bluuebunny/crossref_metadata_2025 huggingface dataset:
Steroids and hematopoiesis II. The effect of steroids on in vitro erythroid colony growth: Evidence for different target cells for different classes of steroids
Authors: Singer Jack W. , Adamson John W.
Published: February 2005

Androgenic steroids and their non‐androgenic 5p‐H metabolites enhance the number of colonies of hemoglobin synthesizing cells grown from rat bone marrow in response to a standard (0.25 unitlml) concentration of erythropoietin. The target cells for two steroids were found to be different. Cells influenced by the androgen, fluoxymesterone (fluoxy), resembled cells responding to erythropoietin in their cycle characteristics, as measured by tritiated thymidine suicide, and in their physical characteristics, as determined by velocity sedimentation gradient separation. Cells responding to etiocholanolone (etio) had a much lower tritiated thymidine suicide rate and different sedimentation velocities. Reincubation of marrow cells with etio for two hours was sufficient to enhance erythroid colony growth by 84%, whereas a similar incubation with fluoxy produced no increment. These studies demonstrate that different classes of steroids may influence in vitro erythropoiesis by acting on distinct populations of marrow cells. Fluoxymesterone appears to act through cells already committed to respond to erythropoietin, while etiocholanolone appears to act on a separate, perhaps more primitive population of marrow cells.
Electrical properties of developing oocytes of the migratory locust, Locusta migratoria
Authors: Wollberg Zvi , Cohen Ephraim , Kalina Moshe
Published: February 2005

The electrical properties of developing nonfertilized oocytes of Locusta migratoria were studied, using intracellular microelectrodes. The inseries potential of the combined oomembrane and of the follicular cells was about 20 mV in the youngest oocytes. It increased as the oocytes developed and it reached a plateau of about 50 mV before full maturation, generally four to seven oocytes away from the fully‐developed terminal oocyte. Current‐voltage relations were always linear for hyperpolarizing currents. Most oocytes exhibited, however, rectification to outward current. Input resistance values varied with oocyte size from about 5 × 106 ohm for young oocytes to about 0.2 × 106 ohm for the more developed ones. Some oocytes displayed a transient depolarization on turning off a hyperpolarizing step of current. This depolarization was not correlated with the size of the oocyte or with any observed morphological feature. Any two adjacent oocytes were electrotonically coupled. A single ovariole thus represented a longitudinal chain of developing oocytes which were connected electrically. This was supported by electron microscope observations which revealed junctions partially impermeable to lanthanum and gap junctions between the follicular cells themselves and between follicular cells and oocytes. The coupling coefficient was dependent on the direction of current flow. The attenuation of voltage along an ovariole was always greater at the distal than at the proximal side.
The transport of chloride in ehrlich ascites tumor cells
Authors: Levinson Charles , Villereal Mitchel L.
Published: February 2005

The steady state transport and distribution of chloride between the intracellular and extracellular phases was investigated when the extracellular chloride concentration was varied by isosmotic replacement with nitrate, bromide and acetate. The results of these experiments show that chloride transport, measured by uptake of 36Cl, is sensitive to the replacement anion. In the presence of nitrate, chloride transport is a linear function of the extracellular chloride concentration. The relationship between chloride transport and extracellular chloride in the presence of bromide is concave upward which suggests that this anion inhibits chloride movement. However, when acetate replaces chloride, the relationship between chloride transport and extracellular chloride is concave downward. The chloride distribution ratio of cells incubated in 145–155 mM chloride medium is 0.386 and is not effected by the replacement of chloride with nitrate, bromide or acetate.

These findings are consistent with the assertion that chloride transport is composed of two parallel pathways, a diffusional plus a saturating, mediated component. Of the total chloride flux (9.1 mmoles Cl‐/kg dry weight per minute) measured in chloride medium (145–155 mM Cl ‐), the mediated component represents 40% and the diffusional component 60%.
Effect of 5‐bromodeoxyuridine on the appearance of cytoplasmic poly‐A containing RNA
Authors: Pawlowski Philip J.
Published: February 2005

Cytoplasmic poly‐A containing RNA, synthesized by cultured chick embryo cells, was examined during growth in 5‐bromodeoxyuridine. The kinetics of 3H‐adenosine incorporation into this species of RNA, when compared to the rest of the cytoplasmic RNA, and to control cells, indicates that the rate of synthesis of this RNA is slower in BrdU treated cells. An examination of the rate at which a steady state distribution of radioactivity, between the poly‐A segment and non‐poly‐A portion of poly‐A containing RNA is reached also indicated that this species is synthesized at a lower rate in BrdU treated cells.
Growth abnormalities of cultured human skin fibroblasts derived from individuals with hereditary adenomatosis of the colon and rectum
Authors: Pfeffer Lawrence , Lipkin Martin , Stutman Osias , Kopelovich Levy
Published: February 2005

A heritable propensity to develop malignant lesions is found in individuals with familial adenomatosis of the colon and rectum (ACR) and the Gardner's syndrome variant, an autosomal dominant trait. In the present study, the growth characteristics of cultured skin fibroblasts (SF) derived from normal‐appearing flat skin biopsies of ACR families, representing all phenotypes, and appropriate controls were investigated. SF were obtained from stocks between the second and fifth passages and grown to confluency in Eagle's Minimal Essential Medium (EMEM) supplemented with 15% fetal calf serum (FCS). Following trypsinization, cells were replanted in EMEM supplemented with either 1% or 15% FCS at an initial density of 4 × 103 cells/cm2 and counted daily for five days. Normal SF representing several age groups (both sexes) and those obtained from non‐afflicted individuals of ACR families grew only in 15% FCS. In contrast, SF from ACR subjects and from embryonal skin grew both in 1% and 15% FCS. SF from several clinically asymptomatic adults, children of ACR patients, grew in 1% FCS as well. Cell cultures from ACR individuals showed regions of criss‐crossed arrays and multilayered pattern. These growth properties were not observed in normal cell cultures. The SF from ACR individuals did not grow in methocel, nor did they form tumors in athymic mice. These results suggest the occurrence of previously undetected biochemical alterations in SF taken from ACR genotypes.
Effect of ESF preparations on different types of erythroblasts
Authors: Hegemann F. , Döurmer P.
Published: February 2005

A new method of quantitative 14C‐autoradiography was applied for evaluating possible effects of erythropoietin (ESF) on the DNA synthesis rate of differentiated erythroid murine bone marrow cells identified as proerythroblasts, basophilic and polychromatic erythroblasts. Eosinophilic myelocytes were used as a control cell population. ESF was prepared from the urine of a patient with chronic aplastic anemia; an inactive urinary preparation served as control. The potency of the preparations was estimated by the 59Fe‐incorporation assay. The materials to be tested were injected into polycythemic mice 4, 8 and 16 hours before in vitro short‐term incubation of the bone marrow cells with 14C‐thymidine and Methotrexate. Animals without test material were taken as additional controls. Autoradiographic grains were counted by an incident light microscope photometer.

Eight hours after injection of ESF a significant increase in the mean 14C‐thymidine incorporation was found in all three erythroid cell types when compared either with the inactive control preparation (excess incorporation 30–40%) or with the untreated control animals (excess incorporation 10–20%). It could be shown that the increase is due to an immediate action of ESF on already differentiated cells and cannot –‐ at least not solely –‐ be attributed to its action on hemopoietic stem cells. The control preparation which was inactive in terms of 59Fe incorporation exerted a slight inhibition of DNA synthesis rate in all erythroid cells as well as in cells not committed to erythroid differentiation.
Argon laser microirradiation of mitochondria in rat myocardial cells in tissue culture. VII. Fibrillation in ventricle and auricle cells
Authors: Waymire Katrina , Kitzes Margarita , Meredith Stephanie , Twiggs Gary , Berns Michael W.
Published: February 2005

Rat myocardial cells in vitro were irradiated in individual mitochondria with an argon ion laser microbeam. The contractile response termed fibrillation in single and multicellular groups of both ventricle and auricle cells were compared. Specific correlations were made between fibrillation duration, the number of cells in the group, and the number of times the cells had fibrillated. Correlations were also made for the number of laser shots needed to induce fibrillation and the number of cells in the group. Another set of correlations were made between the pre‐irradiation beat frequency and the beat frequency following recovery. Several differences and similarities of the above parameters were detected between auricle and ventricle cells. A comparison of the morphology and ultrastructure of auricle and ventricle cells also revealed significant differences.
Endotoxin‐induced size change in bone marrow progenitors of granulocytes and macrophages
Authors: Metcalf D. , Wilson J. W.
Published: February 2005

Injection of 5 μg endotoxin to adult C57BL mice caused a marked increase in the sedimentation velocity of granulocytic and macrophage progenitor (colony‐forming) cells in the bone marrow. This change was maximal two days after injection and was not accompanied by corresponding changes in total marrow nucleated cell populations. The endotoxin‐induced shift was not dependent on the presence of the thymus but did not occur in mice challenged after preinjection with endotoxin. No changes in buoyant density, cell cycle status, pattern of differentiation and responsiveness of granulocytic and macrophage progenitor cells were observed after the injection of endotoxin. The increased sedimentation velocity of progenitor cells appears to indicate an increase in cell volume but the mechanisms involved have not been identified.
Potassium transport in normal and transformed mouse 3T3 cells
Authors: Spaggiare Santa , Wallach Michael J. , Tupper Joseph T.
Published: February 2005

The components of unidirectional K influx and efflux have been investigated in the 3T3 cell and the SV40 transformed 3T3 cell in exponential and stationary growth phase. Over the cell densities used for transport experiments the 3T3 cell goes from exponential growth to density dependent inhibition of growth (4 × 104 to 4 × 105 cell cm−2) whereas the SV40 3T3 maintains exponential or near exponential growth (4 × 104 to 1 × 106 cell cm−2). In agreement with previous observations, volume per cell and mg protein per cell decrease with increasing cell density. Thus, transport measurements have been expressed on a per volume basis. Total unidirectional K influx and efflux in the 3T3 cell is approximately double that of the SV40 3T3 cell at all cell densities investigated. Both cell types have similar volumes initially and show similar decreases with increasing cell density. Thus, in this clone of the 3T3 cell SV40 transformation specifically decreases unidirectional K flux. The magnitude of the total K flux does not change substantially for either cell line during transition from sparse to dense cultures. However, the components of the K transport undergo distinct changes. Both cell lines possess a ouabain sensitive component of K influx, presumably representing the active inward K pump. Both also possess components of K influx and efflux sensitive to furosemide. The data suggest this component represents a one‐for‐one K exchange mechanism. The fraction of K influx mediated by the ouabain sensitive component is reduced to one half its value when exponential versus density inhibited 3T3 cells are compared (63% versus 31% of total influx). No comparable drop occurs in the SV40 3T3 cell at equivalent cell densities (64% versus 56% of total influx). Thus, the pump mediated component of K influx would appear to be correlated with growth. In contrast, the furosemide sensitive component represents approximately 20% of the total unidirectional K influx and efflux in both cell lines in sparse culture. At high cell densities, where growth inhibition occurs in the 3T3 cell but not the SV40 3T3, the furosemide sensitive component doubles in both cell lines. Thus, the apparent K‐K exchange mechanism is density dependent rather than growth dependent.
Carbohydrate uptake by escherichia coli
Authors: Kornberg Hans L.
Published: February 2005

In contrast to active transport, the uptake of carbohydrates via the phosphoenolpyruvate‐dependent phosphotransferase system (PTS) leads to the appearance in the cell of the sugar initially as a 1‐ or 6‐ phosphate ester. The components of the PTS that transfer phosphate to the sugar are not absolutely specific for any one sugar. Both their synthesis and their activity are controlled; in the latter, “fine” control, glucose‐6‐phosphate appears to play an important role. Studies of growth on, and uptake of, galactose by E.coli mutants devoid of components of the PTS and also devoid of active transport systems for galactose, suggest that proteins effecting facilitated diffusion of hexoses may be part of, or be closely associated with, the sugar‐specific components of the PTS.
Regulation of amino acid and glucose transport activity expressed in isolated membranes from untransformed and SV 40‐transformed mouse fibroblasts
Authors: Lever Julia E.
Published: February 2005

Membrane vesicles isolated from untransformed Balb/c and Swiss mouse fibroblasts and their SV 40‐transformed derivatives were shown to catalyze carrier‐mediated, intravesicular uptake of α‐aminoisobutyric acid and D‐glucose. Concentrative uptake of α‐aminoisobutyric acid required the presence of a Na+‐gradient (external >internal) and could occur independently of endogenous (Na+ + K+)ATPase activity. A K+ diffusion gradient (internal > external) in the presence of valinomycin, or the addition of the Na+ salt of a highly permeant anion, conditions expected to create an interior‐negative membrane potential, stimulated Na+‐gradient‐dependent uptake, suggesting this process is electrogenic. D‐Glucose uptake was nonconcentrative and did not require ion gradients or metabolic conversion.

Na+‐gradient‐dependent transport of α‐aminoisobutyric acid was reduced both in initial rate and extent of uptake in vesicles from confluent untransformed cells and increased in those from SV 40‐transformed cells, compared with activities observed in vesicles from proliferating untransformed cells. No changes in D‐glucose carrier activity were observed when assayed at low glucose concentrations.
Amino acid transport by membrane vesicles of virally transformed and nontransformed cells: Effects of sodium gradient and cell density
Authors: Parnes Jane R. , Garvey Thomas Q. , Isselbacher Kurt J.
Published: February 2005

Mixed membrane vesicle populations composed of plasma membrane and endoplasmic reticulum were prepared from Balb/c 3T3 and simian virus 40‐transformed Balb/c 3T3 mouse fibroblasts. The initial rates of uptake of L‐leucine and α‐aminoisobutyric acid by these vesicles were stimulated by a NaCl gradient (external > internal). Cation specificity for stimulation of L‐leucine uptake was Na+ > Li+ > K+. NaSCN was as effective as NaCl. Stimulation of uptake of both amino acids by a NaCl gradient was twice as great in vesicles from transformed as compared to non‐transformed cells. The NaCl gradient produced transient accumulation of both L‐leucine and α‐aminoisobutyric acid to twice the equilibrium level in vesicles from transformed cells. No such “overshoot” was observed in vesicles from nontransformed cells. In vesicles from the contact‐inhibitable Balb/c 3T3 cells, transport of α‐aminoisobutyric acid, but not L‐leucine, exhibited a density‐dependent decrease in Na+ gradient induced stimulation, from 248% for sub‐confluent to 109% with confluent cells. No density‐related changes in uptake were noted with vesicles from the transformed cells. These studies suggest that variations in amino acid uptake associated with viral transformation may be related, at least in part, to alterations in Na+ permeability of the surface membrane.
Uptake of amino acids in reconstituted vesicles derived from plasma membranes of ehrlich ascites cells
Authors: Johnstone R. M. , Bardin Claudette
Published: February 2005

To obtain a clearer concept of the mechanism of organic solute transport in mammalian cells, we have attempted to reconstitute a functional transport system for amino acids from plasma membranes of Ehrlich ascites cells. Purified plasma membranes were dissolved in 2% Na cholate—4 M urea, a mixture which brought over 85% of the membrane proteins into solution. After centrifugation of the solubilized material for 2 hrs at 100,000 × g, the supernatant was dialyzed in the cold for 20 hrs with additional lipid. The reformed vesicles were tested for the ability to transport amino acids. The preliminary results obtained show that the uptake of α‐aminoisobutyric acid can be inhibited by L‐methionine and much less by L‐leucine as would be predicted from the known properties of α‐aminoisobutyrate transport in the intact cells. In addition, it has been possible to show accelerated efflux of intravesicular phenylalanine when phenylalanine is added to the trans side (medium side). The data are consistent with the conclusion that there is carrier mediated transport in the reconstituted vesicles.
Nature of lectin‐induced alteration of potassium transfer in Ehrlich ascites tumor cells
Authors: Aull Felice , Nachbar Martin S. , Oppenheim Joel D.
Published: February 2005

The way in which the lectins concanavalin A (Con A) and Ricinus communis agglutinin (Ricin) alter the K+ content of Ehrlich ascites tumor cells was investigated. Unidirectional and net fluxes were determined in unwashed cells during a time course following lectin addition. Total influx, ouabain sensitive influx, Mg++‐ and Na+‐K+‐ATPase activity were all unaffected. Cell ATP content was normal for at least 19 minutes after exposure to Con A. Early after contact with Ricin or Con A efflux was stimulated 2‐3‐fold, resulting in net K+ loss, but after 20 minutes efflux had returned to normal. Ricin and Con A acted similarly although Ricin was present at only 1/50 the concentration of Con A. When the findings are evaluated together with previous work it is suggested that a particular membrane glycoprotein may be concerned in the efflux alteration observed.
Studies on intercellular adhesion. II. Promoting effect of serum and proteins on adhesion of neural retina cells to isotypic aggregates
Authors: Pessac Bernard , Alliot Françoise , Cornet Monique , Girard Arlette
Published: February 2005

The quantitative assay described in the preceding paper (Pessac et al., 1977) was used to study the effects of serum and various proteins on isotypic adhesion of chick embryo neural retina cells. Fetal bovine, chicken, horse, rabbit and human sera promoted cell adhesion to the same extent. The same sera also enhanced isotypic adhesion of cells from other organs showing that the cell adhesion promoting activity of sera was not organ specific. Neural retina (NR) cell collection in serum supplemented medium was not modified by protein synthesis or metabolic inhibitors and was temperature dependent with a maximum at 38°C. The higher temperature does not seem to be required for repair of the cell surface after dissociation, but for the process of adhesion itself. Various serum fractions and egg albumin showed a cell adhesion promoting activity similar to that of sera.
Glycoprotein synthesis in a temperature‐sensitive Chinese hamster cell cycle mutant
Authors: Tenner A. , Zieg J. , Scheffler I. E.
Published: February 2005

A temperature‐sensitive mutant of Chinese hamster cells is described which has two interesting properties: (1) it is a cell cycle mutant and (2) glycoprotein synthesis appears to be affected at the non‐permissive temperature (40°C). Synchronized cells shifted to 40°C in the beginning of their G1 phase do not incorporate [3H]‐thymidine into DNA during the expected S‐phase, but once DNA synthesis has been initiated (∼ 10 hours after termination of serum starvation) a shift to 40°C no longer leads to an arrest of DNA synthesis. Flow microfluorimetric analysis of DNA content/cell supports this conclusion and indicates that a majority of cells become arrested in the G1 phase of the cell cycle when a non‐synchronized population of cells is transferred to 40°C. Apparently at all times in the cell cycle there is a drastic reduction of incorporation of labeled sugars (particularly fucose) into glycoproteins. The uptake of fucose and its conversion to GDP‐fucose appears to be normal at 40°C. Chromatographic analysis indicates that all classes of glycoproteins are affected, and we do not find any evidence for partially completed oligosaccharides at 40°C. Overall protein synthesis is not reduced at the nonpermissive temperature during the time interval under consideration and the number of polysomes attached to membranes (RER) is also normal at 40°C. This suggests that the defect is at an early step in the synthesis or regulation of synthesis of glycoproteins. The mutation is a recessive mutation in hybrid cells and mutagen induced revertants can be obtained which grow normally at 40°C and in which glycoprotein synthesis at 40°C is restored to normal, wild type levels.
Rapid decline in thymidine kinase activity during differentiation of Naegleria
Authors: Bols N. C. , Corff Sondra , Yuyama Shuhei
Published: February 2005

A logarithmically growing population of Naegleria gruberi differentiate into flagellates synchronously in about 85 to 90 minutes after the stimulation. Using this system, two separate enzymes that phosphorylate thymidine were assayed. Thymidine kinase activity decreases rapidly after the stimulation, while nucleoside phosphotransferase activity remains approximately constant. The possibility that this observed decrease in thymidine kinase activity is due to an expanded thymidine pool or to an increased phosphatase activity has been ruled out. Mixing experiments indicated that there is neither an activator of thymidine kinase in growing cells nor an inhibitor in differentiating cells. Thus, the observed decrease is likely to be due to an inactivation of the enzyme. Addition of actinomycin D or cycloheximide does not prevent the decrease in thymidine kinase activity suggesting that neither transcription nor translation is involved in the putative inactivation of thymidine kinase enzyme. There is very good correlation between the degree (or rate) of decrease in thymidine kinase activity and the percent of cells differentiating into flagellates. Thus, it appears certain that the residual thymidine kinase activity is primarily due to some portion of cells which did not respond to the stimulation and did not differentiate. Since there is an early termination of nuclear DNA synthesis in differentiating Naegleria, the decrease in thymidine activity (but no change in nucleoside phosphotransferase) suggests a correlation between thymidine kinase and DNA synthesis in Naegleria.
Action of FdUrd and dCyd on the incorporation of brdurd in Chinese hamster somatic cell DNA and the isolation of auxotrophic mutants
Authors: Labrecque Romain , Thirion Jean‐Paul
Published: February 2005

Chinese hamster somatic cells grown in the presence of bromodeoxyuridine, deoxycytidine and fluorodeoxyuridine incorporate more bromodeoxyuridine in their DNA than cells grown in the presence of bromodeoxyuridine alone. Thus they become more sensitive to light irradiation. Our data suggest that 0.05 mM–0.2 mM bromodeoxyuridine, 0.05 mM deoxycytidine and 10 mμg/ml fluorodeoxyuridine is one of the best possible combinations for the selection of Chinese hamster somatic cells mutants. Auxotrophs for proline, inositol or both were thus isolated at high frequency.
Altered morphology and increased cell adhesiveness of Chinese hamster ovary cells cultured on fibrin
Authors: Nozawa Ryushi T.
Published: February 2005

Chinese hamster ovary cells cultivated on fibrin exhibited different characteristics from cells growing on plastic.

While sparsely plated cells on plastic dishes had an epithelioid morphology, cells on fibrin assumed a round shape and then converted to a stretched form with protruded processes that increased with cell density. Within a few days, cells fibrinolysed adjacent fibrin and returned to the morphology seen in plastic dishes. When fibrinolysis was inhibited by ϵ‐aminocaproic acid (EACA), cells continued to grow on the fibrin for a longer period and showed dense, criss‐crossed fibroblast‐type congestion. Whereas, cells on plastic maintained pavement‐like epithelioid appearance when they grew to a confluent monolayer.

The other altered characteristics on fibrin was increased accumulation of cells in multilayers. Normally as Chinese hamster cells on plastic proliferate, many cells float into the medium instead of piling up after they form a monolayer. On the other hand, cells on fibrin, being maintained by the addition of EACA, remained adherent, piling up multilayers instead of floating into the medium. A possible explanation of these findings is that the surface properties of the stretched cells on fibrin are altered to make them more adhesive.

A possible link of these characteristics of the cells on fibrin to tumor cell behavior in vivo is discussed.
Early and late volume changes during erythroid differentiation of cultured friend leukemic cells
Authors: Loritz Frank , Bernstein Alan , Miller Richard G.
Published: February 2005

Friend erythroleukemic cells (FLC) can be induced to differentiate in vitro by addition of dimethylsulfoxide (DMSO). We have studied the kinetics of induction by measuring cell volume, volume coefficient of variation and cell doubling time. Two distinct volume changes (early and late) are observed after the addition of the inducing agent. The early change occurs after ten hours and consists of a 10–20% decrease in volume compared to an untreated control population. This shift persists for two days and its magnitude is proportional to both the concentration of DMSO and the number of differentiated cells seen on day 5. FLC lines which induce weakly or not at all with DMSO exhibit a reduced or absent early volume shift. Inclusion of a local anaesthetic in the culture prevents the appearance of differentiated cells and also counteracts the early volume shift. The exact time of the early volume change is a function of cell growth rate and appears to be cell cycle related. Synchronized cell populations exposed to DMSO during G2 and S phase undergo one round of mitosis before expression of the volume change whereas cells in G2‐M express the change only after a second mitosis.

A later, more gradual decrease in volume is observed in those cultures which begin to produce hemoglobin. It occurs after approximately five doubling times and coincides with the first appearance of hemoglobin‐containing cells. Volume distribution parameters indicate that only a proportion of the population becomes smaller in size.
Microfluorometric analysis of cellular DNA following incorporation of bromodeoxyuridine
Authors: Swartzendruber Douglas E.
Published: February 2005

Bromodeoxyuridine (BrdU) incorporation into cellular DNA has a differential effect on the cell‐associated fluorescence of several DNA‐specific dyes. After cells were treated with BrdU, flow microfluorometry was used to study the relative increase or decrease in fluorescence of stained cells. Bromodeoxyuridine incorporation into CHO cells increased the fluorescence of mithramycin‐, olivomycin‐, or chromomycin‐stained cells, decreased that of propidium iodide‐stained cells, and had little, if any, effect on the fluorescence of acriflavine Feulgen‐stained cells. Changes in relative fluorescence of cell‐associated dyes are due to changes in the amounts of dye bound to cells with BrdU‐substituted DNA. Colorimetric and absorbance measurement of DNA content showed that BrdU does not alter the diploid DNA content of CHO cells; however, BrdU induces perturbations in the distribution of cells about the cell cycle which cause an increase in average DNA content.
Sulphonated polystyrene as an optimal substratum for the adhesion and spreading of mesenchymal cells in monovalent and divalent saline solutions
Authors: Maroudas N. G.
Published: February 2005

Cell adhesion and spreading were studied on sulphonated polystyrene dishes in serum‐free saline (Mn, Na, Cl, buffer) i.e., without an intervening protein layer. Spreading as a function of surface charge density, SCD, peaked around 2–10 negative charges per square nanometer, corresponding to a monomolecular layer of sulphonate ions. At optimal SCD, macrophages, BHK‐C13 and whole mouse embryo secondary cells all showed considerable spreading, even in monovalent saline–more so than on a conventional tissue‐culture surface. But outside this narrow range of SCD, or on protein‐coated surfaces, the divalent cation was indispensable.

The biphasic effect of sulphonation on cell adhesion is consistent with the theory that a substratum need not be biochemically specific, provided it is physicochemically polar, rigid and dense. According to this theory, polystyrene of sub‐optimal SCD would not be sufficiently polar, while supra‐optimal sulphonation would produce a hydrogel surface, lacking in local rigidity and density, due to osmotic swelling. The principle of polymer exclusion, by a surface hydrogel layer, is also consistent with observations on the inhibitory effects of adsorbed proteins–viz., albumin, collagen, serum and cellular exudate, respectively–contrasted with the ready attachment of cells to a bare, optimally charged substratum, in this minimal in vitro system.
Cell density‐dependent secretion of plasminogen activator by 3T3 cells
Authors: Chou Iih‐Nan , O'Donnell Sara P. , Black Paul H. , Roblin Richard O.
Published: February 2005

The expression of extracellular fibrinolytic activity in untransformed 3T3 cell cultures depends on the growth state of the cells. Actively growing 3T3 cultures exhibit a relatively high level of fibrinolysis, which decreases progressively as the cells become confluent and density‐inhibited. The low level of fibrinolytic activity in confluent 3T3 cultures is due to a diminution in secretion of plasminogen activator since the intracellular level of plasminogen activator remains high. The amount of plasminogen activator observed in growing 3T3 cultures varies depending upon whether the cells are passaged with trypsin/EDTA solution, or with the Ca++ selective chelating agent, ethylene‐bis (oxyethylenenitrilo) tetraacetic acid (EGTA). However, in cells passaged using either agent, the amount of plasminogen activator secreted is always greatest when the cells are actively growing and decreases thereafter. In contrast to confluent 3T3 cultures, dense cultures of SV40‐virus transformed 3T3 cells continued to secrete relatively large amounts of plasminogen activator. The ability to decrease secretion of plasminogen activator as cells become dense may be an important characteristic of cells which demonstrate density‐dependent inhibition of cell multiplication in vitro.
Transport enhancement and reversal: Glucose and 3‐O‐methyl glucose
Authors: Musliner Thomas A. , Chrousos George P. , Amos Harold
Published: February 2005

In chick embryo fibroblast cultures the 15‐ to 30‐fold enhancement of D‐glucose uptake observed when cells are starved of glucose for 24 hours is not duplicated for derivatives of glucose that compete effectively for uptake and have generally been considered to use the same carrier. 2‐deoxy‐D‐glucose, D‐mannose, D‐galactose and D‐glucosamine are derepressed progressively less sharply in that order with glucosamine uptake never more than doubled by starvation. D‐glucose at a concentration of 5.5 mM in the 24‐hour conditioning medium is a strong “repressor” resulting in low “transport” behavior for each of the five sugars cited. D‐glucosamine is equally effective at the same concentration. A 10‐fold reduction in the concentration of glucosamine (0.55 mM) allows for the escape from repression of mannose, glucose, and deoxyglucose uptake while the others remain repressed. Mannose uptake escapes as well when the glucose concentration in the “conditioning” medium is similarly reduced.

Under certain conditions of starvation and cell density dramatic effects of supplemental stimulation by insulin can be achieved. Insulin withdrawal interrupts the supplemental stimulation process. Cycloheximide, actinomycin D and cordycepin block both non‐insulin and insulin‐induced derepression. Short exposure (15–30 minutes) of 24‐hour starved cells to glucose (5.5 mM) reduces glucose sharply but does not affect 3‐O‐methyl glucose uptake. If the exposure is to 2‐deoxyglucose (5.5 mM) further derepression of glucose uptake results.
Regulation of intracellular calcium in chick embryo fibroblast: Calcium uptake by the microsomal fraction
Authors: Moore Leon , Pastan Ira
Published: February 2005

The total membrane fraction of a chick embryo fibroblast (CEF) homogenate accumulates calcium in an energy‐dependent manner. This activity can be dissociated into azide‐sensitive and azide‐insensitive components. The azide‐sensitive component of calcium uptake is believed to represent mitochondrial calcium uptake. The azide‐insensitive component of calcium uptake is enhanced by the presence of a calcium trapping agent such as oxalate, and cannot utilize, ADP, inorganic phosphate and a Krebs cycle substrate to support uptake. The distribution of the azide‐insensitive calcium uptake in subcellular fractions suggests that this uptake occurs in other than mitochondrial membranes. The membranes most likely to contribute to the azide‐insensitive component of calcium uptake are the endoplasmic reticulum and plasma membrane. A microsomal preparation from CEF cells is essentially devoid of the azide‐sensitive calcium uptake activity. This microsomal activity is similar in characteristics to the sarcoplasmic reticulum of skeletal muscle. However the specific activity of CEF microsomal calcium uptake system is much less than that found in the skeletal muscle system. The transport of calcium by these membranes provide a mechanism for the regulation of cytosol calcium levels and may play a role in the control of movement and growth of cultured cells.
Proline oxidase in cultured mammalian cells
Authors: Downing Sylvia J. , Phang James M. , Kowaloff Edward M. , Valle David , Smith Robert J.
Published: February 2005

We sought a cultured cell line with Proline Oxidase activity to study the regulation and physiologic role of the enzyme in mammalian tissues. Among the cell lines tested, only LLC‐RK1 cells, derived from rabbit kidney, had significant Proline Oxidase activity; the Km for proline of the enzyme from these cells was similar to that for the liver enzyme. LLC cells, Proline Oxidase positive, were able to convert proline to CO2. In contrast, CHL cells, Proline Oxidase negative, did not have this capability. The presence of Proline Oxidase in LLC cells and the absence of the enzyme in fibroblasts suggest that Proline Oxidase may serve as a marker enzyme for distinguishing parenchymal kidney cells from fibroblasts in culture. Cells transformed by SV40 virus and cells transformed by methylcholanthrene had activities higher than the parent cell line, but this effect of transformation could not be generalized to all transformed cells. Finally, L‐hydroxy proline at 100‐fold greater concentration than substrate L‐proline failed to decrease proline oxidation. This finding suggests distinct degradative enzymes for these two amino acids.
Control of proliferation of bovine vascular endothelial cells
Authors: Gospodarowicz Denis , Moran John S. , Braun Debra L.
Published: February 2005

The effects of Fibroblast Growth Factor (FGF) and Epidermal Growth Factor (EGF) on the proliferation of bovine vascular endothelial cells has been examined. FGF induces the initiation of DNA synthesis and cell proliferation in cloned endothelial cells of fetal and adult origin at concentrations as low as 1 ng/ml and is saturating at 50 ng/ml. EGF had no effect over the same range of concentrations. The mitogenic effect of FGF is blocked by a crude extract of cartilage. Platelet extract is also mitogenic for vascular endothelial cells although to a lesser extent than the purified FGF. In contrast to vascular endothelial cells, both EGF and FGF are mitogenic for vascular smooth muscle cells although EGF is less mitogenic than FGF at 100 ng/ml. The mitogenic effect of EGF and FGF on vascular smooth muscle is not blocked by the addition of a crude extract of cartilage, thus demonstrating the specificity of the chalone like effect of cartilage crude extract for endothelial cells.
Fibrinolysis and liver regeneration
Authors: Rixon R. H. , Walker P. R. , Whitfield J. F.
Published: February 2005

The plasmin and plasminogen activator proteases of the plasma fibrinolytic system were investigated as potential blood‐borne mediators of the proliferative activation of hepatocytes by partial hepatectomy. Partial (68%) liver resection, as well as proliferatively activating the remaining hepatocytes, rapidly (by 30 minutes) doubled the level (or activity) of circulating plasminogen activator but later (2 hours) greatly depressed this level. This later depression of the activity of circulating plasminogen activator lasted for eight to ten hours before returning to the normal level two to four hours before the hepatocytes in the liver remnant began to synthesize DNA. This sequence of changes in the fibrinolytic potential was not abolished by prior thyroparathyroidectomy which is known to inhibit the initiation of hepatocyte DNA synthesis and to prevent the secretion of the calcium homeostatic hormones, another early systemic consequence of partial liver resection. Since the early rise in plasminogen activator activity did not cause the appearance of active (free) circulating plasmin, and since the injection of large doses of the fibrinolytic and protease inhibitors, EACA and Trasylol®, during this early, post‐operative period of hyperfibrinolytic potential did not prevent hepatocytes from initiating DNA synthesis, it is unlikely that either plasmin or its activator protease are blood‐borne initiators of hepatocyte proliferative development.
Rapid cell surface‐related stimulation of alkaline phosphatase in HeLa cells by dimethyl DL‐2,3‐distearoyloxypropyl‐2′‐hydroxyethylammonium acetate (Rosenthal's inhibitor)
Authors: Melnykovych G. , Lopez I. C.
Published: February 2005

Cultures of HeLa cells, strains S3G (HeLa65) and S3K (HeLa71) were grown in plastic dishes until firmly attached and were then treated with sonic dispersions of Rosenthal's phospholipase inhibitor. A rapid increase in alkaline phosphatase activity occurred following this treatment in the S3G strain (low inducible alkaline phosphatase) but not in the S3K strain (high constitutive alkaline phosphatase). The stimulatory effect was dependent on the presence of bovine serum in the medium. No stimulation of alkaline phosphatase was observed in a variety of soluble preparations of this enzyme.
Phosphate uptake and control of fibroblast growth
Authors: Barsh Gregory S. , Greenberg Deborah B. , Cunningham Dennis D.
Published: February 2005

Previous studies have shown that growth to quiescence of fibroblast‐like cells is accompanied by a large decrease in the rate of phosphate uptake. Since 3T3 cells can be arrested in the G1 (or G0) phase of the cell cycle by lowering the concentration of phosphate in the medium, we examined the possibility that the decline in phosphate uptake observed during growth to quiescence might be a key event in the inhibition of DNA synthesis and cell division.

The experimental approach consisted of controlling the rate of phosphate uptake by varying the phosphate concentration in the medium. Kinetic experiments showed that phosphate uptake in both growing and quiescent cells was partly accounted for by simple diffusion as well as carrier‐mediated uptake. In fact, diffusion of phosphate into the growing cells was 2.5‐fold greater than in the quiescent cells.

When phosphate uptake was measured in 3T3 cells plated at different initial densities, we found an inverse relationship between phosphate uptake and cell density, showing that phosphate uptake was correlated with growth rate and did not decline simply as a consequence of time in culture.

Measurements of phosphate demonstrated that the lowered rate of phosphate uptake by quiescent cells was not due merely to a reduction of phosphate in the medium. To check the possibility that release of a previously described transport inhibitor might account for the decline in phosphate uptake observed as cells grow to quiescence, we removed media from growing and non‐growing cultures and tested its ability to support phosphate uptake. We found that the medium from growing cultures supported a higher rate of phosphate uptake than the medium from the quiescent cultures did, indicating that a transport inhibitor was being released. In addition, we found that the amount of inhibitor released was proportional to the concentration of phosphate in the medium.

To directly determine if the decline in phosphate uptake was a key event in the decline in DNA synthesis as cells grew to quiescence, we switched growing cultures to a medium with low phosphate immediately after cell attachment. This lowered the rate of phosphate uptake to a level below that of quiescent cells grown in the usual concentration of phosphate. This was done for 3T3, Polyoma virus‐transformed 3T3, human diploid foreskin, and secondary chick embryo cells. Measurements of DNA synthesis and cell number showed that this lowered rate of phosphate uptake had virtually no effect on cell growth, directly demonstrating that the decline in phosphate uptake observed during growth to confluency was not causing the decline in DNA synthesis. In addition, measurements of intracellular phosphate pool size showed that changes in phosphate uptake were not directly paralleled by changes in intracellular phosphate pool size, and that intracellular phosphate pool size was not regulating DNA synthesis or cell division during growth to quiescence.
Bacterial, serum and cellular modulation of granulopoietic activity
Authors: Bolin Rex W. , Robinson William A.
Published: February 2005

The interaction of human peripheral blood lymphocytes, monocytes, gram‐positive bacteria and human serum in the release of colony stimulating activity (CSA) has been studied. CSA was assayed by the soft agar technique using human and murine bone marrow cells. It has been demonstrated that gram‐positive organisms and their products can stimulate release of CSA by mononuclear cells. Human serum is also effective in promoting release of CSA. Release is further modulated by interactions between lymphocytes and monocytes, and lymphocytes may serve to control the modulation. The serum component is sensitive to temperature inactivation suggesting that it may have a specific physiologic role in regulation. Bacterial products, on the other hand, are not subject to temperature inactivation and require the presence of human serum for activity to be expressed.
The detection of in vitro monocyte‐macrophage colony‐forming cells in mouse thymus and lymph nodes
Authors: MacVittie T. J. , McCarthy K. F.
Published: February 2005

In vitro moocyte‐macrophage colony‐forming cells (CFC) have been detected in the thymus (30/106 cells) and in the cervical (22/106) and mesenteric (20/106) lymph nodes (LN) of the mouse. Thymus and LN derived CFC differed from bone marrow derived CFU‐c in several characteristic parameters: (1) sole specificity of PMUE to induce colony formation (CF), (2) apparent singular line of monocyte‐macrophage differentiation, (3) a marked 6‐ to 10‐day lag period prior to initiation of CF, and (4) significantly slower rates of appearance of colonies in culture after initiation of CF. Two of these parameters are shared with those CFC detected within alveolar space, peritoneal exudate and pleural effusion. These are the delay prior to CF and the singular monocyte‐macrophage differentiation. These similarities suggested that T‐CFC and LN‐CFC are probably of similar origin and represent, as suggested by Lin and Stewart ('74), a population of progenitor cells exclusively for monocyte‐macrophages.
The control of human WI‐38 cell proliferation by extracellular calcium and its elimination by SV‐40 virus‐induced proliferative transformation
Authors: Boynton A. L. , Whitfield J. F. , Isaacs R. J. , Tremblay R.
Published: February 2005

The proliferative activity of diploid human WI‐38 cells in sparse cultures depends on the extracellular concentration of free (or physiologically available) calcium, and cultivation in a medium having a calcium concentration of 0.1 mM or less gradually, but reversibly, arrest their proliferative development in the prereplicative (G1) phase of the cell cycle. Calcium's proliferative control of this cell type is eliminated by proliferative and morphological transformation by the oncogenic SV‐40 virus, and the proliferative activity of SV‐WI‐38 cells in sparse cultures is unaffected by variation of the extracellular free calcium concentration between 0.00 and 1.25 mM.
Interaction of local anesthetics with the transport system of glucose in human erythrocytes
Authors: Lacko L. , Wittke B. , Lacko I.
Published: February 2005

Local anesthetics inhibit the exchange transport of glucose in human erythrocytes. All compounds tested showed a competitive inhibition except lidocaine and baycaine causing a non‐competitive one. Moreover the transport system can bind two inhibitor molecules to one transport site as described for tetracaine and oxybuprocaine.
Mechanism of Regulation of fibroblastic cell replication. IV. An analysis of the serum dependence of cell replication based on michaelis‐menten kinetics
Authors: Ellem K. A. O. , Gierthy J. F.
Published: February 2005

The quantitative dependence of the initiation of DNA synthesis in nonreplicating cultures of human diploid fibroblasts (HDFs) on the concentration of fresh serum present in the stimulating medium has been analyzed. A simple formal analysis of the system suggested that the theory of dissociation reactions should lead to a model analogous to the Michaelis‐Menten formulation. Accumulation of enough data for statistical analysis became possible by innovations in the culture techniques and isotope incorporation methods. The data were shown to conform to this model, and parameters Vm and K′m were calculated, together with appropriate statistical functions, from a weighted linear regression of the double reciprocal transformation of the data. Several variables in the culture system were assessed for their effects on the kinetic parameters. The results were consistent with the hypothesis that Vm is a measure of the maximum population of cells capable of responding to the stimulation, and K′m is an estimate of the interaction between the mitogenic factors in serum and the cell surface.

Different strains of HDFs had different parameter values. Of several culture variables tested, the Vm was found to be influenced by the type of serum used, and by overall cell density as well as density variations within the monolayer. K′m showed significant changes only in response to cell density, and the evidence suggests that the affinity of cells for serum factors decreases as the cell density increases in confluence.