bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hexose transport in undifferentiated and differentiated BALB/c 3T3 preadipose cells: Effects of 12–0‐tetradecanoylphorbol‐13‐acetate and insulin
Authors: O'Brien Thomas G. 
Published: February 2005

AbstractThe effect of the phorbol diester 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA) on hexose transport in undifferentiated and differentiated BALB/c 3T3 preadipose cells was studied. Additon of TPA to undifferentiated or fully differentiated cultures resulted in an increased rate of both 2‐deoxyglucose uptake and 3‐0‐methylglucose transport; the time course and maximal stimulation differed for each type of culture and for each hexose. In confluent, undifferentiated cells, half‐maximal stimulation of 2‐deoxyglucose uptake occurred at 3 nM TPA, while the half‐maximal stimulation of 3–0‐methylglucose occurred at 30 nM. Epidermal growth factor and fetal bovine serum increased 2‐deoxyglucose uptake in undifferentiated cells, while insulin did not. Insulin did, however, stimulate 3–0‐methylglucose transport in differentiated cells. From dose‐response curves in differentiated cells, halfmaximally effective concentrations were 0.17 nM for insulin and 30 nM for TPA. At optimal concentrations and incubation times for each, TPA was significantly more effective than insulin in stimulating hexose transport in differentiated cells. It was also shown that insulin could further increase hexose transport in maximally stimulated TPA‐treated cells. Cycloheximide inhibited by 75% the increase in hexose transport by TPA in differentiated cells, while having no effect on the response of these cells to insulin. In differentiated cells, chronic exposure to insulin abolished the ability of these cells to respond acutely to insulin addition but they could still respond to TPA. On the other hand, differentiated cells exposed continuously to TPA for 5 days retained the ability to activate 3–0‐methylglucose transport after either TPA or insulin addition. These results demonstrate that TPA can stimulate hexose transport directly in both undifferentiated and differentiated 3T3 cells and suggest that TPA and insulin affect transport by different mechanisms.
Uptake of [3H]ouabain from the cell surface into the lysosomal compartment of HeLa cells
Authors: Cook John S. , Tate Emily H. , Shaffer Carolyn 
Published: February 2005

Abstract[3H]Ouabain specifically bound at sublethal concentrations to Na, K‐ATPase on the surface of HeLa cells is taken up (internalized) by the cells at a rate of three membrane equivalents of labeled sites per generation. Immediately following a pulse label with the glycoside, codistribution of radioactivity with the surface marker 5‐nucleotidase is found in both conventional sucrose‐gradient fractionation and in fractionation following a digitonin treatment. At appropriate concentrations digitonin increases the buoyant density of the HeLa surface membrane and solubilizes the lysosomal marker β‐hexosaminidase (Tulkens et al., 1974). After internalization, [3H]ouabain is also solubilized by digitonin. A shear analysis is described which shows internalized ouabain and β‐hexosaminidase to be codistributed in a particulate fraction that is homogenous with respect to shear; extrapolation to zero‐shear shows that little or none of either marker is found in the soluble fraction of the cytosol. Both markers are coreleased from the particulate fraction by osmotic shock. Although internalized ouabain is subsequently released from these cells with a half‐time of about 70 hr, apparently by exocytosis, the shear sensitivity of the remaining cell‐associated ouabain does not change for up to 72 hr. Thus ouabain (together with Na, K‐ATPase?) appears to be taken up from the surface into a lysosomal compartment and, by at least one criterion, this compartment does not change its physical properties with time, i.e., does not “age.”
Two‐factor requirement for murine megakaryocyte colony formation
Authors: Williams N. , Eger R. R. , Jackson H. M. , Nelson D. J. 
Published: February 2005

AbstractWEHI‐3 cell‐conditioned medium with the capacity to stimulate megakaryocyte colony formation was separated by Sephadex G‐150 column chromatography. The development of colonies containing megakaryocytes was observed only when mixing experiments were performed. Individual fractions did not support megakaryocyte colony growth. The two factors in WEHI‐3 CM required for megakaryocyte colony growth had apparent average molecular weights of 35,000 daltons (megakaryocyte CSF) and 100,000 daltons (megakaryocyte potentiator). The results were confirmed in serum‐free conditions in which colonies were directly identified in the cultures by acetylcholinesterase staining. Two growth factors may be necessary for the genesis of megakaryocytic colonies.
Changes in NAD(P)+‐dependent malic enzyme and malate dehydrogenase activities during fibroblast proliferation
Authors: McKeehan Wallace L. , McKeehan Kerstin A. 
Published: February 2005

AbstractA sensitive isotope exchange method was developed to assess the requirements for and compartmentation of pyruvate and oxalacetate production from malate in proliferating and nonproliferating human fibroblasts. Malatedependent pyruvate production (malic enzyme activity) in the particulate fraction containing the mitochondria was dependent on either NAD+ or NADP+. The production of pyruvate from malate in the soluble, cytosolic fraction was strictly dependent on NADP+. Oxalacetate production from malate (malate dehydrogenase, EC 1.1.1.37) in both the particulate and soluble fraction was strictly dependent on NAD+. Relative to nonproliferating cells, NAD+‐linked malic enzyme activity was slightly reduced and the NADP+‐linked activity was unchanged in the particulate fraction of serum‐stimulated, exponentially proliferating cells. However, a reduced activity of particulate malate dehydrogenase resulted in a two‐fold increase in the ratio of NAD(P)+‐linked malic enzyme to NAD+‐linked malate dehydrogenase activity in the particulate fraction of proliferating fibroblasts. An increase in soluble NADP+‐dependent malic enzyme activity and a decrease in NAD+‐linked malate dehydrogenase indictated an increase in the ratio of pyruvate‐producing to oxalacetate‐producing malate oxidase activity in the cytosol of proliterating cells. These coordinate changes may affect the relative amount of malate that is oxidized to oxalacetate and pyruvate in proliferating cells and, therefore, the efficient utilization of glutamine as a respiratory fuel during cell proliferation.
Phosphate concentration and transport in Ehrlich ascites tumor cells: Effect of sodium
Authors: Bowen Jesse W. , Levinson Charles 
Published: February 2005

AbstractThe effects of extracellular Pi and Na+ on cellular Pi concentration and transport were studied. Steady‐state Pi exchange flux was measured by 32P uptake in the presence and absence of Na+. Model experiments were also conducted to assess the possibility that hydrolysis of organic phosphate esters contributes to the chemically measured intracellular Pi concentration of Ehrlich ascites tumor cells. The results of these experiments indicate that hydroloysis of labile organic phosphate esters does not contribute to the measured intracellular pool of Pi. The Pi transport system exhibits an apparent Ks of 0.115 mM Pi and a maximal flux of 1.73 mmole min−1 (kg dry wt)−1. When incubated in a phosphate‐buffered choline chloride medium (5 mM Pi) the intracellular Pi and the Pi influx fall by 65 and 88%, respectively. At 5 mM extracellular Pi, the Na+‐dependent component of Pi transport fits Michaelis‐Menten kinetics with the maximal flux equal to 2.46 mmole min−1 (kg dry wt)−1 and an apparent Ks of 35.4 mM Na+. In addition, a Na+‐independent component of Pi transport, comprising about 12% of the total Pi flux, was identified. The data support the hypothesis that a Pi transport system, dependent on Na+, plays a principal role in the maintenance of intracellular Pi concentration.
Effects of inhibitors of ornithine and S‐adenosylmethionine decarboxylases on L6 myoblast proliferation
Authors: Stoscheck Christa M. , Erwin Bradley G. , Florini James R. , Richman Robert A. , Pegg Anthony E. 
Published: February 2005

AbstractThe role of polyamines in myoblast proliferation was studied by treating cells of Yaffe's L6 line of rat myoblasts with inhibitors of polyamine synthesis. Both an irreversible inhibitor of ornithine decarboxylase—difluoromethyl‐ornithine (DFMO)—and a competitive inhibitor of S‐adenosyl‐methionine decarboxylase—methylglyoxal‐bis(guanylhydrazone) (MGBG)—depressed spermidine levels and inhibited myoblast proliferation. Spermine levels were not significantly depressed by either inhibitor and putrescine levels were decreased only by DFMO. Putrescine and spermidine, but not magnesium, prevented inhibition of myoblast proliferation by DFMO and MGBG; determination of 14C‐DFMO uptake in the presence and absence of these compounds de monstrated that they did not reduce the rate or extent of inhibitor uptake and thus prevent its inhibition of ornithine decarboxylase. Thus it seems likely that these inhibitors reduce cell proliferation by inhibiting polyamine formation. Addition of spermidine to the cells led to a substantial reduction in the activity of S‐adenosyl‐methionine decarboxylase, suggesting that the enzyme is subject to negative regulation by the products of the polyamine biosynthetic pathway. Unexpectedly, addition of spermidine also increased intracellular putrescine levels; this apparently resulted from conversion of spermidine to putrescine. Addition of putrescine or spermidine in the absence of serum did not increase the rate of myoblast proliferation although it did elevate intracellular polyamine levels as expected. We conclude that some threshold level of one or more polyamines (probably spermidine) is necessary but not sufficient for initiation and maintenance of myoblast proliferation in culture.
The effects of division rate‐limiting amounts of fetal bovine serum on the proliferation and aging of cultured chick cells
Authors: Ryan J. M. , Nielsen P. J. , Nelson L. R. 
Published: February 2005

AbstractChick embryo fibroblasts serially propagated in media containing division ratelimiting amounts of fetal bovine serum underwent premature culture senescence as illustrated by accelerated declines in the number of cells incorporating 3H‐thymidine, increased population doubling times, reduced cell densities at subcultivation, and reduced replicative life‐spans compared to cells grown in medium containing non‐rate‐limiting amounts of serum. Low serum serially propagated “senescent” cultures returned to 10% serum containing medium had proliferative rates, incorporated 3H‐thymidine, and attained saturation densities at confluency similar to younger cells. “Senescent” cells serially propagated in low serum and returned to 10% serum achieved life‐spans similar to cells continuously grown in the presence of 10% serum. The results of these and other studies show that cells serially propagated in the presence of division rate‐limiting amounts of fetal bovine serum, or at high inoculation densities, accumulate a substantial number of cells in the population during exponential growth conditions that are not senescent but are prevented from entering DNA synthesis becuase of mitogen limitations. Our results indicate that the amount of serum mitogen in the growth medium affects only the rate at which cells express their genetically predetermined replicative potential and not the replicative lifespan per se. These results are discussed in relation to the techniques that should be employed for studying cellular aging and the mechanism of senescent cell formation.
Clonal growth of normal human epidermal keratinocytes in a defined medium
Authors: Tsao Mary C. , Walthall Ben J. , Ham Richard G. 
Published: February 2005

AbstractColony formation by normal human epidermal keratinocytes (HK) has been achieved in a medium that contains no deliberately added undefined supplements. The term “defined” is used to describe this medium, although the possibility that trace contaminants in its components could be contributing to the multiplication that it supports cannot yet be ruled out completely. The defined medium consists of a basal medium, MCDB 152, supplemented with 5 ng/ml epidermal growth factor (EGF), 10 μg/ml transferrin, 5 μg/ml insulin, 1.4 × 10−6 M hydrocortisone, 1.0 × 10−5 M ethanolamine, 1.0 × 10−5 M phosphoethanolamine, and 2.0 × 10−9 M progesterone. MCDB 152 differs from MCDB 151, previously developed for multiplication of HK with small amounts of dialyzed serum (Peehl and Ham, 1980b), only by addition of the trace element mixture from human fibroblast medium MCDB 104 (McKeehan et al., 1977). Most of the requirement for transferrin, which is the least defined component of the defined medium, can be replaced by adding freshly dissolved and sterilized ferrous sulfate to the final medium after it has been filter sterilized. Insulin and EGF are clearly needed for optimal multiplication and hydrocortisone is mildly beneficial. Either ethanolamine or phosphoethanolamine must be present in the defined medium for HK multiplication. There is a greater need for EGF and less for hydrocortisone in the defined medium than in previous partially defined systems that we have worked with. Very large colonies of flattened epithelial cells are obtained in the defined medium, which has a low calcium concentration (0.03 mM) and does not favor keratinocyte differentiation. Less growth and more differentiation are obtained with higher calcium concentrations. The defined medium is highly selective for keratinocyte growth from a mixed inoculum of keratinocytes and fibroblasts.
Effects of retinoic acid on the binding and mitogenic activity of epidermal growth factor
Authors: Jetten Anton M. 
Published: February 2005

AbstractIn this study the effects of retinoic acid on the binding and mitogenic activity of epidermal growth factor (EGF) in mouse fibroblast Balb/c 3T6 cells are further examined. Retinoic acid treatment of 3T6 cells results in a sixfold enhancement of 125I‐labeled mouse EGF binding when assayed at 37°C. In both retinoic acid‐treated and control cells, cell‐associated 125I‐EGF is rapidly internalized, degraded, and secreted. Retinoic acid treatment does not seem to have a significant effect on the rate of internalization and degradation of EGF. At 0°C, internalization of EGF is strongly inhibited in both retinoic acid‐treated and control cells. Under these conditions retinoic acid‐treated cells still exhibit a tenfold higher level of EGF binding compared to control cells. When exposed to high concentrations of EGF both retinoic acid‐treated and control cells “down‐regulate” their EGF receptors. And although the growth rate of retinoic acid‐treated cells is about half that of control cells, the rate at which EGF binding capacity is restored after down‐regulation is about three times as fast as in control cells. No direct antagonism on EGF binding was observed between the tumor promoter 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA) and retinoic acid. EGF is a potent mitogen for 3T6 cells in serum‐free medium; retinoic acid inhibits the mitogenic activity of EGF even though it increases EGF binding. Retinoic acid also inhibits cell proliferation induced by sarcoma growth factor (SGF) and insulin.
Lectin‐induced rearrangement of an immature hematopoietic cell surface marker
Authors: Phelps Bonnie M. , Williamson Patrick , Schlegel Robert A. 
Published: February 2005

AbstractExperiments were carried out to examine the possible physiological role of disordered membrane domains in hematopoietic cell surface differentiation. The hematopoietic stem cell line 416B has been shown to bind the dye merocyanine 540 (MC540), a fluorescent probe which may be specific for disordered regions of lipid bilayers (Williamson et al., 1981). The surface receptors for the lectins Concanavalin A (Con A) and wheat germ agglutinin (WGA) exhibit patchy distributions on the surface of 416B cells which correspond to the distribution of MC540 binding regions. Appropriate incubation of these cells with either of the two lectins results in the induced formation of a cap. The binding regions for MC540 and the receptors for the other lectin become localized to the same region of the membrane by this process. Such coordinated rearrangements of surface glycoproteins associated with disordered lipid domains may play a role in the cocapping of disparate surface molecules (Raz and Bucana, 1980) or in differentiation‐related cell surface rearrangements (Schlegel et al., 1980).
Ion regulation in potassium‐sensitive mutants of paramecium tetraurelia
Authors: Cronkite D. L. , Burg M. 
Published: February 2005

AbstractTwo recessive mutations of Paramecium tetraurelia confer sensitivity to potassium: While wild‐type cells survive when up to 30 mM KCI is added to their growth medium, mutants cease to grow and die when levels of added KCl reach 20–25 mM. Similar sensitivities are seen to Rb+ and Cs+, but not to Na+. Swimming behavior of mutants is indistinguishable from wild type when place in stimulating solutions containing Na+, K+, or Ba2+. Behavioral adaptation to low levels of K+ also is indistiguishable from wild type. Flame photometry reveals that one mutant is unable to keep out K+ when that ion is at high levels in the medium, while the other mutant readily leaks K+ and Na+ when those ions are at low levels in the medium. Both mutants have markedly lower internal Na+ than does wild type. Problem with K+ permeability account for the sensitivity of the one mutant to elevated external K+, but the basis of sensitivity in the other mutant is unclear. These mutants expand the range of ion regulation mutants in Paramecium and demonstrate that lesions in cellular ion regulation in this organism need not result in changes in swimming behavior.
Complement receptor phenotypes of culture‐derived murine macrophages
Authors: Walker William S. , Yen Shing‐Erh 
Published: February 2005

AbstractThe distribution of complement receptors CR1 and CR3 among macrophages derived from cultures of bone marrow, blood, and elicited or normal peritoneal cell population was studied. Cells and colonies from the first three sources had a common phenotype, CR1 + 3 −, whereas those from the noram peritoneal populations had either CR1 + 3 − or CR1 + 3 +. The former phenotype characterized spindle‐shaped as well as epithelial‐like macrophages; the latter was essentially restricted to colonies made up of the epithelioid cells. Both morphologic features and the CR phenotypes remained stable throughout the culture period. These phenotypic differences might be explaniend by the presence of at leas two clonally derived types of macrophages.
Glucagon and choleragen stimulation of glycogenolysis in primary cultures of adult rat liver parenchymal cells: Lack of involvement of the glucocorticoids
Authors: Kletzien Rolf F. , Weber Charles A. , Butcher Fred R. , Stumpo Deborah J. 
Published: February 2005

AbstractThe influence of glucagon, choleragen, and the adrenal glucocorticoids on glycogenolysis in primary cultures of adult rat liver parenchymal cells has been studied. Both glucagon and choleragen caused a twofold to threefold stimulation of glucose production from endogenous reserves of glycogen. The effect of glucagon on glucose production was noted at the earliest time point examined and the stimulation of glucose production was preceded by an elevation of cyclic AMP. Choleragen did not produce a significant stimulation of glucose production until 45 minutes after addition of the agent. Choleragen effects on glucose production were preceded by an elevation of cyclic AMP, but in contrast with glucagon, choleragen did not significantly elevate cyclic AMP until 30 minutes after addition to the culture. One ng of choleragen per ml of medium was sufficient to produce an effect on glucose production. Glucagon‐ or choleragen‐treated cultures mobilized glycogen more rapidly than did untreated cultures incubated in glucose‐free medium. In addition, both agents produced a stong inhibition of lactate production. Thus, the stimulation of glucose production by these agents was partially due to increased glycogen mobilization and partially due to redirection of carbon units from glycolysis. That glucose production in the hepatocytes is regulated in part by a cyclic AMP‐dependent mechanism is strongly supported by the observation that both agents elevate cyclic AMP and cause an increase in glucose production and inhibition of lactate production. The possibility that the glucocorticoids participated in the regulation of glycogenolysis either in a direct or indirect (permissive) fashion was assessed. It was found that when the direct effect of the glucocorticoids on glycogenesis was taken into account, the glucocorticoids had no direct effects on glycogenolysis, nor did they alter the stimulation of glycogenolysis by glucagon or choleragen.
Clonal preadipocyte cell lines with different phenotypes derived from murine marrow stroma: Factors influencing growth and Adipogenesis in vitro
Authors: Lanotte M. , Scott D. , Dexter T. M. , Allen T. D. 
Published: February 2005

AbstractWe have isolated continuously growing cell lines derived from mouse bone marrow stroma. These cell lines were independently obtained, and though they showed morphologies ranging from the epithelioid to the fibroblastoid patterns, they all differentiated into adipocytes. Subclones obtained from two cell lines had a very high frequency (90‐100%) of differentiation into adipocytes after two or three weeks of arrested growth. Though extensive accumulation of lipid often mechanically impaired mitosis, the cells committed to adipocytes did not suffer an irreversible loss of proliferative capacity. Adipogenesis was obtained in conditions similar to those required for fat cell formation in long‐term bone marrow culture. The cell lines were found to be insensitive to insulin as a signal of adipocyte differentiation. The ultrastructural characteristics of the preadipocytes and fat cells are also similar to those of the fat cells developing in long‐term bone marrow culture. As such, these cell lines should prove useful for analysing cell/cell interactions in haemopoiesis.