverse and DNA synthesis, whereas cholesterol or coenzyme Q was ineffective. Taken together, the results suggest that a certain level of de novo synthesis of dolichol is required to maintain normal cell‐cycle traverse and growth of 3T6 cells.
Production of platelet‐derived growth factor by cultured Wilms' tumor cells and fetal kidney cells
Authors: Fraizer Gail E. , Bowen‐Pope Daniel F. , Vogel Arthur M. 
Published: February 2005

AbstractWe have cultured human Wilms' tumors (nephroblastomas) in vitro and examined their growth properties, tumorigenicity in nude mice, karyotypes, and production of a platelet‐derived growth factor (PDGF)‐like activity (PDGFc). The cultured Wilms' tumor cells do not exhibit transformed growth properties, senesce after 15–25 passages, and are not tumorigenic in nude mice. Two of the three Wilms' tumors examined had karyotypic abnormalities but none contained a deleted 11p region. The Wilms' tumor cells and control human fetal kidney cells produce significant amounts of PDGFc and express few PDGF receptors on the cell surface. Analysis of Wilms' tumor cell RNA using probes specific for PDGF A‐chain and B‐chain (c‐sis) detects A‐chain but not B‐chain transcripts in the tumor cells.
Mouse embryonic stem cells exhibit indefinite proliferative potential
Authors: Suda Yoko , Suzuki Misao , Ikawa Yoji , Aizawa Shinichi 
Published: February 2005

AbstractThe proliferative potential of embryonic stem cells was examined. In contrast to the current concept of the finite life‐span being the hallmark of normal cells, we have been able to maintain these embryonic stem cells in vitro up to about 250 cumulative doublings with no indication of “crisis” or transformation. These cells could be considered normal on the basis of: (1) their apparently normal diploid karyotype, (2) their ability to extensively colonize embryos without causing tumors and developmental anomalies, and (3) their ability to form normal gametes when differentiated into the germ‐line. These results suggest that embryonic stem cells prior to differentiation into germ and somatic cells are indeed immortal.
Size‐dependent hyaluronate degradation by cultured cells
Authors: McGuire Paul G. , Castellot John J. , Orkin Roslyn W. 
Published: February 2005

AbstractHyaluronate degradation was examined in cultures of vascular wall cells (bovine aortic endothelial cells, rat aortic smooth muscle cells) and in nonvascular cells (chick embryo fibroblasts). The three cell types examined all produced hyaluronidase activity in culture which had a strict acidic pH requirement for activity. This suggested that the enzyme was active only within an acidic intracellular compartment and therefore that hyaluronate degradation occurred at an intracellular site. This was supported by the observation that the presence of hyaluronidase activity alone was not sufficient to ensure degradation of extracellular hyaluronate. Rather, the key limiting factor in this process appeared to be hyaluronate internalization, and this was found to be hyaluronate size‐dependent and to a degree, cell‐specific. The relationship of these results to morphogenesis and tissue remodeling is discussed.
Expression of gamma‐interferon receptor in murine bone marrow‐derived macrophages associated with macrophage differentiation: Evidence of gamma‐interferon receptors in the regulation of macrophage proliferation
Authors: Chen Ben D.‐M. , Chou Ta‐Hsu , Ratanatharathorn Voravit 
Published: February 2005

AbstractThe effect of purified, recombinant murine gamma interferon (IFN‐γ) on the regulation of macrophage proliferation induced by colony‐stimulating factor 1 (CSF‐1) was investigated. Although both hemopoietic stem cells (GM‐CFC) and tissue‐derived peritoneal exudate macrophages (PEM) proliferated in response to CSF‐1, the more mature PEM were much more sensitive to an antiproliferative effect of IFN‐γ. The role of IFN‐γ receptor expression and its relationship to growth inhibition was examined. Bone marrow cells as a whole did not exhibit an appreciable amount of IFN‐γ receptor binding activity. Likewise, nonadherent (NA) cells derived from CSF‐1‐stimulated bone marrow cultures displayed low levels of IFN‐γ receptor binding activity. On the contrary, more mature adherent (AD) cells (monocytes/macrophages) from the same culture exhibited high levels of IFN‐γ receptor binding activity which continued to increase with culture time. The elevated IFN‐γ binding activity is due to an increase in total receptor number rather than the binding affinity as judged by Scatchard analysis. Similar to the relationship between PEM and GM‐CFC, more mature AD cells were also more susceptible to the inhibitory effect of IFN‐γ on CSF‐1‐induced proliferation than their less mature NA counterparts. The fact that the sensitivity to IFN‐γ correlated well with the expression of existing IFN‐γ receptors strongly suggests that the inhibitory effect is mediated through IFN‐γ receptors. This study shows that the expression of IFN‐γ receptors in mononuclear phagocytes may not only represent one of the phenotypic parameters acquired by the growing macrophages during the process of differentiation, but may play some role in controlling proliferation.
Induction of cytokeratin expression in human mesenchymal cells
Authors: Von Koskull Harriet , Virtanen Ismo 
Published: February 2005

AbstractWe studied the phenotypic features of some typical human mesenchymal cells, including decidual stromal cells and adult and fetal fibroblasts under different cell culture conditions by using antibodies to intermediate filament proteins and desmoplakins. In cell culture, the decidual stromal cells rapidly acquired typical fibroblastoid appearance with abundant arrays of vimentin filaments while the cytokeratin‐positive epithelial cells, occasionally found in typical epithelioid colonies, lacked vimentin positivity and showed desmoplakin positivity. Within a few days, many of the stromal cells started to present cytokeratin positivity when cultured either in Condimed® or in Chang® medium. The cytokeratin positivity was first detected in small, scattered cytoplasmic dotted fibrils or in perinuclear dotlike aggregates with fibrillar projections. Later, denser cytokeratin‐positive fibrillar arrays could also be seen in stromal cells, which lacked desmoplakin positivity as judged by two monoclonal antibodies. Decidual stromal cells were also cloned and in five out of ten clones some of the cells acquired a similar cytokeratin positivity when transferred into Chang® or Condimed® medium. Immunoblotting results indicated that cytokeratins 8, 18, and 19 can be found in these cultures. Similar cytokeratin positivity could also be seen in the same culture conditions in cultured fetal fibroblasts from skin, chorionic villi, and lung but not in young or adult skin fibroblast cultures. The present results suggest that decidual stromal cells as well as some embryonal mesenchymal cells can acquire epithelial differentiation in vitro as judged by the emergence of cytokeratin proteins. This ability appears to be lost in the corresponding adult cell. The results furthermore suggest that cytokeratin fibrils can be organized in the cytoplasm without an apparent organization center and that neither the appearance of desmoplakins nor the formation of cell‐to‐cell contacts are required for cytokeratin filament assembly.
Induction of alkaline phosphatase in primary cultures of epiphyseal growth plate chondrocytes by a serum‐derived factor
Authors: Ishikawa Yoshinori , Valhmu Wilmot B. , Wuthier Roy E. 
Published: February 2005

AbstractAlkaline phosphatase (AP) activity in epiphyseal growth plate cartilage increases markedly during differentiation of the chondrocytes, and reaches high levels in the zone of hypertrophy where vascular penetration and provisional mineralization begin. A proteinaceous factor has been discovered in serum that stimulates the expression of AP in chicken growth plate chondrocytes when these cells are grown in serum‐free media. Sera from a variety of vertebrate species (goat, fetal bovine, horse, human, and chicken) all contained detectable levels of the inducing activity. The chondrocyte AP‐induction factor (CAP‐IF) from fetal bovine serum was precipitated with ammonium sulfate between 33% and 50% saturation, and purified by dye‐ligand affinity chromatography. The active fraction, which eluted from an Affi‐Gel Blue column between 0.10 and 0.15 M NaCl, was further resolved on a QMA anion exchange column. The most active and almost homogenous fraction contained primarily a 64.5 kDa protein; about 3 μg/ml medium induced 50% of the maximal level of AP induction. CAP‐IF is stable to heat (100°C for 3 min) and dithiothreitol (50mM) treatment, and is only mildly inactivated by 2 h treatment with trypsin. CAP‐IF caused no significant effect on cell division as measured by 3H‐thymidine uptake. Time‐course studies revealed that at least 18–24 h exposure of the chondrocytes to CAP‐IF is required to produce major increases in AP activity. Longer exposure time generally further increases the response. Cycloheximide almost completely blocked the increase in AP activity, indicating that de novo protein synthesis is required for induction.
Metabolic effects of interleukin 3 on 32D cl23 cells analyzed by NMR
Authors: Hasthorpe S. , Carver J. A. , Rees D. , Campbell I. D. 
Published: February 2005

Abstract31P NMR of living 32D cl23 cells and 1H NMR of cell extracts were used to study the metabolic effects of interleukin 3 (IL3). When IL3 was removed from 32D cl23 for 9–10 hours 31P spectra showed a decrease in sugar phosphate, γATP/ADP, αATP/ADP/NAD, and βATP resonances which declined progressively over a time period of up to 16 hours. By comparison, ATP measurements using the luciferin/luciferase method resulted in the decline of ATP levels from 12 hours in the absence of IL3. At this time, viability of the cells was unaffected. For 1H NMR experiments cells were grown in the presence and absence of IL3 for 4 and 24 hours, after which acid cell extracts were prepared. These spectra revealed a four‐fold decrease in lactate 4 hours post‐IL3 removal. Alanine levels were unchanged but glycine was elevated 1.5‐fold whilst various other amino acids were elevated slightly. After 24 hours without IL3, only 22% of cells were viable which was reflected in a general decline of most resonance intensities. These findings suggest that IL3 exerts its effect primarily on glucose metabolism and has a delayed secondary effect on maintenance of ATP levels in the cell. We have demonstrated the applicability of high resolution 1H and 31P NMR to the study of cellular metabolism in hemopoietic cells.