bluuebunny/crossref_metadata_2025 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

The 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

[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

WEHI‐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

A 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

The 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

The 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

Chick 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

Colony 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

In 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

Experiments 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

Two 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

The 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

The 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

We 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.
Clonal origin of murine hemopoietic colonies with apparent restriction to granuclocyte‐macrophage‐megakaryocyte (GMM) differentiation
Authors: Nakahata Tatsutoshi , Ogawa Makio
Published: February 2005

We characterized murine hemopoietic colonies consisting of granulocytes, macrophages, megakaryocytes, and blast cells and yet lacking erythroid elements. Mouse marrow or spleen cells were cultured in methylcellulose media in the presence of 10% (v/v) pokeweek mitogen‐stimulated spleen cell‐conditioned medium (PWM‐SCM) and 2 units/ml erythropoietin for 8 days. Granulocyte‐macrophage‐megakaryocyte (GEMM) colonies could be distinguished from granulocyte‐erythrocyte‐macrophage‐megakaryocyte (GEMM) colonies because the former lacked the typical appearance of bursts with red color. Analysis of Y‐chromosomes in mixing experiments with male and female marrow cells confirmed the clonal nature of the GMM colonies. Differential counts of GMM colonies revealed varying, but significant, numbers of blast cells in all of the day‐8 and day‐12 colonies and in seven out of ten day‐14 GMM colonies. In general, the percentages of blast cells were inversely related to the length of incubation in culture. Replating experiments confirmed the absence of late erythroid precursors such as CFU‐E and normoblasts in all of the 50 day‐8 GMM colonies. However, six out of the 50 GMM colonies contained early progenitors capable of erythroid expression, such as BFU‐E, CFU‐EM, CFU‐GEM, and CFU‐GEMM. In contrast, the three day‐14 GMM colonies which did not reveal blast cells failed to produce secondary colonies. Thus, while the progenitors for the latter colonies are restricted to only granulocyte‐macrophage‐megakaryocyte differentiation, some of the apparent GMM colonies containing blast cells may have originated in early progenitors close to pluripotent stem cells. Detailed cytological analyses and replating experiments are necessary for characterization of true differentiation potentials of mixed colonies in culture.
Clonal analysis of progenitor cell commitment to granulocyte or macrophage production
Authors: Metcalf D. , Burgess A. W.
Published: February 2005

Granulocyte‐macrophage colony formation by C57BL bone marrow cells was initiated in agar cultures either by the granulocyte‐macrophage stimulus, GM‐CSF, or by the predominantly macrophage stimulus, M‐CSF. After 24 hours, paired daughter cells of granulocyte‐macrophage colony‐forming cells (GM‐CFC) were separated by micromanipulation and one cultured in GM‐CSF, the other in M‐CSF. From the differentiation pattern of the resulting colonies, irreversible commitment of some cells occurred during the first 24 hours and completion of the first cell division. A similar result was obtained using granddaughter cells present after 24 hours of incubation. However, when intact developing day 2 and days 3 clones were cross‐transferred to GM‐CSF or M‐CSF recipient cultures, irreversible commitment was more obvious. Most M‐CSF‐initiated clones exhibited irreversible commitment to macrophage formation in GM‐CSF cultures and a high proportion of GM‐CSF‐initiated clones continued to produce granulocyte progeny after transfer to M‐CSF.

The results indicated that GM‐CSF and M‐CSF can irreversibly commit the progeny of GM‐CFC respectively to granulocyte or macrophage production. While for some GM‐CFC this occurs within 24 hours and one cell division, for many cells, the process is slower and requires an incubation period of up to 48 hours and/or several cell divisions.

Calculations from the data indicated that two‐thirds of GM‐CFC in adult C57BL marrow are biresponsive and respond to stimulation both by GM‐CSF and M‐CSF.
Parallel changes in protein synthesis and messenger RNA content in growing and resting epithelial cells of Xenopus laevis
Authors: Budorick Nancy E. , Miller Leo
Published: February 2005

The regulation of cell growth can be achieved at many levels but ultimately the regulatory factors must alter protein synthesis since growing cells always exhibit an increased rate of protein synthesis compared to resting cells. Some studies using growing and nongrowing mammalian cells have shown that the rate of protein synthesis compared to resting cells. Some studies using growing and nongrowing mammalian cells have shown that the rate of protein synthesis is directly dependent on mRNA content. Other studies have shown that growing and resting cells have similar amounts of mRNA and that protein synthesis is regulated by the proportion of mRNA in polysomes. We have analyzed mRNA content in growing and resting epithelial cells of Xenopus laevis. Quantitation of poly(A)+ mRNA by uniform labeling with 3H‐uridine and by 3H‐poly(U)hybridization demonstrated a direct relationship between mRNA content and the relative rate of protein synthesis in growing and resting cells. Likewise, after serum stimulation of resting cells the increase in mRNA content closely paralleled the increase in protein synthesis. Our results suggest that control of protein synthesis in growing and nongrowing cells is exerted before the translational level.
The increase in hormone‐stimulated adenylate cyclase activity following rous sarcoma virus transformation
Authors: Howard Randall F. , Friis Robert R. , Sheppard J. R. , Glossmann Hartmut
Published: February 2005

Rous sarcoma virus (RSV)‐infected chicken embryo cells were used to study the effect of viral transformation on the hormone‐stimulated synthesis of cyclic AMP. Transformation by RSV in response to the β‐adrenergic agonist isoproterenol as compared to untransformed cells. This enhancement was observed in both intact cells and in membranes prepared from these cells. The inclusion of guanosine 5'‐0‐(3‐thiotriphosphate), a nonhydrolyzable analogue of GTP, in assays of adenylate cyclase activity did not abolish the quantitative differences between the transformed and normal cell membranes. Infection of cells by Rous‐associated virus, which lacks the oncogene src, did not induce this hyperresponsiveness thus indicating the probable involvement of the src gene product in this phenomenon. The duration of the isoproterenol‐induced cyclic AMP elevation was longer in the transformed than in the untransformed cells; transformed cells, unlike untransformed cells, required at least 120 min before full desensitization became established. Membranes prepared from transformed cells specifically bound more than 5 times the quantity of the β;‐adrenergic radiolabeled antagonist (‐)3H‐dihydroalprenolol and 125I‐iodocyanopindolol compared to the untransformed cell membranes. Thus, it appears that major differences between the transformed and normal phenotypes reside in the concentration of membrane β‐adrenergic receptors and the inability of RSV‐transformed cells to self‐limit their response to specific external stimuli.
Contact‐stimulated proliferation of cultured mouse epidermal cells by 3T3 feeder layers: Inhibition of proliferation by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)
Authors: Miller Don R. , Hamby Kathy M. , Slaga Thomas J.
Published: February 2005

Mouse epidermal cells can be subcultured at 31°C onto an irradiated BALB/c 3T3 clone A31 feeder layer. A31 cells (supposedly derived from embryonic fibroblasts) were found to be specifically required for the optimal production of keratinizing epidermal colonies in secondary culture. This effect was not transmitted through the medium nor by the culture surface, since A31 cells plated on one end of a flask did not stimulate epidermal cell proliferation at the other end, even if the other end had previously held A31 cells. Epidermal cell contact with metabolizing A31 cells was probably necessary for the effect; fixed or freeze‐thawed A31 cells were ineffective. The tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate, recently shown to interfere with contact‐mediated transfer of label (metabolic cooperation) between Swiss 3T3 cells and cells of an established epidermal line in vitro, also blocked epidermal colony formation. The A31‐epidermal cell interaction is apparently not a typical mesenchymal‐epithelial interaction, since the basement membrane would prevent this contact in intact skin.
Signals causing change in morphological phenotype, growth mode, and gene expression of vascular endothelial cells
Authors: McAuslan B. R. , Hannan G. N. , Reilly W.
Published: February 2005

Comparison of three different lines of bovine aortal endothelial cells provides a clear demonstration of reversible morphologic phenotype coincidental with change in expression and growth mode. These phenotypic forms can be externally controlled so that cells may exist either in an epithelioid contact‐inhibitable state or as a fibroblastoid non‐contact‐inhibitable state.

Clonal cell line N (normal) shows a strong tendency to maintain the epithelioid phenotype. Clonal cell line Sp (sprout) can readily and reversibly adopt the epithelioid or fibroblastoid phenotype. A factor in normal serum is responsible for maintaining the cells in the epithelioid phenotype. This factor could be a growth factor since several polypeptide growth factors are shown to drive cells from the fibroblastoid phenotype to the epithelioid phenotype within 11 hours. This growth factor‐induced change is not mediated through induced DNA synthesis. Clonal cell line V (variant) normally maintains the fibroblastoid phenotype but can be directed to the epithelioid phenotype provided cells are on an appropriate collagenous matrix.

Associated with these changes in morphological phenotype are depression of the expression of the pro α2 chain of collagen type I which may be characteristic of the contact‐inhibited state and of an 80,000 mol wt polypeptide synthesized only by cells in the fibroblastoid phenotype. An endothelial cell collagen ECl (mol wt 177,000) was synthesized by all cell lines regardless of phenotype whereas a suspected breakdown product EC3 (mol wt 100,000) was found only in the epithelioid phenotype. Other differences and similarities between cell lines include expression of a 135,000 mol wt glycoprotein GP (V and N), the procollagen of collagen type III (N) of fibronectin (N, V, Sp), and of the pro α1 chain of collagen type I (Sp, V). The characteristic expression of each line and its response to signals controlling morphologic phenotype impinges on the question of whether there exist several distinct types of vascular endothelial cells with different functional potentials controlled by extracellular signals.
Topography of the predifferentiation GD growth arrest state relative to other growth arrest states in the G1 phase of the cell cycle
Authors: Wille John J. , Scott Robert E.
Published: February 2005

The differentiation of 3T3 T proadipocytes is preceded by growth arrest at a state in the G1 phase of the cell cycle (GD) which is distinct from other G1 growth arrest states including those induced by serum deprivation (GS) or nutrient deprivation (GN) (Scott et al., 1982). We now report that the GD, GS, and GN arrest states are also distinct in their topography in G1. This conclusion was derived from kinetic studies which measured the time required for cells arrested at various G1 states to synthesize DNA and from studies on the ability of GD‐, GS‐, or GN‐arrested cells to be converted from one arrest state to another in the absence of DNA synthesis. The results suggest that relative to the start of the G1 phase of the cell cycle, the following arrest state topography exists: GD, GS, GN.
Concentration of mast‐cell progenitors in bone marrow, spleen, and blood of mice determined by limiting dilution analysis
Authors: Sonoda Takashi , Ohno Takako , Kitamura Yukihiko
Published: February 2005

When hematopoietic cells of congenic +/+ mice were injected into the skin of genetically mast‐cell‐depleted (WB × C57BL/6)F1‐W/Wv mice, mast cells appeared at the injection site. The donor origin of developing mast cells was confirmed by using granules of C57BL/6‐bgl/bgl mice as a marker. When the number of injected cells was decreased, the proportion of injection sites at which mast cells did not appear increased according to the expected frequency of null response in a Poisson distribution. Therefore, such proportions were used to calculate the concentration of mast‐cell precursors in the bone marrow, spleen, and peripheral blood. The relative concentration of mast‐cell precursors in these tissues was similar to that of spleen‐colony‐forming cells. The present method seems useful as a semiquantitative in vivo assay for a population of progenitor cells which are committed to differentiate into mast cells.
Role of stimulation of arachidonic acid release in the proliferative response of 3T3 mouse fibroblasts to platelet‐derived growth factor
Authors: Shier W. T. , Durkin J. P.
Published: February 2005

Human platelet‐derived growth factor (PDGF) stimulates release of arachidonic acid from cellular phospholipids, synthesis and release of prostaglandins from the cell, and initiation of DNA synthesis in cultures of 3T3 Swiss mouse fibroblasts at similar concentrations with four independent preparations representing a million‐fold range of purification. Stimulation of archidonic acid and prostaglandin release is an early event (beginning within minutes) in the response to PDGF treatment. Incubating cells with PDGF at 4°C followed by washing leads to activation of archidonic acid release on warming the cells to 37°C, consistent with binding of the factor to the cell surface. PDGF‐stimulated arachidonic acid release, prostaglandin release, and initiation of DNA synthesis are all inhibited by phenylglyoxal at similar concentrations. These results suggest that activation of arachidonic acid release from phospholipids plays an essential role in the mechanism by which PDGF stimulates the initiation of DNA synthesis in 3T3 cells. The stimulation of initiation of DNA synthesis by PDGF does not appear to be mediated by the synthesis of prostaglandins or other known arachidonic acid metabolites because neither indomethacin (a fatty acid cyclooxygenase inhibitor) nor phenidone (a lipoxygenase inhibitor) inhibit initiation of DNA synthesis at concentrations which inhibit arachidonic acid metabolism. Although the activation of arachidonic acid release by PDGF is a calcium‐dependent process, a simple calcium flux appears unimportant to the mechanism of activation. Evidence was also obtained against an involvement of sodium fluxes or proteolytic activity in the mechanism of stimulating arachidonic acid release by PDGF or serum.
Distinct bone marrow precursors for mononuclear phagocytes expressing high and low 5′ ‐nucleotidase activity
Authors: Bursuker Isia , Goldman Rachel
Published: February 2005

Using a cytochemical assay we were able to show that the peritoneal macrophage population of normal nontreated mice (resident peritoneal macrophages) exhibits a heterogeneity with regard to the expression of the activity of the ecto‐enzyme 5′‐nucleotidase (5′‐N). About 75% of the macrophages express high enzymic activity whereas the remaining 25% express low 5′‐N activity. Macrophages accumulating in the peritoneum as a result of an inflammatory response are predominantly of the low activity type. In vitro activation of resident peritoneal macrophages by lymphokines does not result in a decrease in the number of macrophages expressing high enzymic activity though the level of the enzymic activity of these cells is reduced by about 36%. Bone marrow derived mononuclear phagocyte colonies developing in vitro, under liquid culture conditions, from bone marrows of normal mice can be divided into three types with respect to their expression of 5′‐N activity: (1) high activity colonies–relatively small colonies in which all the cells express high 5′‐N activity (about 20% of the colonies); (2) low activity colonies – relatively large colonies in which all the cells express low 5′‐N activity (about 70% of the colonies); and (3) mixed colonies–relatively large colonies in which all the cells express low enzymic activity except for about 8% of cells located at the periphery of the colonies which express high enzymic activity (about 10% of the colonies). During an inflammatory response the frequency of the high activity colonies is significantly reduced. Our results provide evidence for distinct bone marrow precursors for mononuclear phagocytes expressing high and low 5′‐N activity and suggest that (1) the resident macrophages derive from a subpopulation of bone marrow precursor cells developing in vitro into high 5′‐N activity mononuclear phagocytes, and (2) during an inflammatory response there is a preferential expansion of clones of the low enzymic activity phenotype.
Synergistic stimulation of DNA synthesis by cyclic AMP derivatives and growth factors in mouse 3T3 cells
Authors: Rozengurt Enrique
Published: February 2005

Addition of the cAMP derivatives butcAMP or 8BrcAMP to quiescent cultures of Swiss 3T3 causes synergistic stimulation of DNA synthesis with insulin, phorbol esters, vasopressin, epidermal growth factor, or fetal bovine serum (2–5%). In the presence of insulin, 8BrcAMP, and butcAMP stimulate [3H]‐thymidine incorporation into acid‐precipitable material in a dose‐dependent manner. The effect of these agents in specific since 8Br5´AMP, 5′AMP, butyrate, or 8BrcGMP fail to stimulate DNA synthesis under identical experimental conditions. Furthermore, the mitogenic effects of the cAMP derivatives were markedly potentiated by 1‐methyl‐3‐isobutyl xanthine and 4‐(3‐butoxy‐4‐methoxy benzyl)‐2‐imidazolidine, both of which are potent inhibitors of cyclic nucleotide phosphodiesterase activity. The growth‐promoting effects of the cAMP derivatives were demonstrated by [3H]‐thymidine incorporation (either by scintillation counting or by autoradiography), by flow cytofluorometric analysis, and by increase in cell number. When quiescent Swiss 3T3 cells were exposed to butcAMP and insulin, DNA synthesis began after a lag of 17 h. The result of sequential additions of cAMP derivatives and insulin to quiescent 3T3 cells suggest that these agents must act simulaneously in G0/G1 to stimulate entry into DNA synthesis in these cells. The findings support the proposition that an increase in cellular levels of cAMP (but not cGMP) acts as a mitogenic stimulus for confluent and quiescent Swiss 3T3 cells.
Cyclic AMP stimulation of Na‐K pump activity in quiescent Swiss 3T3 cells
Authors: Paris Sonia , Rozengurt Enrique
Published: February 2005

Recently, we have found that an increase in the intracellular level of cAMP acts as a mitogenic signal for Swiss 3T3 cells (Rozengurt et al., Proc. Natl. Acad. Sci. USA, 78:4392, 1981). The results presented in this paper demonstrate that addition of cAMP‐elevating agents to confluent and quiescent cultures of Swiss 3T3 causes a marked increase in the rate of 86Rb+ uptake but has no effect on the rate of cation efflux. The stimulation of ion uptake is mediated by the Na‐K pump as shown by the ouabain sensitivity of the 86Rb+ fluxes. The increase in Na‐K pump activity occurs whether cAMP is generated endogenously by stimulation of adenylate cyclase activity by cholera toxin, adenosine agonists, or PGE1 or added exogenously as 8BrcAMP. The stimulatory effect of these compounds on 86Rb+ uptake is potentiated by inhibitors of cyclic nucleotide phosphodiesterase activity. Cholera toxin stimulates the Na‐K pump in a dose‐dependent manner; half‐maximal effect is achieved at 0.7 ng/ml.

The stimulation of ouabain‐sensitive 86Rb+ uptake by cAMP‐elevating agents reaches a maximum after 2‐3 h of incubation. This contrasts with the rapid (within minutes) stimulation of the Na‐K pump caused by serum and other mitogenic agents. Further, cAMP‐elevating agents fail to increase in Na+ influx, under identical experimental conditions. These findings suggest that the stimulation of Na‐K pump activity caused by increased cAMP levels contrasts mechanistically with the rapid control of pump activity by serum which is primarily mediated by increased Na+ entry into the cells.
β‐Galactosidase—An indicator of the maturational stage of mouse and human mononuclear phagocytes
Authors: Brusuker Isia , Rhodes Joan M. , Goldman Rachel
Published: February 2005

Resident, elicited, and activated mouse peritoneal macrophages exhibit a differential expression of the activity of the enzyme b̃‐galactosidase; freshly harvested resident macrophages express a remarkably high activity whereas the latter two populations are almost void of enzymic activity. During in vitro cultivation there is an enhancement in the level of the enzyme in the three populations, and a significant proportion of both thioglycollate‐elicited and Corynebacterium parvum‐activated macrophages acquire b̃‐galactosidase activity. Cells within in vitro differentiated bone marrow‐derived mononuclear phagocyte colonies are heterogeneous with respect to expression of b̃‐galactosidase activity. The percentage of cells expressing medium to intense enzymic activity is augmented with time in culture. Essentially the same pattern is observed in colonies differentiated from bone marrow of mice bearing acute or chronic inflammation. Freshly isolated human peripheral blood monocytes are essentially void of detectable b̃‐galactosidase activity. Eighty to ninety percent of the monocytes acquire medium to intense activity during a 7‐day cultivation period. The data support the suggestion that b̃‐galactosidase expression in mononuclear phagocytes is a correlate of their maturational stage both in vivo and in vitro and does not reflect the state of elicitation or activation of these cells.
Developmental restriction of mobility of concanavalin a receptors
Authors: Lipke Peter N. , Lilien Jack
Published: February 2005

Trypsinized cells from embryonic chick neural retina redistributed concanavalin A receptors to patches and caps. Between 12 and 16 days of development, the ability to redistribute concanavalin A receptors declined. This restriction in mobility of the receptors was accompanied by changes in susceptibility to the capping‐inhibitory drugs colchicine and cytochalasin B.
Oxygen‐stimulated cytochrome oxidase assembly in hepatocyte monolayer cultures
Authors: Hare James F. , Hodges Randall
Published: February 2005

Isolated rat liver hepatocytes exhibited a twofold to threefold increase in both specific cytochrome oxidase activity and heme a concentration during their first 24 h in monolayer culture. Other mitochondrial respiratory enzyme activity did not increase over this same period. Decreasing the oxygen content of the gas phase to 5% of ambient eliminated the stimulation in heme a content without having an effect on cell protein synthesis or viability. These results indicate that oxygen concentration can effect the level of cytochrome oxidase (heme a) in these differentiated cells. The effect of oxygen may be on the translation or assembly of subunits, synthesis of heme, or incorporation of heme into holoenzyme. Oxygen stimulation of cytochrome oxidase content was not accompanied by increased incorporation of pulsed radiolabelled methionine into immunoprecipitated holoenzyme subunits. Moreover, chloramphenicol, a translation inhibitor of three mitochondrially synthesized cytochrome oxidase subunits, did not diminish the increase in heme a seen over the first 24 h of culture. These latter results suggest that the increasing enzyme content is not due to stimulated translation.
Role of extracellular Na and K in lymphocyte activation
Authors: Deutsch C. , Price M.
Published: February 2005

The transmembrane electrical potential has been measured across human peripheral blood lymphocytes under culture conditions using equilibrium distributions of the lipophilic cation 3H‐tetraphenylphosphonium (TPP). The TPP equilibrates to a steady‐state level that gives calculated voltages of −80 mV for unstirnulated lymphocytes. This value of Ihe potential is constant during the first few hours of succinyl concanavalin A stimulation. When the transmembrane electrical potential is lowered by isotonic replacement of Na with K, this neither by itself stimulates proliferation nor does it inhibit mitogen‐stimulated proliferation. Lymphocytes with similar membrane potentials, such as those incubated in normal‐Na MEM and low‐Na‐mannitol MEM, exhibit drastically different pro‐liferative responses to mitogen stimulation (Deutsch et al., 1981). These results show that isotonic replacement of K for Na prevents low Na inhibition of DNA synthesis and that at least during the first 2.5 hours of lymphocyte activation transmembrane electrical potential per se does not play a significant role in the activation process.
Migration inhibitory action of bacterial lipopolysaccharide on progenitor cells of monocyte/macrophage lineage growing in culture in the presence of colony‐stimulating factor (CSF‐1)
Authors: Ishii Y. , Shinoda M. , Shikita M.
Published: February 2005

Addition of lipopolysaccharide (LPS) to the culture of mouse myeloid stem cells (CFUc) increased the incidence of compact colonies and decreased that of dispersed ones in the presence of colony‐stimulating factor (CSF‐1) which had not such an effect by itself even in high concentrations. Although colony morphology was thus changed, nearly all colonies were composed of monocytes. The incidence of compact colonies increased with the increase of LPS concentration but plateaued at about 50%. Bone marrow cells of LPS‐tolerant mice responded to LPS in vitro to a slightly decreased extent. The activity of LPS was decreased by alkaline or acid hydrolysis of the LPS molecule and inhibited by polymixin B, but not by indomethacin, α‐L‐fucose, nor by α‐methyl‐D‐mannoside. Other immunopotentiating substances, such as OK‐432, Lentinan, and Levamisole, had no effect on the colony morphology. Both muramyl dipeptide and poly(I)poly(C) were also ineffective. Furthermore, the action of LPS was not abolished by the use of heat‐inactivated serum in the culture. LPS was no longer stimulative for the induction of lysosomal enzymes in the CSF‐stimulated culture, although it greatly enhanced the enzyme induction in the unstimulated culture. These results indicate that the cells of monocyte/macrophage lineage develop the capacity for migration before they become responsive to LPS, and that the LPS‐responding monocytic cells can proliferate even in a state of confluence induced by LPS.