bluuebunny/crossref_metadata_2025 huggingface dataset:
Transmembrane potential and intracellular potassium ion activity in fetal and maternal liver
Authors: Chapman Lisa M. , Wondergem Robert
Published: February 2005

We 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

Endothelial 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

Na+,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

Kinetic 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

The 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

Membrane 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

Splenic 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

At 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.
The regulation of mononuclear phagocyte entry into S phase by the colony stimulating factor CSF‐1
Authors: Tushinski Robert J. , Stanley E. Richard
Published: February 2005

CSF‐1 is a hemopoietic growth factor that specifically regulates the survival, proliferation, and differentiation of mononuclear phagocytic cells. Populations of adherent bone marrow‐derived macrophages (BMM) devoid of CSF‐1 producing cells were used to study regulation by CSF‐1 of macrophage entry into S phase. More than 95% of BMM possess the CSF‐1 receptor. It was shown that 93–98% of BMM are cycling (S phase 8–9 hr, doubling time 24–28 hr) when cultured in the presence of CSF‐1. BMM incubated with 15% FCS in the absence of CSF‐1 or in the presence of CSF‐1 concentrations inducing survival without proliferation enter a quiescent state. This state is characterized by a reduction in the synthesis of DNA (98%), total protein (35%), ribosomal protein (76%), and histone (96%) compared with the synthetic rate of these components in exponentially growing cells. Addition of CSF‐1 to BMM rendered quiescent by removal of CSF‐1 stimulated entry into S phase with a lag period of ∼12 h. This lag period is reduced to 8 hr in BMM made quiescent at concentrations of CSF‐1 inducing survival without proliferation, an effect which may be related to the expected higher protein content of these cells (Tushinski and Stanley, J. Cell. Physiol., 116:67–75). Neutralization of CSF‐1 by antibody at different times during the lag period indicates that CSF‐1 is required for almost the entire lag period for the entry of any cells into S phase. In BMM rendered quiescent by removal of both serum and CSF‐1, purified CSF‐1 without serum stimulated entry of cells into S phase, whereas serum alone was ineffective. The results are consistent with a primary regulatory role of CSF‐1 in mononuclear phagocyte proliferation, survival, and function.
Synergism between hemopoietic growth factors (HGFs) detected by their effects on cells bearing receptors for a lineage specific HGF: Assay of hemopoietin‐1
Authors: Bartelmez S. H. , Stanley E. Richard
Published: February 2005

The preceding paper describes a new approach to the detection and assay of growth factors for developmentally early multipotent hemopoietic cells (Bartelmez et al., J. Cell. Physiol., 1985). This approach, involving measurement of the increase in the number of receptors for the mononuclear phagocyte specific hemopoietic growth factor (HGF), colony stimulating factor‐1 (CSF‐1), in cultures of developmentally early murine cells incubated with putative HGFs, has been used to define and assay hemopoietin‐1. Hemopoietin‐1 (Mr ∼ 20,000) is found in the medium derived from serum‐free cultures of cells of the human urinary bladder carcinoma line 5637. In contrast to both hemopoietin‐2 and CSF‐1, which also stimulate an increase in CSF‐1 receptor numbers in cultures of developmentally early hemopoietic cells, hemopoietin‐1 alone has no detectable effect. However, hemopoietin‐1 exhibits dramatic synergism wth CSF‐1. In the presence of CSF‐1, hemopoietin‐1 stimulates the proliferation of developmentally earlier cells than those that respond to either CSF‐1 alone or hemopoietin‐2 alone or their combination. These cells proliferate for at least 3 days with no alteration of the average CSF‐1 receptor density. However, by 5 days of incubation, the progeny of developmentally early hemopoietic cells that have proliferated in response to hemopoietin‐1 + CSF‐1 exhibit an approximately tenfold increase in the average CSF‐1 receptor density per cell, which immediately precedes their differentiation to adherent mononuclear phagocytes. As hemopoietin‐1 does not possess colony stimulating or burst promoting activities for murine bone marrow cells, but acts on multipotent hemopoietic cells, the analysis of the mechanism of its synergistic effects with HGFs such as CSF‐1 are of special relevance to the regulation of early events in hemopoiesis.
Selenium deficiency in cultured adrenocortical cells: Restoration of glutathione peroxidase and resistance to hydroperoxides on addition of selenium
Authors: Hornsby Peter J. , Pearson Doran W. , Autor Anne P. , Aldern Kathy A. , Harris Sandra E.
Published: February 2005

Cultured bovine adrenocortical cells were previously shown to be functionally deficient in selenium and vitamin E when grown in medium supplemented with fetal bovine serum. In the present experiments, the lack of significant bioavailable amounts of selenium in the medium was demonstrated by the finding of only low levels of glutathione peroxidase in the cultured cells (0.008 U/mg protein compared with 0.045 U/mg protein in fresh adrenocortical tissue). When 20 nM selenium as selenite was added to the cultured adrenocortical cells, glutathione peroxidase activity increased continuously over 72 h, with a total increase of about eightfold over this period. Over the same time‐course, the highest concentration of cumene hydroperoxide tolerated by the cells without cell death increased progressively from 10 μM to 50 μM. Addition of 1μM α‐tocopherol also increased the amount of cumene hydroperoxide tolerated to 50 μM. Cell death was measured by cloning efficiency after removal of cumene hydroperoxide. Addition of either selenium or α‐tocopherol had little effect on the growth rate of the cells over six passages, even when residual vitamin E was removed from the serum by extraction with ether and residual low molecular weight selenium compounds were removed by dialysis. It is concluded that combined deficiency of selenium and vitamin E, at least in the presence of other components of fetal bovine serum, has little effect on the ability of the cells to survive under normal conditions, as evidenced by continued long‐term proliferation. However, the low levels of glutathione peroxidase resulting from selenium deficiency cause an increase susceptibility to peroxide‐mediated toxicity. The combined deficiency of selenium and vitamin E impairs the ability of cells to survive under adverse conditions, as well as altering mitochondrial functions, as previously demonstrated.
Utilization and formation of amino acids by chicken epiphyseal chondrocytes: Comparative studies with cultured cells and native cartilage tissue
Authors: Ishikawa Yoshinori , Chin J. E. , Hubbard H. Lee , Wuthier Roy E.
Published: February 2005

Utilization and production of amino acids by primary cultures of chicken growth plate epiphyseal chondrocytes grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum were investigated in both short‐term (6–72 h) and long‐term (3–24 day) cultures. Comparative studies were made on levels of free amino acids in chicken blood plasma and serum, and in extracellular fluids from different regions of growth plate cartilage and from two types of muscle. Chondrocytes rapidly consumed glutamine from the medium, and to lesser extents, various other amino acids. In contrast, free ammonia, alanine, glycine, glutamate, proline, and aspartate were released into the medium. The utilization of certain amino acids changed, depending on the stage of culture. Initially glutamate was released into the medium but after confluency was consumed. Conversely, histidine, lysine, and phenylalanine were initially utilized but later were released into the medium. Levels of total free amino acids in extracellular fluids of cartilage and muscle were higher than those in plasma and serum, while in cartilage the levels increased progressively from the resting to the hypertrophic zones. In these sequential regions certain amino acids increased proportionally, whereas others decreased. These interrelationships generally correlated closely with metabolism of amino acids by the cultured chondrocytes. They indicate that significant differences in amino acid metabolism exist between tissue areas and are reflected in the extracellular fluid composition. Accordingly, adjustment of specific amino acids may optimize culture conditions, enabling more normal phenotypic expression in vitro.
Analysis of a mutant expressing temperature‐sensitive changes of cell size in Tetrahymena
Authors: Palissa Harriet , Seyfert Hans‐Martin , Cleffmann Günter
Published: February 2005

A temperature‐sensitive mutant of Tetrahymena expresses an increase in cell volume by a factor of 2.5 upon shift to restrictive temperature. Cellular amounts of protein, RNA, and DNA increase at roughly the same proportions. The mutant cell size is attained by cessation of divisions immediately after temperature shift for a period of time which is about equal to one generation time. During this time cell growth and DNA replication continue at virtually unchanged rates. Maintained at the restrictive temperature the mutant cells divide at the same rate as the wild‐type cells. Upon return to the permissive temperature, cell size is reduced by the combined effects of an accelerated division rate together with a decelerated growth rate.
Retinoids antagonize the induction of ornithine decarboxylase activity by phorbol esters and phospholipase C in rat tracheal epithelial cells
Authors: Jetten Anton M. , Shirley Jill E.
Published: February 2005

In this study we examined the action of phorbol esters, several phospholipases and retinoids on the induction of ornithine decarboxylase (ODC) activity in rat tracheal epithelial cells. 12‐O‐Tetradecanoylphorbol‐13‐acetate (TPA) induces ODC activity in these cells in a dose‐and time‐dependent manner. This induction is inhibited by cycloheximide indicating a requirement for protein synthesis. Tracheal epithelial 2C5 cells contain two binding sites for phorbol esters, one with a high affinity KD,1 = 4.58 nM and one with a low affinity KD,2 = 344.8 nM. The ability of several phorbol esters to induce ODC correlates well with the described efficacy with which they bind to the receptor and is in agreement with the concept that phorbol ester receptors are involved in the induction of ODC. There is strong evidence that the phorbol ester receptor is the protein kinase C for which diacylglycerol is the physiological ligand. Treatment of cells with phospholipase C generates diacylglycerol and induces ODC activity in a dose‐ and time‐dependent manner. Treatment with phospholipase A2 or D has no effect on ODC activity. These results support the concept that activation of protein kinase C is related to the induction of ODC activity. The induction of ODC by TPA as well as by phospholipase C is inhibited by retinoids. Specific cytosolic binding proteins for retinoids might be involved in at least some of the responses to these compounds. To examine whether the binding proteins are involved in the inhibition of ODC we determined the presence of these binding proteins and the structure‐activity relationship of retinoids. Both retinol and retinoic acid‐binding proteins can be detected in 2C5 cells, their levels are 1.06 and 3.36 pmoles/mg protein, respectively. The ability of several retinoids to inhibit ODC induction correlates well with their binding activity and support a role for these binding proteins in the action of retinoids on ODC induction.
Transport by the (Na+, K+) ATPase: Modulation by differentiation inducers and inhibition of protein synthesis in the MDCK kidney epithelial cell line
Authors: Kennedy Brian G. , Lever Julia E.
Published: February 2005

MDCK kidney epithelial cell cultures exposed to the differentiation inducer hexamethylene bisacetamid (HMBA) for 24 hours exhibited a 50% decrease in transport activity per (Na+, K+)‐ATPase molecule (turnover number) but an unchanged number of pump sites (Kennedy and Lever, 1984). Inhibition of protein synthesis by either 10 μM cycloheximide or 2 μM emetine blocked the inhibitory effects of HMBA on Na+/K+ pump efficiency assessed by measurements of [3H]‐ouabain binding to intact cells, (Na+, K+) ATPase activity of detergent‐activated cell extracts, and ouabain‐sensitive Rb+ uptake. In the absence of inducer treatment, inhibition of protein synthesis increased Na+/K+ pump turnover number by twofold while maintaining Na+/K+ pump activity per cell at a constant level. Intracellular Na+ levels were decreased after cycloheximide treatment; therefore, pump stimulation was not due to substrate effects. Furthermore, cycloheximide effects of Rb+ uptake could be dissociated from effects on tight junctions. These observations suggest that the transport activity of the (Na+, K+) ATPase is tightly regulated by factors dependent on protein synthesis.
Variable expression of Qa‐m7, Qa‐m8, and Qa‐m9 antigenic determinants on primitive hemopoietic precursor cells
Authors: Harris R. A. , Sandrin M. S. , Sutton V. R. , Hogarth P. M. , McKenzie I. F. C. , Penington D. G.
Published: February 2005

Using monoclonal antibodies, we have analysed the distribution of three recently described Qa antigenic determinants (Qa‐m7, Qa‐m8 and Qa‐m9) on murine clonable hemopoietic progenitor cells and spleen colony‐forming units (CFU‐S). Cytotoxicity experiments showed that Qa‐m7 was expressed on almost all the progenitor cells (colony‐forming cells, CFC) of megakaryocytes (MEG‐CFC), erythroid cells (E‐CFC), B lymphocytes (BL‐CFC), and mixed colonies (MIX‐CFC) as well as day 13 CFU‐S, and a major proportion of granulocyte‐macrophage colony‐forming cells (GM‐CFC) and day 8 CFU‐S. Experiments using four sources of granulocyte‐macrophage colony‐stimulating activity suggested differential expression of Qa‐m7 on subpopulations of GM‐CFC, those preferentially forming macrophage colonies having lowest Qa‐m7 antigen density. Immune rosetting techniques demonstrated the selective expression of Qa‐m8 on approximately 50% of MEG‐CFC, MIX‐CFC and day 13 CFU‐S, a pattern similar to that of Qa‐m2. In contrast, Qa‐m9 was not detected on any of the primitive hemopoietic precursors assayed. The results demonstrate the complexity of the Qa antigenic system, and suggest a possible role for these antigens in hemopoietic differentiation.
Two distinct mechanisms for the interaction of cells with fibronectin substrata
Authors: Schwarz M. A. , Juliano R. L.
Published: February 2005

Fibronectin (Fn) was adsorbed onto neutral, sulfonated, imine‐conjugated or gelatin coated polystyrene latex beads. In all cases, the Fn coated beads bound effectively to Chinese hamster ovary (CHO) cells in suspension. However, the binding of Fn coated neutral or positively charged imine conjugated bead was inhibited by low concentrations of heparin or heparan sulfate or by treatment of the cells with Flavobacterium heparanase. By contrast, binding of Fn coated sulfonated or gelatin beads was insensitive to inhibition by heparin and to heparanase treatment of cells. Adhesion of CHO cells to Fn coated tissue culture plastic was not sensitive to heparin, whereas adhesion of CHO cells to Fn‐coated imine‐conjugated plastic was sensitive to heparin. These observations imply that the functional status of Fn can be modulated by the nature of the surface to which the Fn is adsorbed. They further imply that, under some circumstances, the heparin/heparan sulfate binding domains of Fn can play a role in the attachment of Fn to the cell membrane via membrane proteoglycans. Under other circumstances, the interaction of Fn with the cell may primarily involve other receptors for Fn, presumably cell surface glycoproteins.
Production of insulin‐like growth factor‐II (MSA) by endoderm‐like cells derived from embryonal carcinoma cells: Possible mediator of embryonic cell growth
Authors: Nagarajan Lalitha , Anderson Wayne B. , Nissley S. Peter , Rechler Matthew M. , Jetten Anton M.
Published: February 2005

The present study was carried out to determine if an insulin‐like growth factor (IGF) type activity might be produced by embryonal carcinoma‐derived cells. The cell line used to condition growth medium for the isolation of secreted growth factors was a newly established Dif 5 cell type. Dif 5 cells are a differentiated endoderm‐like cell type derived from F9 embryonal carcinoma cells (which possess properties similar to mouse embryonic stem cells) following extensive exposure to retinoic acid. When growth medium conditioned by Dif 5 cells is chromatographed on Sephadex G‐75 in 1 M acetic acid two peaks of activity are observed which compete for specific [125I]iodo multiplication stimulating activity (MSA) binding to PYS cells. MSA is the rat homologue of human IGF‐II. The high molecular weight fraction (Mr ˜ 60K) apparently corresponds to IGF‐binding protein as determined by its ability to bind [125I]iodo‐MSA. The low molecular weight fraction (Mr ˜ 8K) is biologically active as this fraction stimulates [3H]thymidine incorporation into serum‐starved chick embryo fibroblasts. Radioimmunoassay data indicate that the IGF‐like activity produced by Dif 5 cells is more closely related to IGF‐II than to IGF‐I. Undifferentiated embryonal carcinoma stem cell lines (F9, Nulli, and PCC4) produced little of this MSA‐like activity, while PYS‐2 (parietal endoderm‐like) cells produced about 16 ng MSA/106 cells/24 hr as determined by radioimmunoassay. Dif 5 and PSA‐5E (visceral endoderm‐like) cells, are found to secrete significant amounts of MSA into the growth medium (30–50 ng MSA/106 cells/24 hr). These findings offer further support to a proposal that MSA (IGF‐II) produced by endoderm cells, particularly visceral endoderm, may serve as an early embryonic growth factor.
Residual inhibition of epidermal growth factor binding by pancreatic secretagogues and phorbol ester in rat pancreas
Authors: Korc Murray , Magun Bruce E.
Published: February 2005

Cholecystokinin‐octapeptide (CCK8) inhibits 125I‐labeled epidermal growth factor (EGF) cell‐associated radioactivity in pancreatic acini, ostensibly as a result of its ability to mobilize cellular Ca2+. The phorbol ester tetradecanoyl phorbol acetate (TPA), a compound that activates protein kinase C, mimics the inhibitory action of CCK8. In the present study we examined the relationship between occupancy of the cholecystokinin (CCK) receptor, the subsequent inhibition of EGF binding, and the potential role of C‐kinase activation in mediating this inhibition. Proglumide and dibutyryl cyclic GMP (dbGMP), two distinct competitive antagonists of CCK8, reversed the inhibitory actions of CCK8. Analysis of steady‐state saturation kinetics of 125I‐EGF binding indicated that CCK8 decreased the apparent affinity of the EGF receptor, mainly as a result of a marked decrease in the amount of internalized ligand. TPA also inhibited 125I‐EGF internalization. Removal of CCK8 and TPA from incubation medium did not abolish their inhibitory actions. Carbachol, but not bombesin, exerted a similar residual inhibitory effect. It is suggested that in addition to acting via Ca2+, certain pancreatic secretagogues may also act through C‐kinase to regulate EGF binding.
Phosphate uptake by primary renal proximal tubule cell cultures grown in hormonally defined medium
Authors: Waqar M. Anwar , Seto Janny , Chung Soon Dong , Hiller‐Grohol Sue , Taub Mary
Published: February 2005

The uptake of labeied inorganic phosphate into primary rabbit kidney proximal tubule cells has been examined. Phosphate was accumulated into the primary proximal tubule cells against a concentration gradient. This accumulation was sensitive to inhibition by metabolic inhibitors. The dependence of phosphate uptake on the extracellular phosphate concentration was examined. Similarities were observed between primary proximal tubule cells and the LLC‐PK1 cell line in these regards. These phosphate uptake data were then plotted on a Lineweaver‐Burke plot. A nonlinear plot was obtained, which suggested that phosphate uptake occurs by means of a Na+ dependent, carrier mediated process, as well as by another Na+ independent mechanism. The pH dependence of phosphate uptake was also examined. Unlike previous observations with LLC‐PK1 cells, optimal phosphate uptake occurred at pH 6.5. However, this difference between the two cell culture systems may possibly be explained by differences in uptake conditions. The dependence of phosphate uptake on the extracellular NaCl concentration was examined at three different pH values. The rate of phosphate uptake at pH 7.0 was observed to saturate at a lower NaCl concentration than at either pH 6.0 or pH 6.5. Furthermore, the optimal rate of phosphate uptake at pH 7.0 was observed to be higher than at the other two pH values studied when the NaCl concentration was below 120 mM. However, when the NaCl concentration was raised to 150 mM, optimal phosphate was observed to occur at pH 6.5 rather than at pH 7.0. These observations may be explained if the pH affects not only the rate of phosphate uptake but also the affinity of the phosphate uptake system for sodium. Phosphate uptake was also observed to be sensitive to several agents, Na2·SO4 and NaSCN, which affect the membrane potential. As observed with phosphate uptake by LLC‐PK1 (and renal brush border membrane vesicles), phosphate uptake was highly sensitive to inhibition by the phosphate analogue arsenate. Novel observations were that the phosphate analogue vanadate and its cellular metabolite vanadyl stimulated the initial rate of phosphate uptake.
Amiloride in differentiation and commitment of Friend erythroleukemia cells
Authors: Lubin Martin
Published: February 2005

Dimethylsulfoxide (DMSO) converts almost all of the undifferentiated murine erythroleukemia cells (MEL or Friend cells, clone 745A) in a culture to differentiated cells that contain high levels of hemoglobin and that stop growing after a limited number of cell divisions. Contrary to other reports—that amiloride strongly inhibits DMSO‐induced differentiation in MEL cells—in this laboratory, inhibition by amiloride, tested with DMSO over a range of concentrations in two kinds of media and at various cell densities, was found to be only weak or absent. Similarly, amiloride did not inhibit induction by N, N′‐hexamethylene bis‐acetamide (HMBA). As expected from previous findings with other cell systems, amiloride inhibited protein synthesis and cell multiplication.
Voltage‐sensitive cyanine dye fluorescence signals in lymphocytes: Plasma membrane and mitochondrial components
Authors: Wilson H. Alexander , Seligmann Bruce E. , Chused Thomas M.
Published: February 2005

The origin of the cyanine dye fluorescence signal in murine and human peripheral blood leukocytes was investigated using the oxa‐ and indo‐carbo‐cyanines di‐O‐C5(3). and di‐l‐C5(3). Fluorescence signals from individual cells suspended with nanomolar concentrations of the dyes were measured in a flow cytometer modified to permit simultaneous four‐parameter analysis (including two‐color fluorescence or fluorescence polarization measurements). The contributions of mitochondrial membrane potential (Ψm) and plasma membrane potential (Ψpm) to the total voltage‐sensitive fluorescence signal were found to depend on the equilibrium extracellular dye concentration, manipulated in these experiments by varying the ratio of dye to cell density. Hence, conditions could be chosen that amplified either the Ψm or the Ψpm component. Selective depolarization of lymphocytes or polymorphonuclear leukocytes (PMN) in mixed cell suspensions demonstrated that defining the partition of dye between cells and medium is requisite to assessing the heterogeneity of cell responses by cyanine dye fluorescence. At extracellular dye concentrations exceeding 5 nM in equilibrated cell suspensions, both mitochondrial and plasma membrane dye toxicity were observed. In murine splenic lymphocytes, plasma membrane toxicity (dye‐induced depolarization) was selective for the B lymphocytes. Certain problems in calibration of Ψpm with valinomycin at low dye concentrations and perturbations of Ψpm by mitochondrial inhibitors are presented. These findings address the current controversy concerning Ψm and Ψpm measurement in intact cells by cyanine dye fluorescence. The finding of selective toxicity at low cyanine dye concentrations suggest that purported differences in resting Ψm among cells or changes in Ψpm with cell activation may reflect varible susceptibility to dye toxicity rather than intrinsic cell properties.
Lymphocyte membrane potential and Ca2+‐sensitive potassium channels described by oxonol dye fluorescence measurements
Authors: Wilson H. Alexander , Chused Thomas M.
Published: February 2005

A method is described for quantitative measurement of lymphocyte transmembrane electrical potential difference (Ψ) by flow cytometric recording of the oxonol dye fluorescence of single cells. Both the simultaneous collection and analysis of multiple optical parameters and the use of a negatively charged oxonol probe allowed more accurate measurement of Ψ than may be obtained by bulk cell suspension techniques employing cationic voltage indicators. Mouse spleen and human blood lymphocyte Ψ was calculated to be −70 mV. T and B lymphocytes maintain a constant Ψ as extracellular K+ is varied from 2 to 10 mM and the deviation from K+ equilibrium potentials (Ek) is shown to result from Na+ permeability. At [K+]o values greater than 10 mM, lymphocytes behave as K+ electrodes. Examination of lymphocyte subsets showed that hyperpolarization induced by the Ca2+ ionophore A23187 occurs only in T cells. This response was identified as activation of a Ca2+‐sensitive K+ channel by pharmacologic manipulations. Hence, T cells depolarized by 4‐aminopyridine (4‐AP, 10 mM) were observed to return to resting Ψ by A23187‐induced elevation of [Ca2+]i. Cells depolarized by quinine (100 μM) were unaffected by A23187. The Ca2+‐activated channel does not contribute to resting Ψ in T cells since it may be selectively blocked by quinine (20 μM) or modulated by calmodulin antagonists (5 μM trifluperazine) without affecting resting Ψ.
Induction of metallothionein synthesis in cultured cells derived from rabbit kidney
Authors: Koizumi Shinji , Sone Toshio , Kimura Masami
Published: February 2005

Metallothionein (MT) synthesis in rabbit kidney‐derived RK‐13 cells was studied. In response to Cd2+, RK‐13 cells synthesized proteins closely similar in chromatographic and electrophoretic behaviors to the liver MTs induced in Cd2+ ‐injected rabbit. These proteins were specifically immunoprecipitated by anti‐mouse liver MT‐II serum. The rate of RK‐13 thionein (apoprotein of Mt) synthesis rapidly increased after exposure to 1μg/ml of Cd2+, and reached the maximum in 7 h. The dose‐response curve for the synthesis was biphasic; a sharp increase up to 0.5 μg/ml Cd2+ and a slower increase at higher concentrations. RK‐13 cells retained kidney‐specific properties in terms of responsiveness of thionein synthesis to inducers; The MTs were inducible also by Zn2+ and probably by Hg2+, but not by dexamethasone. This system would therefore be a useful model in vitro for studying the regulation of MT synthesis in kidney cells.
Adenosine uptake, transport, and metabolism in human erythrocytes
Authors: Plagemann Peter G. W. , Wohlhueter Robert M. , Kraupp Martin
Published: February 2005

Using rapid kinetic techniques, we have determined the kinetics of zero‐trans influx and equilibrium exchange of adenosine, and its uptake and in situ phosphorylation at 25°C in human erythrocytes which were pretreated with 2′‐deoxycoformycin to inhibit deamination of adenosine. Both the Km and Vmax for adenosine transport were about 300 times higher than those for the in situ phosphorylation of adenosine (Km about 0.2 μM), so that the first order rate constants for both processes were about the same. In contrast, the first order rate constant for adenosine deamination by untreated, intact cells was about 20% of that of adenosine transport or phosphorylation. These kinetic properties of the various steps, in combination with substrate inhibition of adenosine phosphorylation above 1 μM adenosine, assure that, at extracellular concentrations of physiological relevance ( < 1 μM), adenosine is very rapidly and efficiently salvaged by the erythrocytes and converted to ATP, whereas at extracellular concentrations of 10 μM or higher, practically all adenosine transported into the cells is deaminated. When the concentration of adenosine was 0.1 μM, a 10% (v/v) suspension of erythrocytes depleted the extracellular fluid of adenosine within 1 min of incubation at 25°C.
Growth requirements and characterization of rat cervical epithelial cells in culture
Authors: Wright Thomas C. , Nahabedian Christine
Published: February 2005

The extended culture of rat cervical epithelial cells can be achieved in the absence of a fibroblast feeder layer by utilizing collagen gels and a complex growth medium. The medium contains a 1:1 mixture of RPMI‐1640 and Ham's F12 supplemented with 7.5% porcine serum and epidermal growth factor, cholera toxin, transferrin, insulin, and hydrocortisone, Under these culture conditions the cells show rapid log‐phase growth and high saturation densities while retaining the ultrastructural characteristics of immature squamous metaplastic cells of the rat uterine cervix even after extended passage. In a manner similar to epithelial cells from a variety of sources, rat cervical epithelial cells form hemicysts at confluence in vitro when cultured on impermeable substrates. The development of these methods for culturing cervical epithelial cells provides an experimental system for the study of factors important in regulating the growth and differentiation of metaplastic squamous epithelial cells.
Isolation and characterization of hexose transport mutants in L6 rat myoblasts
Authors: D'Amore Tony , Duronio Vincent , Cheung Matthias O. , Lo Theodore C. Y.
Published: February 2005

A method for the selection and isolation of hexose transport mutants in undifferentiated rat myoblast L6 cells is reported; 2‐deoxy‐D‐glucose (2‐DOG)‐and 2‐deoxy‐2‐fluoro‐D‐glucose (2FG)‐resistant mutants were selected after mutagenization of L6 cells with ethyl methanesulfonate. Of these, D18 and D23 (selected with 0.1 mM 2‐DOG) and F72 and F76 (selected with 0.1 mM 2FG) exhibited the lowest hexose transport activity. Uptake of 0.06 mM 2‐DOG, 2FG, or 3‐O‐methyl‐D‐glucose (3‐OMG) by mutants grown in fructose medium supplemented with 0.05 mM 2FG was about four‐ to five‐fold lower than the parental L6 cells. These mutants contain normal levels of ATP and glycolytic enzyme activities. They also exhibit normal transport activities for α‐aminoisobutyric acid and fructose. Furthermore, hexose transport was observed to be decreased in plasma membrane vesicles prepared from these mutants. Kinetic analysis of 2‐DOG and 3‐OMG transport in mutant F72 demonstrated that the Vmax for 2‐DOG uptake was significantly reduced, whereas the Vmax for 3‐OMG transport was not affected. In all cases, the affinity for these hexose analogues was unaffected. In addition mutant F72 was found to be only slightly affected by treatment with various energy inhibitors and sulfhydryl reagents. The results suggest that this mutant is defective in, or has low levels of, a plasma membrane component(s) involved in the high‐affinity hexose transport system.
Proteoglycans synthesized by gingival fibroblasts derived from human donors of different ages
Authors: Bartold P. Mark , Boyd Robin R. , Page Roy C.
Published: February 2005

The proteoglycans synthesized by fibroblasts derived from human donors of ages ranging from 12 years to 68 years have been studied. In addition, the in vitro proliferation rates of the various cell strains were studied and demonstrated that increasing donor age correlated with a decrease in proliferative activity. The incorporation of [35S]‐sulfate into proteoglycans decreased with increasing donor age with cells from the oldest donor demonstrating a 50% reduction compared with cells from the youngest donor. Analysis on Sepharose CL‐4B of isolated [35S]‐labeled proteoglycans for molecular size distribution revealed few differences between the cell‐layer‐associated proteoglycans of all cell strains studied. However, analysis of the medium‐associated [35S]‐labeled proteoglycans demonstrated an increase in the amount of small molecular size proteoglycans with increasing age. More specific analysis of the glycosaminoglycan composition revealed an increase in heparan sulfate from 52% to 73% in the cell‐layer‐associated proteoglycans of cells from the youngest and oldest donors, respectively. Accompanying this increase was a relative decrease in dermatan and chondroitin sulfate content from 24% to 13% and 25% to 16%, respectively, with increasing donor age. Additionally, the degree of N‐sulfation of cell layer heparan sulfate increased with age. Heparan sulfate levels increased in the medium as well with increasing age, with a concomitant decrease in chondroitin sulfate. The quantity of medium‐derived dermatan sulfate remained relatively evenly distributed throughout the various ages studied. The various differences noted are considered to reflect the general metabolic changes associated with aging. In particular the increase in heparan sulfate content with age is considered to be related to the decreased proliferative activity of the fibroblasts with increasing age.
Studies of DNA polymerases alpha and beta from cultured human cells in various replicative states
Authors: Krauss Sharon Wald , Linn Stuart
Published: February 2005

DNA polymerase activities from HeLa cells and from cultured diploid human fibroblasts in various growth states were compared. α‐Polymerase activities from log phase fibroblasts treated with sodium butyrate and from stationary phase HeLa cells had DEAE‐cellulose elution patterns that differed from those of polymerases from dividing cells. Moreover, α‐ and β‐polymerases from nondividing cells replicated synthetic polymers less faithfully. Although similar changes were observed previously for polymerases from late‐passage and postconfluent early passage fibroblasts, amounts of α‐polymerase activity recovered from nondividing cells in this study did not dramatically decline as they had in the former cases. The α‐polymerase activities from HeLa cells and fibroblasts in various growth states sedimented near 7.5S in 0.4 M KCl and could be inhibited by a monoclonal lgG fraction prepared against KB cell α‐polymerase. By several criteria, there was no significant differences in levels of UV‐stimulated repair synthesis observed in early or late‐passage postconfluent fibroblasts or in log phase fibroblasts treated with sodium butyrate. In summary, levels of α‐polymerase do not necessarily correlate either with replicative activity or with apparent levels of repair synthesis. However, cells with decreased replicative activity always yielded enzyme with decreased fidelity in vitro and altered chromatographic behavior. It appears, therefore, that the alterations observed for α‐polymerase from late‐passage cells may be attributed more generally to the nondividing nature of these cells.