bluuebunny/crossref_metadata_2025 huggingface dataset:
Population density and regulation of cell division in 3T3 cells. I. Inorganic phosphate levels, uptake and release
Authors: Gray Peter N. , Cullum Malford E. , Griffin Martin J.
Published: February 2005

Triggering mechanisms for initiating density dependent inhibition of cell division in 3T3 cell monolayers are activated approximately two to three population doublings prior to cessation of cell division at monolayer confluency. This activation occurs at a critical contact cell density of approximately 8 × 103 cells/cm2. During this period there are selective controls on transport and storage of required low molecular weight nutrients. A possible correlation between orthophosphate and rates of cell division has been investigated. We have demonstrated a relationship between cellular concentrations of orthophosphate and initiation of density dependent inhibition of cell division. Prior to critical intercellular contact, the [Pi] in 3T3 cells is 10 mM. During critical contact, this concentration is quickly reduced to approximately 2 mM and remains at this concentration to confluency. Similar alterations do not occur in Py 3T3 cells, which maintain a concentration of approximately 2 mM Pi regardless of cell density. After confluent 3T3 cells are released from inhibition of cell division the [Pi] must increase several‐fold before DNA synthesis commences. These are physiological changes in 3T3 cellular [Pi] as a function of cell density, and cannot be attributed to nutrient depletion, altered transport of Pi into the cell, increased [ATP], or increased [PPi] levels. The controlled modulation of [Pi] may regulate glycolysis and coordinate counter‐ion changes (Ca++) may regulate mitochondrial activity.
The effect of oxygen tension on the growth and metabolism of WI‐38 cells
Authors: Balin Arthur K. , Goodman David B. P. , Rasmussen Howard , Cristofalo Vincent J.
Published: February 2005

The effect of oxygen tension on cellular growth and metabolism was studied in actively growing WI‐38 cells [>90% labeled nuclei (LN)] grown under atmospheres containing 5% CO2 and various combinations of O2 and N2. Cells grown under a partial pressure of oxygen (P) of 7.8 ± 3.5 mm Hg had a significantly slower growth rate, lower saturation densities and higher rates of glucose consumption and lactate production than did cells grown under a P of 44 ± 7 mm Hg. There were no significant differences in saturation density or the rates of glucose consumption or lactate production between cells grown under P 26 ± 4 mm Hg, 44 ± 7 mm Hg, or 134 ± 11 mm Hg. Population doubling time was slightly prolonged at a P of 134 mm Hg compared to a P of 44 mm Hg. Cells grown under a P of 291 ± 25 mm Hg showed only 20–30% of the growth rate and 10–20% of the saturation density of cells grown under a P of 134 mm Hg. Despite this reduced growth, cells grown under a P of 291 mm Hg consumed four to six times as much glucose and produced four to six times as much lactate per cell as cells grown at a P of 134 mm Hg. Cells grown under a P of 560 ± 38 mm Hg attached but did not proliferate. This toxic effect of oxygen on cell proliferation was reversible and was not due to an effect of oxygen on the media.
Inhibition of sulfate transport in ehrlich ascites tumor cells by 4‐acetamido‐4′‐isothiocyano‐stilbene‐2,2′‐disulfonic acid (SITS)
Authors: Villereal Mitchel L. , Levinson Charles
Published: February 2005

The effects of the nonpenetrating amino reactive reagent 4‐acetamido‐4′‐isothiocyano‐stilbene‐2‐2′‐dilsulfonic acid (SITS) on anion transport (sulfate, chloride, and inorganic phosphate) were investigated in Ehrlich ascites tumor cells. Short time exposure to SITS produces a reversible inhibition (92%) of sulfate transport. The kinetics of interaction suggest that reversibly bound SITS competitively inhibits sulfate transport, Ki = 3 × 10−6 M. Incubation of tumor cells with SITS (1 × 10−4 M) for longer periods of time results in a time dependent irreversible inhibition of sulfate transport which obeys first order kinetics. The rate coefficient for the inactivation process is 0.040 min−1. The kinetics of irreversible inhibition is best explained by the irreversible binding of SITS to the sulfate transport site, and therefore makes SITS a potentially useful probe for the quantitation of these sites in the tumor cell. The lack of effect of irreversibly bound SITS on either chloride or inorganic phosphate transport points to a specificity in the interaction of SITS with the tumor cell membrane, as well as indicating that an alternate pathway exists for the movement of these anions across the membrane.
Endosymbiosis in amoebae: Recently established endosymbionts have become required cytoplasmic components
Authors: Jeon K. W. , Jeon M. S.
Published: February 2005

A strain of large, free‐living amoeba that became dependent on bacterial endosymbionts which had infected the amoebae initially as intracellular parasites, was studied by micrurgy and electron microscopy. The results show that the infected host cells require the presence of live endosymbionts for their survival. Thus, the nucleus of an infected amoeba can form a viable cell with the cytoplasm of a noninfected amoeba only when live endosymbionts are present. The endosymbiotic bacteria are not digested by the host amoebae and are not themselves used as nutritional supplement. While the host amoebae are dependent specifically on the endosymbionts, the latter can live inside amoebae of different strains, indicating that their dependence on the host cells is not yet strain specific.
α‐Aminoisobutyric Acid Transport into Human Glia and Glioma Cells in Culture
Authors: Ronquist G. , Ågren G. , Ponten J. , Westermark B.
Published: February 2005

The AIB transport into human glia and glioma cells in culture has been studied. Because of the high affinity of AIB to the plastic culture dishes, a special washing technique had to be developed. With this technique, it was possible to perform transport experiments in a single plate containing about one million cells. The cells were viable, intact and adhered to the supporting medium throughout the experiment.

The AIB transport into both types of cells was Na+‐dependent and showed saturation kinetics when the small component of the transport due to diffusion had been subtracted.

The AIB transport capacity of neoplastic glioma cells was 3.6 times higher than that of glia cells. This difference was related to the Vmax‐values for the two types of cells. The apparent Km‐values were the same.

Inhibition experiments with other amino acids support the view that AIB is transported via System A in both glia and glioma cells.

Sulfhydryl reagents (ethacrynic acid and NEM) and cytochalasin B clearly inhibited the AIB transport into glia cells whereas the effect on glioma cells was minimal.
Different recovery patterns of mouse haemopoietic stem cells in response to cytotoxic agents
Authors: Blackett N. M. , Millard R. E.
Published: February 2005

The response and subsequent recovery of mouse haemopoietic progenitor cells (spleen colony forming cells and agar colony forming cells) has been studied following two cytotoxic agents. Busulphan was administered to normal mice and vinblastine to mice where the progenitor cell proliferation rate had been increased by a period of continuous γ‐irradiation. With both these agents there is a difference between the response of the spleen colony forming cells and the agar colony forming cells during the first five days. They then recover together, but much more slowly after busulphan than after vinblastine even though their proliferation rate is increased.

The rate of progenitor cell recovery after busulphan is increased if the progenitor cells are depleted further by vinblastine. However, methotrexate, which severely depletes the peripheral blood count and bone marrow cellularity but not the progenitor cells, has no effect on the recovery following busulphan.

These results suggest that following cytotoxic agents the agar colony forming cells (“committed” stem cells) are not self‐maintaining but are dependent on a supply of cells from the pluripotential spleen colony forming cells. In addition it appears that the depletion of the progenitor cells of the bone marrow and not the depletion of the maturing cells, provides a stimulus for stem cell recovery.
Induction of specific amino acid binding proteins preceding onset of tyrocidine synthesis in Bacillus brevis
Authors: Lipmann Fritz , Lee Sung G.
Published: February 2005

A report on the induction of amino acid binding proteins prior to or together with the formation of enzymes for the biosynthesis of tyrocidine by Bacillus brevis.
The H+/site ratio of mitochondrial electron transport
Authors: Brand Martin D. , Reynafarje Baltazar , Lehninger Albert L.
Published: February 2005

The number of H+ ejected during passage of 2e− through each energy‐conserving site of the mitochondrial respiratory chain (the H+/site ratio) was measured in three ways. In each case transmembrane movements of endogenous phosphate were minimized. (1) Measurement of the uptake of weak acids during loading of mitochondria with Ca2+ demonstrated that 2.0 weak acid anions were accumulated per Ca2+ ion. Since 1.7 to 2.0 Ca2+ ions were taken up per site, these data correspond to an H+/site ratio of 3.5 to 4.0. (2) More direct measurement of H+ ejection using the oxygen pulse technique demonstrated that the H+/site ratio was 3.0. In these experiments phosphate movements were prevented by addition of N‐ethylmaleimide to inhibit phosphate‐hydroxide antiport, by washing the mitochondria to remove endogenous phosphate, or by working at 5°C to reduce the rate of phosphate transport. When phosphate movements were allowed, H+/site ratios of 2.0 were observed. (3) Measurement of the initial steady rates of oxygen consumption and H+ ejection following addition of substrate to aerobic, substrate‐limited mitochondria yielded H+/site ratios of 2.0, which were elevated to 4.0 when phosphate transport was prevented as described above.

Previous determinations of the H+/site ratio were thus underestimates due to the unrecognized movements of endogenous phosphate; our results show that the H+/site ratio is at least 3.0 and may be as high as 4.0.
Is glucose transport enhanced in virus‐transformed mammalian cells? A dissenting view
Authors: Romano Antonio H.
Published: February 2005

Much of the literature on the uptake of glucose by untransformed and transformed animal cells is based on experiments carried out with 2‐deoxy‐D‐glucose (2‐DOG). Results obtained with this analog can be ambiguous, since 2‐DOG can be phosphorylated by hexokinases of animal cells. An intracellular trapping mechanism is thus provided. Therefore, the total flux of 2‐DOG into the cell is a resultant of both transport and hexokinase action, and the measurement of total 2‐DOG incorporation is a valid measurement of transport only if 2‐DOG is phosphorylated as rapidly as it enters the cell. Evidence is presented here that this is not necessarily the case; significant levels of free intracellular 2‐DOG approaching external concentrations were found in untransformed and transformed mouse 3T3 cells even at early times during uptake. Differences in total intracellular 2‐DOG between untransformed and transformed cells were accounted for entirely by 2‐deoxyglucose phosphate. Thus, it appears the apparent increase of 2‐DOG uptake accompanying transformation in these cell lines is not due to an effect on the transport process, but on enhanced phosphorylation, which is a reflection of an alteration in the regulation of glycolysis. The ambiguity introduced by phosphorylation can be obviated by the use of an analog that cannot be phosphorylated, such as 3‐O‐methyl‐D‐glucose. The rate of transport and efflux of this sugar was not found to be different in untransformed versus transformed 3T3 cells. Moreover, deficiencies of this analog as a substrate for the glucose transport system are pointed out.
High‐affinity cytochalasin b binding to normal and transformed BALB/3T3 cells
Authors: Atlas Susan J. , Lin Shin
Published: February 2005

To study the molecular basis of changes in sugar uptake rate in cultured mouse fibroblasts with different physiological states, we have measured the high affinity binding of [3H] cytochalasin B, a potent sugar transport inhibitor, to actively growing and contact inhibited Balb/3T3 cells as well as to 3T12 and SV3T3 cells. Binding was the same whether the cells were detached from dishes with EDTA or trypsin. The amount of drug bound to intact cells measured with a centrifugation assay was essentially the same as that bound to cell sonicates measured with equilibrium dialysis. Cytochalasin B binding to intact cells was extremely rapid and reversible over a wide range of drug concentrations, and was not affected by 0.1 M D‐glucose or L‐glucose in the assay medium. Actively growing and contact inhibited 3T3 cells had a similar number of high affinity cytochalasin B binding sites per cell, while 3T12 and SV3T3 cells had one third to one fourth the number of sites per cell. However, the number of sites per μg cellular protein appeared to be similar for cells in all of the physiological states examined.
Neoplastic potentials and regulation of uptake of nutrients. I. A glutamine independent variant of polyoma bhk with a very high neoplastic potential
Authors: Gammon Maureen T. , Isselbacher Kurt J.
Published: February 2005

A variant of polyoma BHK cells is described which is capable of growing in a glutamine‐free medium. These glutamine‐independent variant cells (GIV) have a size and morphologic appearance intermediate between the nontransformed (BHK) and polyoma transformed (Py6) cell line. In contrast to Py6, the GIV cells grow more slowly, produce less lactic acid and do not grow in suspension. However, their adherence to substrate and release of galactosyl‐transferase into the medium is comparable to that seen with the parent PyBHK. Most striking is the finding that GIV cells are more readily agglutinated by Concanavalin A and when inoculated into hamsters are much more tumorigenic than the parent Py6 cells.
Differences in neutral amino acid and glucose transport between brush border and basolateral plasma membrane of intestinal epithelial cells
Authors: Hopfer Ulrich , Sigrist‐Nelson Kristine , Ammann Elvira , Murer Heini
Published: February 2005

A comparison of L‐valine and D‐glucose transport was carried out with vesicles of plasma membrane isolated either from the luminal (brush border) or from the contra‐luminal (basolateral) region of small intestinal epithelial cells. The existence of transport systems for both non‐electrolytes was demonstrated by stereospecificity and saturability of uptake, as well as tracer coupling. Transport of L‐valine and D‐glucose differs markedly in the two types of plasma membrane with respect to stimulation by Na+. The presence of Na+ stimulated initial L‐valine and D‐glucose uptake in brush border, but not in basolateral membrane. Moreover, an electro‐chemical Na+ gradient, oriented with the lower potential on the inside, supported accumulation of the non‐electrolytes above medium concentration only in the brush border membrane. L‐Valine and D‐glucose transport also were saturated at lower concentrations in brush border (10‐20 mM) than in basolateral plasma membranes (30‐50 mM). A third difference between the two membranes was found in the effectiveness of known inhibitors of D‐glucose transport. In brush border membranes phlorizin was more potent than phloretin and 2′,3′,4′‐trihydroxy‐4‐methoxy chalcone and cytochalasin B did not inhibit at all. In contrast, with the basolateral plasma membranes the order of potency was changed to phloretin = 2′,3′,4′‐trihydroxy‐4‐methoxy chalcone > cytochalasin B > phlorizin.

These results indicate the presence of different types of transport systems for monosaccharides and neutral amino acids in the luminal and contra‐luminal region of the plasma membrane. Active transepithelial transport can be explained on the basis of the different properties of the non‐electrolyte transport systems in the two cellular regions and an electro‐chemical Na+ gradient that is dependent on cellular metabolism.
Heparan sulfates of mouse cells. Analysis of parent and transformed 3T3 cell lines
Authors: Underhill Charles B. , Keller John M.
Published: February 2005

Heparan sulfate from the surface of a variety of mouse cells at different cell densities was examined by ion‐exchange chromatography. The results of this analysis show that:

The heparan sulfate from new isolates of Swiss 3T3 cells transformed SV40 virus (a DNA tumor virus) elutes from DEAE‐cellulose at a lower ionic strength than that from the parent cell type. This finding confirms our earlier observation with an established SV40‐transformed cell line (Underhill and Keller, 1975) and eliminates the possibility that this change is caused by extended passage in culture.

For both parent and transformed 3T3 cells, the heparan sulfates from low and high density cultures were the same as judged by chromatography on DEAE‐cellulose. This result demonstrates that the transformation‐dependent change which we have observed is independent of cell density.

The heparan sulfate from Balb/c 3T3 cells transformed with Kirsten murine sarcoma virus (an RNA tumor virus) elutes from DEAE‐cellulose prior to that from parent Balb/c 3T3 cells. This result extends the transformation dependent change in heparan sulfate to the Balb/c 3T3 cell line and to cells transformed with an RNA virus.
Differential effects of ACTH or 8‐Br‐cAMP on growth and replication in a functional adrenal tumor cell line
Authors: Weidman E. Russell , Gill Gordon N.
Published: February 2005

The effects of ACTH and 8‐Br‐cAMP on growth and replication of a functional mouse adrenal tumor cell line (Y‐1) were investigated. ACTH and 8‐Br‐cAMP both inhibited DNA synthesis and replication when added to randomly growing cell cultures. ACTH addition and serum deprivation each arrested cells in G1; an additional point of arrest in G2 occurred with 8‐Br‐cAMP. Cells whose growth was arrested in G1 by ACTH had a significantly larger volume and protein and RNA content compared to cells arrested in G1 by serum deprivation. When ACTH or 8‐Br‐cAMP was added with serum to cells arrested by serum deprivation, the wave of DNA synthesis and cell division seen with serum was abolished. ACTH and 8‐Br‐cAMP had no effect on the serum‐induced increases in protein and RNA content, rates of leucine incorporation into protein and uridine incorporation into RNA, and RNA polymerase I activity observed in cells during the pre‐replicative period. Partial inhibition of the serum‐induced increase in uridine transport occurred. ACTH and cAMP do not appear to inhibit replication by generalized negative pleiotypic effects but rather to inhibit the initiation of DNA synthesis more specifically. The ACTH‐arrested Y‐1 cell resembles an in vivo hypertrophied adrenal cortical cell.
Organ interactions in the regulation of hematopoiesis: In vitro interactions of bone, thymus, and spleen with bone marrow stem cells in normal, Sl/Sld and W/Wv mice
Authors: Cline Martin J. , Le Fevre Carol , Golde David W.
Published: February 2005

Hematopoietic cell differentiation is influenced by organ‐dependent microenvironmental factors as well as humoral regulators. A technique is described for examining certain aspects of the hemopoietic inductive microenvironment in vitro.

Suspension and agar cultures of mouse bone marrow were used to study the effects of organ stromal factors on cellular proliferation and differentiation. Bone, spleen, and thymus fragments from irradiated mice were placed in direct contact with or separated by a Nuclepore membrane from syngeneic marrow cells growing in suspension cultures. Normal adult mouse bone and spleen influenced granulocytic differentiation as well as cell proliferation.

In this system, bone marrow and organ fragments from W/Wv and Sl/Sld mice behaved like those of their non‐anemic littermates. The most prominent difference between W/Wv and Sl/Sla mice and their normal counterparts was observed in the induction of CFU‐C from splenic precursors under the influence of CSA. In both types of anemic mice, in vitro generation of CFU‐C from spleen was abnormal in young animals but was corrected by four months of age.
Messenger RNA regulation in human diploid fibroblasts
Authors: Meedel Thomas H. , Levine Elliot M.
Published: February 2005

In resting, non‐growing human diploid fibroblasts the amount of rRNA is reduced 1.8‐fold, cytoplasmic polysomes are disaggregated, and the level of poly‐A RNA (mRNA) is reduced 1.8‐fold in relation to growing cells. The distribution of poly‐A RNA is altered in resting, non‐growing cells so that an average of 64% of the total cytoplasmic poly‐A RNA sediments along with particles lighter than 80S (prepolysomal) in sucrose density gradients. By comparison, in growing cells only 30% of the cytoplasmic poly‐A RNA sediments in the prepolysomal region. In SDS sucrose gradients, the sedimentation profile of the prepolysomal poly‐A RNA from resting cells resembles that of polysomal poly‐A RNA from those cells. In contrast, the average size of prepolysomal poly‐A RNA from growing cells is much smaller than that of the polysomal poly‐A RNA from those cells. These data are compatible with the possibility that resting cell prepolysomal poly‐A is untranslated mRNA. Also consistent with this interpretation are experiments which demonstrate that one‐quarter to one‐third of the prepolysomal poly‐A RNA of resting cells is recruited into polysomes in the presence of cycloheximide.
Differential effects of inhibitors of cell division upon the growth stimulating activities of insulin and serum in nutritionally depleted human and mouse cells
Authors: Kamely Daphne
Published: February 2005

Cultured mouse and human cells were arrested in their growth by artificially depriving them of phosphate. The quiescent cells could be stimulated to synthesize DNA and to divide by addition to the growth medium of insulin, dialyzed serum and/or the full concentration of phosphate. In order to gain insight into mechanisms by which insulin and serum stimulate growth, the inhibitory effects of antimitotic agents were examined. Of the inhibitors tested, vinblastine and cytocalasin B abolished the growth promoting activity of insulin, while colchicine inhibited the activity of both serum and insulin. The present results suggest that insulin‐stimulated growth is mediated by a different pathway than serum‐stimulated growth and is sensitive to mechanisms that occur at various times prior to insulin addition.
The effects of erythropoietin in vitro on spleen colony‐forming cells
Authors: Van Zant Gary , Goldwasser Eugene , Pech Nancy
Published: February 2005

Erythropoietin (epo) added to liquid cultures of mouse bone marrow cells affected both the numbers of spleen colony‐forming cells (CFU) in the cultures and the types of spleen colonies formed from these cells in irradiated hosts. Epo caused an increase in the numbers of CFU detected in cultures on the second day; this effect persisted through day 10, with the maximal increase occurring on the seventh day. The magnitude of the rise on day 7 was proportional to the amount of epo added. The increase in spleen colonies found with cells cultured in the presence of epo was due solely to erythroid colonies. After seven days in culture without epo, there was a peak of cells that formed non‐erythroid colonies. This peak did not appear when the cells were cultured in the presence of epo.
The effects of biotin and fatty acids on SV3T3 cell growth in the presence of normal calf serum
Authors: Messmer Trudy O. , Young Delano V.
Published: February 2005

The growth of SV3T3 cells in medium containing a low concentration (0.20% v/v) of normal calf serum is enhanced by the addition of biotin or certain unsaturated fatty acids. The biotin effect on the final viable cell density is 5‐ to 10‐fold over the control and is extremely potent, exerting a saturating response at a concentration of approximately 200 pg/ml. The optimal growth response observed with fatty acids is 5‐fold over the control and requires the combination of nervonic acid, palmitoleic acid, and arachidonic acid. The fatty acids are probably not replacing the function of biotin since these two substances are additive in their growth effects.
Secondary activities of diverse inhibitors potentiate the response of hamster embryo cultures to a mitotic stimulus
Authors: Mironescu Stefan , Ellem Kay A. O.
Published: February 2005

The reversal of the density dependent inhibition of replication (DDIR) of Syrian hamster embryo cultures by fresh medium containing 30–50% fetal bovine serum was poorly synchronized. There were two waves of DNA synthesis eight to nine hours apart, which by examination of autoradiograms of cultures pulse‐labeled with 3H‐thymidine, were found to involve 55% and 45% of the cell population. The second wave was found to be due to a subpopulation of epithelioid cells set in the predominantly fibroblastic cell cultures.

Pretreatment of the DDIR cultures with hydroxyurea (HU) or arabinosyl cytosine (ara‐C), followed by serum stimulation in the absence of the drugs, led to an enhancement of the synchrony. The effect increased with lengthening of the contact with antimetabolites, to a maximum after 20 hours' pre‐exposure, and was in part due to the shortening of the G1 phase of the epithelioid elements, and in part to increasing the synchrony of the fibroblastic cells. The resulting synchrony involved some 95% of the cells in simultaneous DNA synthesis after a median G1 period of 12 hours. The effect had no relationship to the role of HU and ara‐C as specific inhibitors of DNA synthesis since the cultures were mitotically quiescent, and a similar enhanced response could be induced in DDIR cultures by prestimulation exposure lasting only two hours to cycloheximide (cyx), an inhibitor of protein synthesis. Pre‐exposure of DDIR cultures to actinomycin D did not potentiate the cell response. A survey of the known secondary inhibitions caused by these three antimetabolites suggests that they all may cause deficiencies in the glycolipid or glycoprotein moieties of the cell surface. These observations provide a useful, simple means of improving synchrony in these systems and may prove to be a useful probe for investigating the role of the cell surface in regulating cell replication.
Replication kinetics of three DNA sequence families in synchronized mouse cells
Authors: Dooley Douglas C. , Ozer Harvey L.
Published: February 2005

Balb/C 3T3 cells entered the quiescent G0 state following serum deprivation. On addition of fresh serum, more than 95% of the culture resumed growth, but with asynchronous kinetics. If hydroxyurea were added just before the first cells reached S phase, at least 90% of the cells accumulated at the Gl/S border over the next ten hours. When the block was removed, the culture moved synchronously into S phase. As the cells traversed S, the replication kinetics of three classes of rapidly renaturing DNA were analyzed. Main band highly repeated DNA and foldback DNA replicated continuously. In contrast, satellite DNA replication did not commence until three hours into S, whereupon its rate of synthesis increased very rapidly, reaching a maximum within the next two hours. These results are discussed in the light of earlier work utilizing other methods of cell synchronization.
Comparative studies of glucose‐fed and glucose‐starved hamster cell cultures: Responses in galactose metabolism
Authors: Christopher C. William , Colby Wendy W. , Ullrey Donna , Kalckar Herman M.
Published: February 2005

The metabolic flow of trace amounts of D‐[14C]‐galactose was followed in cultures of transformed and untransformed hamster cells over a period ranging from five minutes to two hours. The results of chromatographic and enzymatic analyses of the soluble pools are described. Non‐glycolytic cells (previously deprived of sugar for periods of up to 24 hours) convert D‐galactose to galactose‐1‐phosphate and uridine diphosphoglucuronic acid in 10 to 20 minutes. In the same short assay time, glycolytic cells which have been maintained for 24 hours in media containing glucose or galactose convert D‐galactose to uridine diphosphogalactose and uridine diphosphoglucose (ratio 1.4:1). Longterm deprivation of sugar also results in 3‐ to 4‐fold increases in the uptake of galactose. In addition, the incorporation of galactose label into chloroform‐methanol soluble material appears to be influenced by the culture conditions of the untransformed cells while incorporation in the transformed cells appears unaffected. When cycloheximide is included in the maintenance medium for extended periods, the non‐glycolytic cells also show increases in galactose uptake rates but the glucose‐fed, glycolytic cells lose uptake ability. UDPhexose is the main galactose metabolic peak in the soluble pools of the cycloheximide‐treated, glycolytic and the cycloheximide‐treated, non‐glycolytic cells. The results of these experiments suggest that uptake of galactose and its subsequent metabolism are under separate control.
Characterization of the cell cycle of cultured human diploid cells: Effects of aging and hydrocortisone
Authors: Grove Gary L. , Cristofalo Vincent J.
Published: February 2005

Age‐related changes in the cytokinetics of human diploid cells in vitro have been compared in normal cultures and in cultures in which lifespan has been prolonged by the addition of hydrocortisone to the medium. For both cultures, with advancing age the fraction of cells in the actively proliferating pool decreased and the intercellular variation in cell cycle times increased. The average cell cycle time was prolonged during aging due almost entirely to changes in the duration of G1. The duration of S remained constant, while a small delay in G2 was observed in late passage cells near the end of their lifespan. Although the same pattern of change in proliferative parameters occurred in both control and hydrocortisone‐treated cultures, the changes were somewhat delayed in the presence of the steroid. The results are interpreted in terms of several cell cycle models and suggest that the events controlling cell proliferation are sensitive to hydrocortisone modulation during the G1 and possibly the G2 periods.
Surface membrane differentiation of hemopoietic cells as observed by radioactive labeling
Authors: Fehlmann M. , Lafleur L. , Marceau N.
Published: February 2005

Bone marrow cells from normal rats were separated by velocity sedimentation into three distinct populations corresponding to granulocytes, lymphocytes and reticulocytes. Cells from each population were surface labelled via the lactoperoxidase radioiodination or the tritiated borohydride reduction. Distinct labeling patterns were observed on SDS‐PAGE for each cell population. Separation of bone marrow cells from anemic rats injected with TAB vaccine led to four populations corresponding to successive stages of erythroid cell maturation. Labelled protein patterns were not in this case as different from one population to the other, except for one species which increased in intensity with the degree of maturation. The tritiated glycoprotein profiles show a shift from high to low molecular weight species during the process of maturation.
Coordinate regulation of protein synthesis and messenger RNA content during growth arrest of suspension Chinese hamster ovary cells
Authors: Levis Robert , McReynolds Larry , Penman Sheldon
Published: February 2005

We have found Chinese Hamster Ovary cells, cultured in suspension, are subject to growth control by serum. When suspended in medium containing 0.5% serum the cells become reversibly arrested in the beginning of the G1 phase of the cell cycle and can be maintained in this viable, nonproliferating state for several days.

This system was used to examine the regulation of protein synthesis with growth rate. In particular, the experiments addressed the question whether mRNA content is the principal controlling factor determining the rate of protein synthesis. The rate of leucine incorporation in resting cells in low serum is 2‐ to 2.5‐fold lower than that of cells growing in 10% serum. The steady‐state number of cytoplasmic poly A (+) RNA molecules shows a proportional decrease, consistent with it being a determining factor controlling the rate of protein synthesis. Furthermore, the rate of production of poly A (+) and poly A (−) RNA appears to be regulated coordinately.

Regulation of the rate of initiation of translation would result in fewer ribosomes bound per active message and/or a lower proportion of total mRNA's being active. Our measurements indicate that the fraction of cytoplasmic poly A (+) mRNA in polyribosomes and the relative degree of loading of each active poly A(+) mRNA with ribosomes is the same in resting and growing cells. Thus these cells resemble 3T6 and translational control does not appear to be an important part of the change in protein synthetic rate with the state of growth.
Chloride‐Stimulated sulfate efflux in Ehrlich ascites tumor cells: Evidence for 1:1 coupling
Authors: Villereal Mitchel L. , Levinson Charles
Published: February 2005

The kinetics of Cl−‐SO4−2 exchange in Ehrlich ascites tumor cells was investigated in an attempt to determine the stoichiometry of this process.

When tumor cells, equilibrated in Cl−‐free, 25 mM SO4−2 medium are placed in SO4−2‐free, 25 mM Cl− medium, both the net amount and rate of Cl− uptake far exceeds SO4−2 loss. Addition of the anion transport inhibitor SITS (4‐acetamido‐4′‐isothiocyano‐stilbene‐2,2′‐disulfonic acid) greatly reduces sulfate efflux (97%), but has no measurable effect on chloride uptake. Addition of furosemide, a Cl− transport inhibitor, reduces chloride uptake 94% but is without effect on sulfate efflux. These findings suggest that a chloride permeability pathway exists distinct from that utilized by SO4−2.

SITS, when added to furosemide treated cells, further reduces chloride uptake as well as inhibiting sulfate efflux, and under these experimental conditions, a linear relationship exists between SITS‐sensitive, net chloride uptake and sulfate loss. The slope of this line is 1.05 (correlation coefficient = 0.996) which suggests the stoichiometry of Cl−‐SO4−2 exchange is 1:1. Assuming a 1:1 stoichiometry, measurement of the initial chloride influx and initial sulfate efflux indicate that 92% of net chloride uptake is independent of sulfate efflux.

Taken altogether, these results support the contention that the tumor cell possesses a permeability pathway which facilitates the exchange of one sulfate for one chloride. Under conditions where anion transport is not inhibited, this coupling is obscured by a second and quantitatively more important pathway for chloride uptake. This pathway is SITS‐insensitive, although partially inhibited by furosemide.
Variations in prolactin and growth hormone production during cellular growth in clonal strains of rat pituitary cells
Authors: Haug Egil , Tjernshaugen Hans , Gautvik Kaare M.
Published: February 2005

A permanent, clonal strain of rat pituitary tumor cells (GH3‐cells) spontaneously synthesizes and secretes prolactin (rPRL) and growth hormone (rGH) into the culture medium. The rates of hormone production (μg extracellular hormone/mg cell protein/24 hours) and synthesis (vida infra) as well as the rate of [3H]thymidine incorporation into DNA (DNA synthesis) have been studied.

During logarithmic growth rPRL and rGH production increased to 160 and 250% of the value at day 2 after plating, while during the plateau phase of cell growth hormone production decreased to initial values. The fluctuations in rPRL production could be fully explained by variations in the rate of rPRL synthesis: [3H]leucine incorporated into rPRL as measured with immunoprecipitation and polyacryl‐amide gel electrophoresis. Also the rates of synthesis and production of rGH showed parallel changes during exponential and plateau phase of growth, but this hormone was probably degraded intracellularly. The relative reduction in the rate of synthesis of rPRL and rGH during the plateau phase of growth corresponded closely to the fall in the rate of DNA synthesis. The reduction in rPRL synthesis could not be explained through an inhibition by extra‐cellular rPRL accumulation or by cell to cell interaction occurring in dense cultures.

The intracellular concentrations of both hormones were unaltered during logarithmic growth, but rose to 500% for rPRL and 200% for rGH during the plateau phase.

In spite of the marked variations in basal rPRL and rGH production, the GH3 cultures of different ages were equally able to increase rPRL and decrease rGH production in response to thyrotropin releasing hormone (3 X 10−7 M) and 17β‐estradiol (10−8 M).
The effect of medium pH on rate of growth, neurite formation and acetylcholinesterase activity in mouse neuroblastoma cells in culture
Authors: Bear Mark P. , Schneider F. Howard
Published: February 2005

Cell division, neurite formation and acetylcholinesterase activity were examined in a clone (NBA2) of mouse neuroblastoma cells maintained for up to 120 hours in medium with pH values between 6.6 and 8.0. Growth rate decreased as pH was reduced from 7.8 to 6.6. Generation time at pH 7.4 was 25 hours, while the rate of cell division was negligible at pH 6.6. The total number of cells at stationary phase was less at the lower pH values. Neurite formation was enhanced markedly as the pH was reduced from 7.4 to 6.6. Acetylcholinesterase activity was 5‐ to 8‐fold greater in cells exposed to medium at pH 6.6 than in cells maintained in medium at pH 7.4. The reduction in the rate of cell division and increases in neurite formation and acetylcholinesterase activity at pH 6.6 were reversible upon exposure of the cells to pH 7.4 medium. Cell viability was greater than 90% at all medium pH values over a period of 120 hours. Uncloned T‐59 mouse neuroblastoma cells were affected similarly by changes in pH. These results show that manipulation of the environmental pH can reversibly alter growth, neurite formation, and acetylcholinesterase activity of mouse neuroblastoma cells in culture.
Erythroid colony formation in cultures of human marrow: Effect of leukocyte conditioned medium
Authors: Aye M. T.
Published: February 2005

A method, based on the differing capacities of cells to adhere to a column of polyester fibres, has been described for separating human bone marrow cells into a nonadherent and an adherent fraction. The effect of this cell separation procedure on colony formation by erythroid progenitor cells was investigated. In contrast to the unseparated population, it was found that erythropoietin‐dependent erythroid colony formation by nonadherent cells could be considerably enhanced by the addition of leukocyte conditioned medium to the cultures. Similar erythroid enhancing activity was also detected in a partially purified preparation of granulocytic colony stimulating activity obtained from human embryo kidney culture supernatants.

Erythroid colony formation in the absence of added erythropoietin, by non‐adherent bone marrow cells from patients with polycythemia rubra vera, were also enhanced by the addition of LCM to the cultures. This finding suggests that the enhancing factor in LCM may not be dependent on the presence of erythropoietin in the cultures for its activity.

While the cellular mechanisms by which leukocyte conditioned medium enhances eyrthroid growth remain to be determined, the data presented provides strong evidence for the view that the plating efficiency of erythroid progenitor cells is determined not only be the concentration of erythropoietin, but also by the presence of leukocyte conditioned medium in the cultures.
Hormonal control of hyaluronic acid production in fibroblasts and its relation to nucleic acid and protein synthesis
Authors: Moscatelli David , Rubin Harry
Published: February 2005

Whole serum and elevated pH previously had been found to stimulate both cell multiplication and hyaluronic acid production by chick embryo fibroblasts in culture. In a study to determine whether cell multiplication and hyaluronic acid production both respond to a single well‐defined substance, insulin was found to stimulate, and cortisol to inhibit both processes coordinately. It appears, therefore, that multiplication and differentiated function in fibroblasts respond to a common underlying regulatory signal.

Inhibition of ribosomal RNA synthesis by actinomycin D does not prevent serum stimulation of hyaluronic acid production, but inhibition of total RNA synthesis does. If total RNA synthesis is inhibited only after the hyaluronic acid production has reached a new high level, it continues at that level for the next five hours. The stimulatory treatment causes an increase in the activity of the enzyme hyaluronate synthetase. Inhibition of protein synthesis prevents any increase in hyaluronic acid production, and reduces the basal level of production.

Reduction of the availability of Mg2+ in the medium coordinately inhibits DNA synthesis and hyaluronic acid production. The results are discussed in the light of a model for coordinate control of growth and metabolism based on the availability of Mg2+.
The effect of 2‐deoxyglucose on guinea pig polymorphonuclear leukocyte phagocytosis
Authors: Boxer Laurence A. , Baehner Robert L. , Davis Jacqueline
Published: February 2005

The effects of 2‐deoxyglucose (DOG), an inhibitor of glycolysis, on guinea pig polymorphonuclear leukocytes (PMN) obtained from peritoneal exudates was examined. ATP levels in PMN were reduced by 40% by one hour following an incubation with 2‐deoxyglucose. When complement (C3) coated 14C‐staphylococcus aureus, C3 coated lipopolysaccharide‐paraffin oil droplets (LPSPO), 14C‐pneumococcus opsonized with IgG, or albumin coated paraffin oil droplets opsonized with IgG were added to cell suspensions containing DOG, the phagocytizing rate was 1,310 ± 55 cpm/5 x 106 cells/15 minutes, 6 ± 2 μg paraffin oil (PO)/107 cells/minute, 2,250 ± 175 cpm/1 x 106 cells/20 minutes or 0.037 ± 0.01 mg PO/107 cells/minute compared to control values of 5,970 ± 275 cpm/5 x 106 cells/15 minutes, 35 ± μg PO/107 cells/15 minutes, 4,510 ± 200 cpm/1 x 106 cells/20 minutes and 0.067 ± 0.01 mg PO/107 cells/minute. In parallel studies the phagocytic index for latex was 0.74 ± 0.28 in DOG compared to control of 2.36 ± 1.13 and the phagocytic rate of albumin coated paraffin oil droplets was 0.029 ± 0.01 mg PO/107 PMN/minute in DOG compared to control of 0.048 mg PO/107 cells/minute. When ATP levels were maintained by the simultaneous addition of 5 mM glucose or pyruvate to media containing DOG, latex ingestion was improved to 1.15 ± 0.3 with glucose and 1.59 ± 0.64 with pyruvate and albumin coated particles to 0.045 ± 0.01 mg PO/107 PMN/minute with pyruvate. There was no improvement in the uptake of either the C3 dependent particles or IgG coated Pneumococci in media containing DOG and glucose and/or pyruvate. Following the removal of DOG from the extracellular medium and the addition of pyruvate or glucose, phagocytosis of C3 dependent LPS‐PO was restored to normal values. Neither the binding of C3 or IgG coated particles to the PMN nor the lateral movement of glycoprotein utilizing concanavalin A capping was affected by DOG. Thus, the presence of DOG in the PMN containing adequate amounts of ATP will selectively and reversibly inhibit those surface events required for phagocytosis of C3 and IgG bound particles but not latex particles or albumin particles which non‐specifically bind to PMN.
Characterization of a depolarizing dopamine response in a vertebrate neuronal somatic cell hybrid
Authors: Myers Paul R. , Livengood David R. , Shain William
Published: February 2005

The physiology and pharmacology of a depolarizing dopamine response was studied in the vertebrate neuronal somatic cell hybrid TCX11. The average resting membrane potential was −50 mV (S.D. = ±7) with a membrane resistance of 40.5 mOhms (S.D. = ±8) as determined from intracellular recordings. Depolarizing current pulses did not elicit an action potential. Cells displayed a linear current‐voltage relationship when artificially depolarized up to +30 mV. Iontophoretically applied dopamine elicited a depolarizing response with a conductance increase and a reversal potential of −15 mV (S.D. = ±4.7). Experiments altering medium ion concentrations demonstrated the conductance increase was to sodium and most likely potassium. The dopamine agonist ET495 (Piribedil) and the analogue epinine mimicked dopamine, while closely related biogenic amines, with the exception of noradrenaline, elicited no response. Apomorphine also elicited a depolarizing response but was much less efficacious than Piribedil. Noradrenaline was less potent than dopamine and appeared to act at the dopamine receptor. Methylation (3‐methoxytyramine) or absence of the 3‐hydroxy group (tyramine) of dopamine resulted in total loss of activity. The dopamine antagonists chlorpromazine, trifluoperazine, promazine, and bulbocapnine reversibly blocked the response to dopamine at medium concentrations less than 5 μM. The adrenergic antagonist phentolamine blocked the response while phenoxybenzamine only reduced the response at higher concentrations. The acetylcholine antagonists α‐bungarotoxin, hexamethonium, and scopolamine did not block the dopamine response. Both d‐tubocurarine and atropine acted as antagonists.

Collectively, these results demonstrate the presence of a receptor on a cultured cell line that is specific for dopamine, mediates a depolarizing and conductance increase response to dopamine, and displays the pharmacology most closely associated with dopamine receptors.
In vitro activation of the in vivo colony‐forming units of the mouse yolk sac
Authors: Perah Gershon , Feldman Michael
Published: February 2005

Experiments were performed to investigate the presence of colony‐forming units (CFU) in the mouse embryonic yolk sac during the developmental period in which the yolk sac is the sole hemopoietic organ. Injection of yolk sac cell suspensions from normal embryos into syngeneic, lethally irradiated adult recipients evoked a very low number of spleen colonies. However, prior cultivation of yolk sacs in vitro caused a dramatic increase in the spleen colony‐forming capacity–as high as 84‐fold–following 48 hours in culture. The yolk sac origin of the spleen colonies was confirmed by: (a) Chromosomal marker analysis; (b) dose‐response analysis; (c) demonstrating that the above colonies were not of endogenous origin induced by the mere injection of grafted cells. We conclude that the yolk sac contains many precursors of colony‐forming cells which though undetectable by immediate grafting apparently become activated in culture by an as yet unknown induction process.
The dissociation of the surface architecture described by enhanced lectin agglutinability and the transformed phenotype expressed as anchorage independence
Authors: Noonan Kenneth D. , Bouck Noël , di Mayorca Giampiero
Published: February 2005

Using a series of cold‐sensitive variants of chemically transformed BHK‐21 cells, revertants to the normal phenotype derived from a dimethylnitrosamine transformed clone of BHK‐21 as well as revertants to the normal phenotype derived from polyoma transformed BHK‐21 cells we have demonstrated that the surface phenotype described by enhanced agglutinability with Con A and WGA can be dissociated from the transformed phenotype described by anchorage independence (growth in semisolid medium). Specifically we have demonstrated that the surface characteristic of enhanced agglutinability may be found in a variety of cell lines which fail to display an ability to grow in agar. Our work clearly shows that the two phenotypes described are not concomitantly controlled and tends to suggest that the phenotype of enhanced lectin agglutinability may be dissociated from the transformed phenotype.