bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rapid changes in bidirectional K+ fluxes preceding DMSO‐induced granulocytic differentiation of HL‐60 human leukemic cells
Authors: Gargus J. J. , Adelberg E. A. , Slayman C. W. 
Published: February 2005

AbstractWhen grown in medium containing 5 mM potassium and 140 mM sodium, HL‐60, a human promyelocytic cell line, maintained a steady‐state intracellular K+ concentration of 145 mmol/L cells and a steady‐state intracellular Na+ concentration of 30 mmol/L cells. Nearly 90% of the unidirectional 42K+ influx could be inhibited by the cardiac glycoside ouabain with a Ki of 5 × 10−8 M. This ouabain‐sensitive component of influx rose as a saturating function of the extracellular K+ concentration with a K1/2 of 0.85 mM. The component of 42K+ influx resistant to ouabain inhibition was a linear function of the extracellular K+ concentration and was insensitive to inhibition by the diuretic furosemide. Unidirectional K+ efflux followed first order kinetics with a half‐time of 55 min. Addition of 1.5% dimethyl sulfoxide (DMSO) to a culture of HL‐60 cells allowed two population doublings followed by the cessation of growth without an impairment of cell viability. Beginning 2 to 3 days after DMSO addition, the cells underwent a dramatic reduction in volume (from 925 μm3 to 500 μm3) and began to take on the morphological features of mature granulocytes. Throughout this process of differentiation there was no change in the intracellular sodium or potassium concentration. However, immediately following the addition of DMSO to a culture of cells, there began an immediate, coordinated reduction in bidirectional K+ flux. The initial rate of the ouabain‐sensitive component of K+ influx fell with a half‐time of 11 h to a final rate, at 6 days induction, equal to one ninth that of the uninduced control, and over the same period, the rate constant for K+ efflux fell with a half‐time of 14 h to a final value one fourth that of the uninduced control. The rapidity with which these flux changes occur raises the possibility that they play some role in the control of subsequent events in the process of differentiation.
Control of enzyme activity levels by serum and hydrocortisone in neonatal rat heart cells cultured in serum‐free medium
Authors: Freerksen Deborah L. , Schroedl Nancy A. , Hartzell Charles R. 
Published: February 2005

AbstractA serum‐free, hormone‐supplemented medium (SFHM) for maintaining neonatal rat heart cells in culture has been developed in this laboratory (Mohamed et al., 1983). Morphological assessment of heart cells grown in SFHM show it to be similar to commonly used serum‐supplemented media. To quantitatively compare cell behavior in SFHM with serum‐supplemented media, the activities of ten regulatory enzymes which represent four metabolic pathways were studied in heart cells cultured in SFHM. The enzyme activities which were measured included hexokinase, glucose‐6‐phosphate dehydrogenase, 6‐phosphogluconate dehydrogenase, phosphofructokinase, pyruvate kinase, NAD+‐linked sn‐glycerol‐3‐phosphate dehydrogenase, malate dehydrogenase, NAD+‐linked isocitrate dehydrogenase, NADH‐cytochrome c reductase, and succinic cytochrome c reductase. Rat heart cells maintained in culture on SFHM are not only qualitatively and quantitatively similar to those maintained in serum‐supplemented medium but also provide a more suitable model system for metabolic studies of neonatal cardiac tissue for several reasons: (1) many enzyme activities that may represent dedifferentiation are elevated by serum; (2) NAD‐linked glycerol‐3‐phosphate dehydrogenase activity in cells maintained on SFHM is similar to the in vivo activity; (3) cells beat at or near the in vivo frequency and can be maintained 3 months on SFHM; (4) the SFHM is chemically defined and thus can be completely manipulated by the investigator. The effects of three concentrations of hydrocortisone (HC) (5,000 ng/ml, 50 μg/ml, 0 ng/ml) on heart cells cultured in SFHM supported our previous conclusion that function (beating) and growth (protein accumulation) are inversely related in cultured neonatal rat heart cells.
25‐Hydroxycholesterol‐induced elevation in 45Ca uptake: Correlation with depressed DNA synthesis
Authors: Boissonneault Gilbert A. , Heiniger Hans‐Jörg 
Published: February 2005

AbstractThe mechanism whereby 25‐hydroxycholesterol, an inhibitor of the synthesis of cholesterol, depresses DNA synthesis in cycling P815 mastocytoma cells was investigated. The uptake of 45Ca into P815 cells treated with 1 μg/ml 25‐hydroxycholesterol began to rise above control levels by 6 hours after initiation of treatment and was increased tenfold by 15 hours. Kinetic data of calcium uptake indicated the presence of at least two components of calcium uptake, fast and slow. The fast phase of calcium exchange at the cell surface was changed little by treatment with 25‐hydroxycholesterol. The slow phase of calcium exchange with the intracellular compartment was markedly affected by treatment with the inhibitor, there being a large increase in the flux and half‐time of uptake, and a fall in the rate constant. This resulted in a large elevation of the intracellular compartment size. Incorporation of [3H]thymidine into DNA began to decline between 9 and 12 hours posttreatment in these cultures. Uptake of calcium and depression of DNA synthesis were shown to be directly reiated to the dose of 25‐hydroxycholesterol used. The changes in 45Ca uptake and DNA synthesis due to 25‐hydroxycholesterol treatment were abolished by addition of exogenous cholesterol to the incubation medium. The results are consistent with the hypothesis that 25‐hydroxycholesterol, by inhibiting cholesterol production, depresses DNA synthesis via an elevation in the uptake of calcium into the cell to a level incompatible with continued DNA replication.
Thermal down‐regulation of exportable rRNA in nuclei
Authors: Wunderlich Frank , Giese Günter , Herlan Gerhard 
Published: February 2005

AbstractThe export of rRNP particles from nuclei isolated from Tetrahymena was investigated after preincubating the nuclei at different temperatures under nonpermissive export‐conditions. We observed a new phenomenon: Temperature elevation from the sublethal cells' growth temperature, 8°C, to the optimal temperature, 28°C, lead to a gradual down‐regulation in the maximal proportion of rRNP particles subsequently exported from nuclei at 28°C. This thermal down‐regulation is apparently not due to qualitative changes in the exported rRNP particles, a derangement in the gross nuclear organization, a degradation and/or nicking of the nuclear rRNA, a gross decomposition of the major nuclear proteins, a random cross‐linking of nuclear components by disulfide bonds, or an elution of nuclear factors possibly required for rRNP export. Moreover, there is a corresponding thermal down‐regulation in nuclear envelope‐free nuclei. Our data indicate that nuclei possess a mechanism that regulates the number of potentially exportable rRNP particles at a level preceding the rRNP passage through the nuclear envelope.
Characterization and partial purification of keratinocyte growth factor from the hypothalamus
Authors: Gilchrest B. A. , Marshall W. L. , Karassik R. L. , Weinstein R. , Maciag T. 
Published: February 2005

AbstractAn extract of bovine hypothalamus is known to be mitogenic for human keratinocytes in vitro. In order to identify the'responsible substance(s), biochemical characterization and subsequent bioassay of the extract in a serum‐free culture system were performed. The keratinocyte growth‐promoting activity of the hypothalamic extract was unaffected by heating (100 C, 10 min); acidification to pH 3.3; or by exposure to lipase, RNAase, or proteolytic enzymes; but was abolished by alkalinization to pH 11. An approximate molecular weight of 1,700 daltons was determined by elution on a calibrated Sephadex G‐25 column, and an approximate pI of 3.5 was determined by isoelectric focusing. Optimal concentrations of the crude extract (150‐300 μg/ ml) increased keratinocyte growth approximately 50‐fold compared to control cultures lacking the extract. Partial purification resulted in a preparation biologically active at 30 ng/ml protein equivalent and was consistent with the presence of a single mitogen which we have termed keratinocyte growth factor (KGF). Mitogenic activity for human melanocytes, dermal fibroblasts, and endothelial cells, present in the crude hypothalamic extract, was lacking in heat‐treated preparations that contained KGF. Optimal concentrations of purified epidermal growth factor and ethanolamine, the only remotely similar substances previously reported to augment keratinocyte growth in vitro, could not substitute for KGF in the serum‐free culture system. Keratinocyte growth‐promoting activity comparable to that observed in bovine hypothalamic extracts was present in human hypothalamic extracts prepared in the same manner.
Transmembrane potential and intracellular potassium ion activity in fetal and maternal liver
Authors: Chapman Lisa M. , Wondergem Robert 
Published: February 2005

AbstractWe have compared transmembrane potentials (Em) of maternal liver with Em of fetal liver, and as an initial step to account for differences in Em, we have measured intracellular potassium ion activities (a) in both tissues. Paired segments of maternal and fetal (day 17) mouse liver were suffused (15 ml/min) with Krebs' physiologic salt solution equilibrated with 95% 02 −5% CO2 (pH 7.3‐7.4) at 37°C. To measure Em, cells were impaled with open‐tip microelectrodes filled with 0.5 M KCI. Intracellular voltage recordings that were stable ± 2 mV for at least 10 s were considered valid impalements. Maternal liver mean Em = − 41 ± 1 (SEM) mV, n = 10 animals. In contrast, fetal liver mean Em = − 23 ± 1 (SEM) mV, n = 10 animals. In the same segments we measured a with potassium‐selective liquid ion‐exchanger microelectrodes. Maternal liver mean a = 95 ± 7 (SEM) mM and fetal liver mean a = 62 ± 4 (SEM) mM. In addition, Em and a of fetal liver increased to values comparable to those of maternal liver during the first 8 days of neonatal life. The differences of Em and a between fetal and maternal liver, and the changes in these values that occur in the neonate, may result from activity of a membrane Na‐K exchange pump that increases with tissue development.
Release of endothelial cell‐derived growth factor (ECDGAF) by heparin
Authors: Gajdusek Corinne M. 
Published: February 2005

AbstractEndothelial cells in vitro produce potent growth‐promoting activities collectively known as endothelial cell‐derived growth factor (ECDGF). These mitogens (including platelet‐derived growth factor [PDGF]‐like and non‐PDGF‐like species) support DNA synthesis and proliferation of smooth muscle cells and fibroblasts in the absence of other defined mitogens. The current study examines interaction of heparin and heparin‐related polysaccharides with bovine aortic endothelial (BAE) cells and the levels of secreted growth‐promoting activity. Heparin, dextran sulfate, and carageenan rapidly release non‐PDGF‐like ECDGF from BAE cell monolayers. The effective heparin concentrations closely correlate with amounts of exogenous 3H‐heparin required for maximal binding to BAE cells. Both heparin binding and ECDGF release occur independently of incubation temperature. Inhibition of heparin binding by dextran sulfate and carageenan and the lack of inhibition by dextran, chondroitin sulfate, and dermatan sulfate imply a high degree of specificity of the interaction. In contrast to quiescent monolayers of BAE cells, migrating cells release lower levels of growth‐promoting activity, and no ECDGF is released in response to heparin, although migrating cells bind the same levels of 3H‐heparin as confluent monolayers. Partial characterization of the endothelial cell released mitogens indicates that the non‐PDGF‐like mitogen is associated with endothelial cell surfaces in contrast to the apparent constitutive release of PDGF‐like mitogens. These results indicate that endothelial cells modulate production and release of specific mitogens in response to growth state.
Regulation of Na+, K+‐ATPase activity in MDCK kidney epithelial cell cultures: Role of growth state, cyclic AMP, and chemical inducers of dome formation and differentiation
Authors: Kennedy Brian G. , Lever Julia E. 
Published: February 2005

AbstractNa+,K+‐ATPase activity was monitored in MDCK kidney epithelial cell monolayers and in cell extracts as a function of cell density, cAMP elevation, and exposure to hexamethylene bisacetamide (HMBA) and dimethylsulfoxide (Me2SO). Ouabain‐sensitive Na+,K+‐ATPase and 86Rb+ uptake activities, and the number of [3H]‐ouabain binding sites were maximal in subconfluent cultures and decreased accompanying the development of a confluent monolayer. A sodium pump density of 8 × 107 pumps/cell was estimated for subconfluent cultures, declining to 9 × 105 pumps/cell at confluence. Previous studies have shown that dibutyryl cyclic AMP (Bt2cAMP), 1‐methyl‐3‐isobutylxanthine (IBMX), or the differentiation inducers HMBA and Me2SO, which also caused cAMP elevation, all stimulated dome formation, a visible manifestation of active transepithelial Na+ and water transport (Lever, 1979). In the present study, all of these inducers were found to elevate intracellular Na+ content, implicating this variable in control of induction of dome formation. Operationally, inducers could be divided into two classes. HMBA and Me2SO partially inhibited ouabain‐sensitive 86Rb+ influx. Ouabain, at a concentration that caused partial sodium pump inhibition and increased intracellular Na+ content, was also effective as an inducer. The second class, exemplified by IBMX and Bt2cAMP caused a furosemide‐sensitive increase in intracellular Na+ content. This class of inducers stimulated ouabain‐sensitive 86Rb+ uptake, presumably by substrate effects due to increased Na+ levels. The Na+ or ATP activation of Na+,K+‐ATPase activity assayed in cell‐free extracts, the affinity of the transport system for Rb+ in intact cells and intracellular ATP levels were unchanged by inducer treatment. Elevation of intracellular Na+ concentration, either by cAMP‐stimulated, furosemide‐sensitive mechanisms or by partial inhibition of the sodium pump may stimulate the induction of dome formation in MDCK cells.
Platelet‐derived growth factor: Morphologic and biochemical studies of binding, internalization, and degradation
Authors: Rosenfeld Michael E. , Bowen‐Pope Daniel F. , Ross Russell 
Published: February 2005

AbstractKinetic studies of binding and internalization of 125 I‐platelet‐derived growth factor (PDGF) demonstrate that up to 15% of membrane‐associated radioactivity is internalized within 2 minutes after warming to 37°C in a variety of cell types. The T 1/2 for internalization is approximately 20 minutes. The T 1/2 for the subsequent appearance of degradation products in the culture medium is between 60–90 minutes following initiation of internalization. Internalization and lysosomal association of 125I‐PDGF were confirmed by EM autoradiography. Quantitative studies using PDGF adsorbed to colloidal gold (gold‐PDGF) demonstrate that 17% of the cell‐associated sites are along coated regions of the plasma membrane (1.0 sites/μm), while 82% are associated with noncoated membrane (0.2 sites/μm). There is a significant redistribution of the gold‐PDGF complexes upon warming. Within 1‐2 minutes at 37°C, gold particles are found within endocytic vesicles, endosomes (0.09‐0.3 μm diameter), and lysosomes (> 0.2 μm diameter). At this time the vesicle/endosome compartment comprises 15% of the total sites and contains 0.9 sites per μm2 of surface area. The lysosomes account for 8% of the total sites and contain 0.8 sites per μm2 of surface area. Simultaneously, there is an increase in the number of gold‐PDGF binding sites within coated‐pits (1.6 sites/μm, 18% of the total sites) and a decrease along noncoated regions of the membrane (0.11 sites/μm, 58% of the total sites). After 15 minutes at 37°C, 26% of the total sites (1.4 sites/μm2) are highly concentrated within lysosomes, while sites in the vesicle/endosome compartment remain constant. At the same time, binding sites within coated pits decrease substantially (0.5 sites/μm, 4% of the total sites), while the number of sites along noncoated regions of the membrane remain constant. Gold‐PDGF was not observed associated with the Golgi complex at any time up to 120 minutes following warming. We conclude that gold‐PDGF is processed via both receptor‐mediated and nonspecific endocytosis and follows an intracellular pathway comparable to that followed by some other protein ligands.
Inhibition of cell communication between Balb/c 3t3 cells by tumor promoters and protection by cAMP
Authors: Enomoto Taira , Martel Nicole , Kanno Yoshinobu , Yamasaki Hiroshi 
Published: February 2005

AbstractThe effect of phorbol ester tumor promoters on the communication between individual cells in confluent culture was studied using a fluorescent dye transfer method. Cell‐cell communication between mouse Balb/c 3T3 cells and between Chinese hamser V79 cells was inhibited almost completely by tumor‐promoting phorbol esters, but not by nonpromoting derivatives; the effect was reversed upon removal of the promoter. Intercellular communication between Balb/c 3T3 cells, but not Chinese hamster V79 cells, was increased significantly in the presence of dbcAMP and caffeine, and these compounds counteracted the effects of tumor promoters. Inhibition of cell communication by phorbol esters appears to be receptor‐mediated, since specific binding of 3H‐phorbol‐12,13‐dibutyrate to Balb/c 3T3 cells was inhibited only by compounds that also inhibit intercellular dye transfer. A study with cycloheximide suggests that the reversible inhibition of intercellular communication by phorbol esters may not need de novo protein synthesis, while upregulation of communication by cAMP requires protein synthesis.
Acetylcholine receptors in monkey and rabbit oocytes
Authors: Caratsch Carlo , Eusebi Fabrizio , Salustri Antonietta 
Published: February 2005

AbstractMembrane potential responses to acetylcholine (ACh, 10−7‐10−3 M) were investigated in monkey and rabbit ovarian oocytes. In monkey oocytes ACh most commonly elicited a short‐latency hyperpolarization concomitant with a decreased membrane input resistance (Rin). Under voltage‐clamp short‐latency ACh currents had an equilibrium potential of approximately −40 mV. In rabbit oocytes responses to ACh consisted of an increase in Rin or of a depolarization with an equilibrium potential of approximately −15 mV. Curare, hexamethonium, and atropine (10−5–10−3 M) did not block these ACh responses. Thus, the oocyte membrane in the rabbit contains ACh receptors that cannot be classified as either muscarinic or nicotinic.
Splenic erythroblasts in anemia‐inducing friend disease: A source of cells for studies of erythropoietin‐mediated differentiation
Authors: Koury M. J. , Sawyer S. T. , Bondurant M. C. 
Published: February 2005

AbstractSplenic erythroblasts obtained from mice during the acute disease caused by either the polycythemia‐inducing (FVP) or anemia‐inducing (FVA) strain of Friend virus were examined for their degree of terminal differentiation. Morphology, benzidine staining, and heme synthesis kinetics showed that many erythroblasts from FVP‐infected mice were undergoing terminal differentiation, while few erythroblasts from FVA‐infected mice showed evidence of terminal differentiation. When cultured in methylcellulose medium, splenic erythroblasts from FVP‐infected mice completed differentiation without the addition of erythropoietin (EP) to the medium. However, splenic erythroblasts from FVA‐infected mice underwent terminal differentiation in vitro only when EP was added to the medium. From spleens of FVA‐infected mice, a population of large, immature‐appearing erythroblasts was obtained by separation with velocity sedimentation at unit gravity. Serial studies of the separated erythroblasts which were cultured with EP showed that despite some heterogeneity in their proliferative capacity, they were relatively homogeneous in their degree of differentiation in that they had not begun to synthesize heme or globin. Morphological changes and syntheses of heme and globins were monitored during terminal differentiation induced in vitro by EP. The accumulation of immature erythroblasts in vivo, their responsiveness in vitro to EP, and availability of large numbers of cells (108 or more) make the splenic erythroblasts of FVA‐infected mice an ideal population of cells with which to study EP‐mediated terminal differentiation. This erythroblast population should permit the biochemical and molecular studies in erythroid differentiation which heretofore had to be done with chemically induced erythroid differentiation in continuous cell lines.
Comparative effects of somatomedin C and insulin on the metabolism and growth of cultured human fibroblasts
Authors: Conover Cheryl A. , Hintz Raymond L. , Rosenfeld Ron G. 
Published: February 2005

AbstractAt concentrations of 25 ng/ml in serum‐free medium, somatomedin C (SM‐C) and insulin stimulated 3H‐thymidine incorporation in adult human fibroblasts 4‐ and 1.5‐fold, respectively. The presence of 0.25% human hypopituitary serum (HHS), which by itself had little effect, enhanced the mitogenicity of both SM‐C and insulin. Furthermore, 10−7 M dexamethasone dramatically potentiated SM‐C stimulation (70‐fold) and insulin stimulation (28‐fold) of 3H‐thymidine incorporation. With dexamethasone and 0.25% HHS, significant stimulation of DNA synthesis was seen at 2.5 ng/ml for both SM‐C and insulin. The effects of SM‐C and insulin on 3H‐thymidine incorporation were additive. These 3H‐thymidine incorporation results were clearly supported by cell replication studies. On the other hand, SM‐C and insulin had equivalent, non‐additive effects on RNA and protein synthesis and protein degradation. Half‐maximal effects were seen for both peptides on all three metabolic processes at 2–5 ng/ml. In contrast to their synergism with SM‐C in the stimulation of DNA synthesis and cell replication, HHS and dexamethasone did not enhance SM‐C stimulation of RNA or protein synthesis or protein degradation. These data indicate that SM‐C and insulin stimulate DNA, RNA, and protein synthesis, protein degradation, and cell replication in adult human fibroblasts at nanomolar concentrations, suggesting that each peptide is capable of acting through its own receptor. Both SM‐C and insulin are also capable of synergism with low concentrations of serum and dexamethasone in the stimulation of DNA synthesis and cell replication. It is proposed that SM‐C and insulin both participate in the regulation of cell growth and metabolism in vivo.