bluuebunny/crossref_metadata_2025 huggingface dataset:
Cystamine augments the stimulation of DNA synthesis by peptide growth factors and microtubule‐disrupting agents in cultures of 3T3 mouse fibroblasts
Authors: Fong Steven , Friedkin Morris
Published: February 2005

Cystamine together with colchicine markedly enhanced the uptake of [3H]‐thymidine into DNA of quiescent cultures of insulin‐stimulated Swiss 3T3 mouse fibroblasts. Flow cytofluorometric analyses showed an increased rate of transition of cells from G0/G1 → S + G2 in response to combinations of insulin, colchicine, and cystamine. Cystamine, the most effective of several thiol compounds, gave maximal augmentation at 200 μM and was toxic at 300–500 μM. Amplification of DNA synthesis by cystamine was also obtained with epidermal growth factor, vasopressin, and 0.5% fetal bovine serum. Combinations of cystamine and other microtubule‐disrupting agents such as nocodazole, maytansine, and podophyllotoxin enhanced DNA synthesis in insulin‐stimulated cells. In experiments involving sequential addition of agents, significant enhancement of DNA synthesis was observed when the addition of colchicine to cystamine‐treated cells was delayed or conversely when the addition of cystamine to colchicine‐treated cultures was delayed. This reciprocal interaction between cystamine and colchicine suggests that a prereplicative intermediate accumulates in response to the action of these dissimilar compounds. We consider the possibility that cystamine may act by forming mixed disulfides with thiol groups of unknown protein(s) that regulate DNA replication.
3‐Aminobenzamide is lethal to MMS‐damaged human fibroblasts primarily during s phase
Authors: Boorstein Robert J. , Pardee Arthur B.
Published: February 2005

3‐Aminobenzamide (3‐AB) interferes with DNA repair and enhances lethality in growing MMS (methyl methane sulfonate)‐treated human fibroblasts. This sensitivity to 3‐AB disappears slowly; MMS‐treated cells are sensitive to 3‐AB for up to 36 hours (Boorstein and Pardee, 1984). Evidence is now presented that 3‐AB potentiates the effects of MMS primarily during S phase. When cells were synchronized at the G1/S boundary, released, and then treated with MMS, 3‐AB caused very substantial lethality in only 4 hours, and a 12‐hour treatment gave maximum lethality. These cells also lost sensitivity to 3‐AB within 12 hours of growth minus 3‐AB. In contrast, MMS‐treated quiescent (G0) cells did not lose sensitivity to 3‐AB nor did 3‐AB cause lethality during G0. Enhanced lethality occurred when damaged G0‐arrested cells were subsequently allowed to proceed through S phase in the presence of 3‐AB; this 3‐AB sensitivity was removed only during growth in the absence of 3‐AB. The lethality of 3‐AB to a population of asynchronously cycling cells treated with MMS is thus the summation of effects on the cells as they traverse S phase. Aphidicolin prevenced lethality of 3‐AB to cells released from G1/S and treated with MMS. It also inhibited the loss of sensitivity to added 3‐AB later. Correlation with the inhibition of DNA synthesis by this drug suggests that DNA synthesis is essential for the lethality enhancement by 3‐AB. Cells treated first with MMS and then with 3‐AB accumulated in G2. This G2 arrest depended on S‐phase events and correlated with cell lethality. Cells treated with a nonlethal dose of MMS at the G1/S boundary were delayed briefly (3 hours) in their passage through S and G2. These cells, when also exposed to 3‐AB, were delayed 6–9 hours in S and they became arrested in G2. There was no G2 arrest when 3‐AB was added only after these cells had reached G2. Treatment with 3‐AB during S phase thus resulted in both enhanced lethality and G2 arrest. 3‐AB inhibited repair of DNA single‐strand damage, shown by alkaline elution analysis, in both S‐phase and quiescent cells. Aphidicolin inhibited disappearance of breaks and eliminated the difference between 3‐AB‐treated and untreated cells. Lethality did not correlate well with the measured single‐strand damage. We propose that there is a class of MMS‐induced lesions whose repair occurs during normal replicative DNA synthesis. When this repair is interrupted by 3‐AB, during S phase, sublethal damage is converted to lesions which arrest the cells in G2 phase and prevent them from forming colonies. DNA synthesis is required to reach the step in DNA repair at which 3‐AB causes lethality.
Induction of microvillar hydrolase activities by cell density and exogenous differentiation inducers in an established kidney epithelial cell line (LLC‐PK1)
Authors: Yoneyama Yoshitaka , Lever Julia E.
Published: February 2005

Several hydrolase activities characteristic of the apical brush border membrane of renal proximal tubule, leucine aminopeptidase, γ‐ glutamyl transpeptidase, alkaline phosphatase, maltase, and trehalase, were identified in cultures of the LLC‐PK1 kidney epithelial cell line. A coordinate increase in activities of these enzymes was observed upon development of a confluent cell density and functional membrane polarization. Further large progressive increases in individual hydrolase activities were induced after the addition of compounds known as differentiation inducers. Hexamethylene bisacetamide preferentially induced increased trehalase and maltase activities. Induced trehalase activity exhibited an increased Vmax but a similar Km compared with activity in control extracts. Induction required protein synthesis and was dependent on inducer concentration and exposure time. Treatment of confluent cultures with N,N′‐dimethylformamide triggered an induction of maltase, trehalase, alkaline phosphatase, and γ‐glutamyl transpeptidase activities, whereas dimethylsulfoxide induced trehalase and γ‐glutamyl transpeptidase activities. Increased leucine aminopeptidase and maltase activities were observed after addition of the phosphodiesterase inhibitor 1‐methyl‐3‐isobutylxanthine. Induction of trehalase activity by N,N′‐dimethylformamide was reversible over a 4‐day period after removal of inducer, but effects of hexamethylene bisacetamide were irreversible. These results suggest that the LLC‐PK1 cell line reproducibly develops differentiation‐specific characteristics under defined conditions in cell culture, which can be individually modulated by chemicals known as inducers of cell differentiation.
An acellular human amnionic membrane model for in vitro culture of type ii pneumocytes: The role of the basement membrane in cell morphology and function
Authors: Lwebuga‐Mukasa Jamson S. , Thulin Gunilla , Madri Joseph A. , Barrett Carolyn , Warshaw Joseph B.
Published: February 2005

To determine whether a preformed basement membrane contributes to the maintenance of morphology and function of type II pneumocytes, we cultured isolated adult rat type II pneumocytes on the basement membrane and stromal surfaces of an acellular human amnionic membrane and on plastic. The presence of lamellar bodies on transmission electron microscopy and epithelial morphology in culture and a characteristic phospholipid profile after incubation with 3H‐acetate identified the cells as type II. When type II cells were cultured on a preexisting basement membrane, they formed a well‐organized monolayer with polarity, centrally located surface microvilli, and more basally located nuclei. Individual cells maintained a cuboidal morphology for 8–10 days. Intracellularly, there were numerous mitochondria, endoplasmic reticulum (ER), and lamellar bodies. The cells secreted a new basal lamina of their own. When cultured on the stromal side of the amnion, the cells became flattened within 48–60 hours, formed small lamellar bodies, and had scanty surface microvilli; they formed clumps and appeared less ordered. These cells did not secrete a visible basement membrane, and the majority detached from the stromal surface after 7–8 days in culture. In addition, culture on the basement membrane aspect of the amnion prevented the rapid decline in the percentage of 3H‐acetate label incorporated in phosphatidylcholine after 72 hours of culture. We conclude that a preformed basement membrane influences the function and morphology of type II pneumocytes, organizes them into a monolayer in culture, and influences deposition of a visible basal lamina. Thus, the acellular human amnion provides an excellent model for the systematic study of basement membrane influence on the biology and pathology of these cells.
Transcriptional induction of urokinase in cultured human kidney carcinoma cells by tetradecanoyl‐phorbol‐acetate
Authors: Ferraiuolo Rosa , Stoppelli M. Patrizia , Verde P. , Bullock Sarah , Lazzaro Pasquale , Blasi Francesco , Pietropaolo Tina C.
Published: February 2005

A cell line derived from a human kidney carcinoma produces in vitro the urinary type of plasminogen activator (urokinase). The synthesis of plasminogen activator is enhanced by 12‐O‐tetradecanoyl‐phorbol‐13‐acetate(TPA); the increase can be followed both in the cell lysate and in the culture medium. The effect requires RNA and protein synthesis as well as the continuous presence of the inducer. Immunofluorescence and immunoprecipitation experiments with monospecific antiurokinase IgG show that kidney carcinoma cells synthesize a 50,000‐dalton urokinase and TPA enhances the synthesis of the same molecular species. Hybridization of the total cellular RNA to a human urokinase cDNA probe shows that TPA increases the urokinase mRNA level.
Ca2+ and hormones interact synergistically to stimulate rapidly both prolactin production and overall protein synthesis in pituitary tumor cells
Authors: Brostrom Margaret A. , Brostrom Charles O. , Bocckino Stephen B. , Green Susan S.
Published: February 2005

Effects of Ca2+ and hormones on short‐term protein synthesis were examined utilizing intact Ca2+‐depleted and Ca2+‐restored GH3 pituitary tumor cells as a model system. Amino acid incorporation by cells in complete growth medium during short incubations was markedly reduced by EGTA concentrations in excess of Ca2+. Thyrotropin‐releasing hormone (TRH) rapidly enhanced amino acid incorporation and prolactin production, with both effects being reserved by EGTA in excess of extracellular Ca2+ or prevented by cellular Ca2+ depletion. Epidermal growth factor and phorbol myristate acetate (PMA) also stimulated amino acid incorporation and prolactin production; absolute increases in protein synthesis provided by these agents were significantly greater in Ca2+‐restored than in Ca2+‐depleted preparations. TRH and PMA concentrations which raised prolactin production were identical to those increasing the rate of amino acid incorporation into overall protein. The extracellular Ca2+ concentration dependencies of amino acid incorporation and prolactin production were similar and were unchanged by hormone. PMA, the most efficacious of the agents tested, and Ca2+ promoted incorporation of amino acid into the same spectrum of proteins. Stimulation of protein synthesis by hormones was not attributable to alterations in amino acid uptake, attachment to substrata, hormone binding, protein catabolism or transcription. Trifluoperazine selectively prevented the stimulation by Ca2+ of amino acid incorporation and prolactin production. Unlike total prolactin, the total protein content of GH3 cells during these short incubations was not altered by Ca2+, hormones or trifluoperazine. It is proposed that hormones and Ca2+, which have been demonstrated to regulate prolactin secretion and prolactin mRNA transcription in GH3 cells, also exert translational controls which serve to facilitate the overall expression of the prolactin gene.
Control of DNA synthesis in polyploid mammalian cells
Authors: Graves Jennifer A. Marshall , McMillan Julie
Published: February 2005

To test the hypothesis that the duration of DNA synthesis is an inverse function of nuclear size or DNA content, the S phase was calculated from PLM analysis for pseudodiploid, tetraploid, and octaploid lines of Chinese hamster cells growing as a monolayer or in suspension. S phase times were found not to be significantly different between polyploid lines and the diploid lines from which they were derived, regardless of the conformation of the nucleus. There is no evidence, therefore, that would implicate the nuclear membrane, or nuclear surface area/volume relationships, in the control of DNA synthesis.
Stimulation of DNA‐mediated transformation by UV irradiation of recipient (mouse FM3A) cells
Authors: Nagata Yoshiho , Takagi Hideki , Morita Toshimi , Oishi Michio
Published: February 2005

DNA‐mediated transformation for thymidine autotrophs (Tk+) was stimulated severalfold when the recipient (mouse FM3A) (Tk−) cells were preirradiated by UV light. The effect was most prominently seen with the cells irradiated 12–16 hours prior to DNA addition. A similar stimulatory effect was observed when the cells were treated with novobiocin or mitomycin C. The effect, however, was blocked when cycloheximide was present during postirradiation period, suggesting that de novo protein synthesis is required for producing the effect. Most of the Tk+ transformants arised from the UV‐irradiated cells carried the donor DNA sequences in their chromosomes.
The effect of the extracellular matrix on the detachment of human endothelial cells
Authors: Gordon Portia B. , Levitt Mindy A. , Jenkins C. S. P. , Hatcher Victor B.
Published: February 2005

Human umbilical vein endothelial cells can be serially passaged by supplementing medium with a partially purified growth factor. Cell‐substratum detachment of early and late passage endothelial cells was examined using trypsin, collagenase, or homocysteine. Late‐passage cells detached more rapidly than early passage cells under all conditions tested. The rate of detachment was dependent upon the specific agent used. Protease‐mediated detachment was most rapid, occurring over minutes, in contrast to homocysteine‐induced detachment, which occurred over hours. When detached cells were collected and replated in the absence of the detaching agent, these cells reattached spread, and continued to proliferate. No significant difference was observed in the rate of adhesion of either early or late passage cells to a gelatin matrix. When early or late‐passage endothelial cells were plated and grown to confluence on a matrix synthesized by the opposite cell type, the rate of protease‐mediated cell detachment resembled the cell type from which the matrix was derived. The ease of endothelial cell detachment was determined by the origin of the extracellular matrix. Examination of the extracellular matrices from early and late passage cells revealed significant differences in the amounts of glycosaminoglycans and sulfated proteins present. These studies demonstrate the importance of the endothelial cell extracellular matrix in protease‐mediated cell detachment. The rate of cell detachment was controlled by the extracellular matrices and not altered by the endothelial cells.
Mitogen‐independent activation of Na+/H+ exchange in human epidermoid carcinoma A431 cells: Regulation by medium osmolarity
Authors: Cassel Dan , Whiteley Brian , Zhuang Ying Xin , Glaser Luis
Published: February 2005

Previous studies have documented the activation of Na+/H+ exchange in A431 cells by the addition of epidermal growth factor or serum (Rothenberg et al., 1983b). Here we show that exposure of A4 31 cells to medium of increased osmolarity also leads to activation of Na+/H+ exchange and to an increase in intracellular pH (pHi), which under a variety of conditions displays similar kinetics to that observed upon addition of mitogens to the cells. Measurements of cell volume using the 3–0‐methylglucose equilibration technique clearly show that mitogens do not activate Na+/H+ exchange by an osmotic mechanism (i.e., a decrease in cell volume). In fact, mitogens can induce further intracellular alkalinization if added to cells which have been shrunken in hypertonic medium. Activation of the Na+/H+ antiport does not lead to an obligatory change in pHi. Addition of epidermal growth factor of hypertonic solution to A431 cells in bicarbonate buffer activates Na+/H+ exchange without a concomitant increase in pHi. Under these conditions the increased proton efflux via Na+/H+ exchange must therefore be compensated by other mechanisms that control cytoplasmic pH.
Effect of oxygen and endotoxin on lactate dehydrogenase release, 5‐hydroxytryptamine uptake, and antioxidant enzyme activities in endothelial cells
Authors: Block Edward R. , Patel Jawaharlal M. , Sheridan Norman P.
Published: February 2005

We compared the effects of 95% O2 (hyperoxia) alone, endotoxin (20 ng/ml) alone, and 95% O2 plus endotoxin on the release of lactate dehydrogenase (LDH), uptake of 5‐hydroxytryptamine (5‐HT), and antioxidant enzyme activities in porcine pulmonary arterial and aortic endothelial cells in monolayer culture. Hyperoxia increased LDH release and decreased 5‐HT in both endothelial cell types. Hyperoxia also caused a decrease in catalase (CAT) activity and an increase in total superoxide dismutase (SOD) and glutathione reductase (GSH‐Red) activities in both cell types. Endotoxin alone had no effect on LDH release, 5‐HT uptake, or antioxidant enzyme activities. However, endotoxin prevented the hyperoxic increase in LDH release and the hyperoxia decrease in 5‐HT uptake. Endotoxin plus 95% O2 had no consistent effect on the antioxidant enzyme profile in pulmonary artery or aortic endothelial cells. These results indicate that (1) hyperoxia injures both pulmonary artery and aortic endothelial cells in culture and causes changes in the antioxidant enzyme profile that are similar in the two cell types; (2) hyperoxia‐induced decreases in CAT activity and increases in SOD activity may be responsible for increased sensitivity of endothelial cells to O2 toxicity; and (3) endotoxin protects against hyperoxic injury to endothelial cells in vitro, but increases in antioxidant enzyme activities are not the mechanism for this protection.
The osmotic response of human erythrocytes and the membrane cytoskeleton
Authors: Heubusch Philip , Jung Chan Y. , Green Floyd A.
Published: February 2005

The volumes of human erythrocytes suspended in solutions of varying concentrations of sodium chloride and sucrose were measured by a Coulter Channelyzer® Model H4 with appropriate corrections. The cells showed greatly restricted volume changes at osmolarities between 200–700 mOsm. At osmolarities outside this limit, on the other hand, the cells showed nonrestricted volume changes following essentially the predictions of an ideal osmometer. This unexpected volume response was not spuriously due to changes in shape or to a changing orientation of the cells as they traversed the aperture. The restricted volume change observed was abolished when the cells had previously been treated with diamide or had been heated for 60 minutes at 50°C, conditions that are known to disturb the spectrin‐actin network. The possibility must be considered that the osmotic behavior of human erythrocytes may be nonideal and that this nonideal behavior is primarily due to mechanical restriction provided by the spectrin‐actin network of the membrane cytoskeleton.
Human endothelial cell cultures: Phenotypic modulation by leukocyte interleukins
Authors: Montesano R. , Orci L. , Vassalli P.
Published: February 2005

We report here that soluble factors from activated mononuclear leukocytes have a dramatic effect on cultured endothelial cells. While human umbilical vein endothelial cells grown under standard conditions show a polygonal, epitheliallike morphology, cells exposed to culture media conditioned by lectin‐activated human mononuclear leukocytes become extremely elongated and/or send out numerous cytoplasmic processes, assuming a dendritic configuration. This effect cannot be mimicked by exogenous cyclic AMP, is reversible upon interruption of the treatment, and appears specific for endothelial cells, since it has not been observed so far with other cell types. The shape changes are accompanied by a reorganization of the endothelial cell cytoskeleton: actin microfilament bundles tend to be disposed in parallel arrays, while intermediate filaments and microtubules penetrate up to the extremity of the cytoplasmic processes. Colchicine prevents endothelial cell elongation but only slightly impairs the formation of lateral cell processes (“dendritic configuration”). Purified interleukins were tested for their ability to induce these changes of cell shape. Escherichia coli‐recombinant human interleukin 2 had no effect, and γ‐interferon only a slight effect on endothelial cell morphology. Interleukin 1 induced moderate cell elongation, while combined treatment with both interleukin 1 and γ‐interferon resulted in shape changes indistinguishable from those elicited by supernatants of activated mononuclear leukocytes. The possible relevance of the observed endothelial cell changes to the reported angiogenic activity of mononuclear cell products is discussed.
Modulation of the platelet‐derived growth factor induced replicative response
Authors: Scher C. D. , Whipple A. P. , Singh J. P. , Pledger W. J.
Published: February 2005

Quiescent cultures of density arrested BALB/c‐3T3 cells have been sensitized to the growth stimulatory action of the platelet‐derived growth factor (PDGF). Sensitization was achieved by depriving the cultures of PDGF prior to growth stimulation and was noted after transfer of cultures from medium supplemented with 10% serum to medium containing either an equivalent concentration of platelet‐poor plasma or a low concentration (0.5%) of serum. Sensitized cultures required less pure PDGF for growth stimulation than non‐sensitized ones. In addition such cultures required less mitogen to synthesize a PDGF modulated major excreted protein (MEP). The mechanism of sensitization was investigated. Sensitized cultures did not bind more PDGF than non‐sensitized ones. Rather, sensitization appeared to result from the loss of cells that occurred when cultures were deprived of PDGF. Such a loss increased the amount of PDGF available per cell, causing a higher percentage of cells to enter the S phase. Similarly, the amount of PDGF per cell regulated MEP synthesis. Furthermore, in non‐sensitized cultures (containing the same number of cells), the absolute quantity rather than the concentration of PDGF regulated DNA synthesis. It appears that the amount of PDGF per cell modulates mitogenesis.
Kinetics of long‐term (72 hr) calcium content during mitogen activation of cultured human T lymphocytes
Authors: Wolff C. H. J. , Åkerman K. E. O. , Andersson L. C.
Published: February 2005

In vitro stimulation of human blood lymphocytes with mitogen resulted in an increased intracellular content of Ca2+ per unit cell volume. This increase in Ca2+ content of lectin‐activated cells reached a maximum after 24 hr of culture and thereafter slowly declined. Brief treatment of cells at 24 hr of culture with the Ca2+ ionophore A23187 in combination with EGTA resulted in a larger release of Ca2+ from cells in mitogen‐stimulated cultures than from cells in control cultures. This indicates that the Ca2+ is accumulated intracellularly but is readily exchangeable. At 24 hr of culture the increase in cellular Ca2+ correlated well with the proliferative response as measured by 3H‐thymidine incorporation. Ca2+ influx at 24 and 48 hr of culture was markedly enhanced in the mitogenically stimulated cells as compared either to cells cultured for 1 and 72 hr or cells cultured without mitogen.
Cell volume regulation of cerebrovascular endothelium in vitro
Authors: Kempski O. , Spatz M. , Valet G. , Baethmann A.
Published: February 2005

Regulation of cell volume as a fundamental cellular function of high biological priority was studied in cultured cerebrovascular endothelium. The use of a multiparameter flow cytometric system allowed simultaneous measurements of cell volume, viability, and membrane potential or intracellular pH. Endothelium, the cellular constituent of the blood‐brain barrier (BBB), swells immediately on exposure to low osmolality. This is associated with membrane depolarization and a fall of intracellular pH. Within 30–60 min, cell volume and membrane potential recover completely, although the extracellular osmolality is kept low. Intracellular pH does not normalize fully. Measurements of intracellular K+ and Na+ concentrations reveal their involvement in the regulatory process. The findings strongly suggest that the cerebrovascular endothelium has a highly effective built‐in capacity for homeostatic control essential for normal BBB function.
Endogenous metabolism by sperm in response to altered cellular ATP requirements
Authors: Inskeep Philip B. , Hammerstedt Roy H.
Published: February 2005

A quantitative description of the relative importance of endogenous metabolism to overall ATP production has not been established for mammalian cells. We report herein results of experiments using sperm (selected because of their simple metabolic potential and absence of biosynthetic pathways) and calorimetry (chosen because it serves as a general monitor of metabolism) to assess the importance of endogenous metabolism to total ATP synthesis under several incubation conditions. In experiments in which sperm were incubated at different temperatures (20°C, 25°C, 30°C or 35°C) and with different substrates (glucose, fructose, lactate, or β‐hydroxbutyrate), endogenous metabolism occurred at a constant rate regardless of the rate of ATP turnover in the cells or the nature of the exogenous substrate available to them. Sperm incubated at 35°C with glycolyzable substrates synthesized more ATP (9 μmol ATP·h−1/108 cells) than did sperm incubated with the non‐glycolyzable substrate, lactate (6.2 μmol ATP·h−1/108 cells). To investigate this substrate‐related difference in the rate of ATP synthesis, the motility of sperm incubated at 35°C with glucose or with lactate was determined. The velocities of the sperm incubated with either substrate were identical, indicating that the rates of ATP consumption for support of motility were identical. Most of the additional ATP synthesized by cells with glycolyzable substrates was consumed in the process of substrate cycling of the metabolic intermediates of glucose.
The effects of osmotic stress on human platelets
Authors: Armitage W. J. , Parmar N. , Hunt C. J.
Published: February 2005

The effect of osmotic stress on human platelets was investigated at 0, 25, and 37°C. The osmolality of the suspending plasma was decreased by adding water or increased by adding sodium chloride or sucrose. After 5 min, isotonicity was restored by dilution with an excess of isotonic phosphate‐buffered saline. After centrifugation, the platelets were resuspended in autologous plasma and then incubated for 1 hr at 37°C before assaying the active transport of 5‐hydroxytryptamine (5‐HT) and the hypotonic stress response. Anisosmotic conditions had a greater effect on the extent of volume reversal in the hypotonic stress test than on 5‐HT uptake. At 25°C, only moderate degrees of hypotonicity (0.25 osmol/kg) or hypertonicity (0.59 osmol/kg) were sufficient to depress the hypotonic stress response. In general, platelets tolerated departures from isotonic conditions better at 0°C than at the higher temperatures. Furthermore, at 0 and 25°C approximately equiosmolal concentrations of sucrose and sodium chloride depressed the hypotonic stress response to similar extents, but at 37°C high osmolalities (> 2 osmol/kg) were tolerated better when the additive was sucrose than when it was sodium chloride. Platelets shrank when subjected to hyperosmotic conditions, but their discoid shape and the peripheral band of microtubules were maintained.
Stimulation of bumetanide‐sensitive K+ transport in Swiss 3T3 fibroblasts by serum and mitogenic hormones
Authors: Amsler Kurt , Donahue Jeremiah J. , Slayman Carolyn W. , Adelberg Edward A.
Published: February 2005

Rapidly growing Swiss 3T3 fibroblasts possess a bumetanide‐sensitive K+ transport system that is dependent on both Na+ and Cl− ions; a smaller bumetanide‐insensitive component of K+ transport is also present. In cells brought to the quiescent state by 8–11 days of incubation without a medium change, the bumetanide‐sensitive rate of transport was reduced by 63%; the bumetanide‐insensitive rate did not change. Removal of dialyzed fetal calf serum from the uptake medium resulted in a substantial reduction in bumetanide‐sensitive uptake in both rapidly growing cells (33% reduction) and quiescent cells (68% reduction) but had no effect on bumetanide‐insensitive uptake. Insulin was almost as effective as dialyzed fetal calf serum in stimulating bumetanide‐sensitive uptake; insulin was maximally stimulatory at 2.5 μg/ml. The combination of insulin, epidermal growth factor, and arginine‐vasopressin was maximally effective in stimulating both bumetanide‐sensitive K+ uptake and 3H‐thymidine incorporation in quiescent cells; bumetanide, however, did not interfere with the hormonal stimulation of DNA synthesis. Thus, the bumetanide‐sensitive K+ transport system is not necessary for such stimulation to occur. Furthermore, concentrations of hormones which stimulated significant levels of DNA synthesis produced no elevation in the intracellular concentration of K+. We conclude that the bumetanide‐sensitive pathway of K+ transport is modulated by serum and by mitogenic hormones, but does not play a role in the stimulation of DNA synthesis by these factors.
Characterization of the Na+/H+ exchanger in human epidermoid carcinoma A431 vesicles
Authors: Mancuso David , Glaser Luis
Published: February 2005

A previous report from this laboratory (Rothenberg et al., 1983a) demonstrated the presence of an Na+/H+ exchanger in human epidermoid carcinoma A431 cells. We now characterize surface‐derived membrane vesicles from this cell line which contain a functional Na+/H+ exchanger. The Na+/H+ exchanger in A431 vesicles shares a number of characteristics in common with previously described Na+/H+ exchangers including the following: (1) Na+ uptake is stimulated by an outward‐directed pH gradient and inhibited by an inward‐directed pH gradient. (2) Na+ uptake is inhibited by amiloride and its analogs and their relative effectiveness is similar in vesicles and A431 cells. (3) The Na+/H+ exchanger uses Na+ or Li+ as a substrate but not K+ or Cs+. (4) H+ efflux is stimulated by an inward‐directed Na+ gradient and inhibited by the amiloride analog 5‐N‐dimethylamiloride. The Na+/H+ exchanger in these membrane vesicles is activated allosterically by low intravesicular pH. The apparent pKa of the activating site is 6.4–6.6, characteristic of the NA+/H+ exchanger before activation by mitogens.
Regulation of deoxyglucose uptake by adrenocorticotropic hormone in cultured neurons
Authors: Daval Jean‐Luc , Anglard Patrick , Gerard Marie‐José , Vincendon Guy , Louis Jean‐Claude
Published: February 2005

The influence of ACTH and some of its N‐terminal related peptides was investigated on the uptake of (3H)‐2‐deoxy‐D‐glucose in pure cultures of neurons from chick embryo cerebral hemispheres. ACTH influences deoxyglucose uptake in a time and dose‐dependent fashion. The stimulation of deoxyglucose uptake is observed after a delay of 6–8 h and requires active protein synthesis. ACTH does not affect deoxyglucose in non‐neuronal cells (astroglial cells, hepatocytes, myoblasts, fibroblasts). The effect of various peptide hormones, neuropeptides and growth factors, active in the central nervous system or other tissues, has also been examined. None of these were able to stimulate deoxyglucose uptake, suggesting that the regulation of hexose uptake in neurons is specific for the ACTH‐related peptides.
Characterization of a BALB/c 3T3 preadipose cell mutant with altered Na+K+Cl− contransport activity
Authors: Sussman Ilene , O'Brien Thomas G.
Published: February 2005

A BALB/c 3T3 cell mutant (3T3‐E12) was isolated by its ability to survive at a low extracellular K+ concentration (0.14 mM). The growth rate of mutant cells was less dependent on external K+ than parental cells. Analysis of potassium transport revealed that 3T3‐E12 cells have a decreased activity of the furosemide‐sensitive Na+K+Cl− contrasport system, both in the efflux and influx modes. This is shown to be a result of a decrease in the apparent affinity of the transport system for K+ and Na+, but not Cl−. Upon exposure to the phorbol ester 12‐0‐tetradecanoyl‐phorbol‐13‐acetate (TPA), BALB/c 3T3 cells exhibited a maximal volume decrease of 20%, while mutant cells shrunk by only 7%, suggesting that regulation of cell volume, at least after exposure to a tumor promoter, is impaired in mutant cells compared to parental 3T3 cells.
The essential role of L‐glutamine in lymphocyte differentiation in vitro
Authors: Crawford Jeffrey , Cohen Harvey Jay
Published: February 2005

The biochemistry of human B lymphocyte differentiation to plasma cells is incompletely understood. L‐glutamine appears to be required for both lymphoblastic transformation and plasma cell formation in pokeweed‐mitogenstimulated human peripheral blood mononuclear cell cultures. Cells cultured with pokeweed mitogen in glutamine‐deficient RPMI‐1640 with 10% heat‐inactivated and dialyzed fetal bovine serum were unable to incorporate 3H‐thymidine or undergo morphologic lymphoblastic transformation assessed at 72 hours. However, 3H‐thymidine incorporation could be maximally restored with as little as 0.08 mM L‐glutamine or by using nondialyzed heat‐inactivated fetal bovine serum, containing approximately .1 mM L‐glutamine. In subsequent cultures, using glutamine‐deficient RPMI‐1640 with 10% nondialyzed heat‐inactivated fetal bovine serum, lymphoblastic transformation was equivalent with or without additional L‐glutamine supplementation. However, only cultures with 2 mM L‐glutamine supplementation underwent plasma cell differentiation as assessed by cytoplasmic staining with fluorescein‐conjugated anti‐immunoglobulin. When the kinetics of cellular immunoglobulin synthesis and secretion were analyzed by 3H‐ leucine incorporation into immunoglobulin, synthesis was 2–5 fold greater, and secretion 3–10‐fold greater in cell cultures with 2 mM L‐glutamine supplementation. By electron microscopy, only the glutamine‐supplemented cells showed development of rough endoplasmic reticulum consistent with active immunoglobulin production. L‐glutamine supplementation had no apparent effect on cell recovery, viability, % B cells, % T cells, % monocytes, or % helper and suppressor T cells. Thus, L‐glutamine is essential for both lymphoblastic transformation and plasma cell differentiation. Future investigation of the selective nutritional requirements of cultured cells should yield further insights into the biochemical control of immune cell differentiation and function.
Binding of 125I‐labeled granulocyte colony‐stimulating factor to normal murine hemopoietic cells
Authors: Nicola N. A. , Metcalf D.
Published: February 2005

The binding of granulocyte colony‐stimulating factor (G‐CSF) to murine bone marrow cells was investigated using a radioiodinated derivative of high specific radioactivity which retaine full biological activity. The binding was time‐and temperature‐dependent, saturable and highly specific. The apparent dissociation constant for the reaction was 60–80 pM at 37°C and 90–110 pM at 4°C, similar to that found for the binding of G‐CSF to murine leukemic cells (WEHI‐3B D+) and significantly higher than the concentration of G‐CSF required to stimulate colony formation in vitro. Autoradiographic analysis confirmed the specificity of binding since granulocytic cells were labeled but lymphocytes, erythroid cells and eosinophils were not. Blast cells and monocytic cells were partially labeled, the latter at low levels. In the neutrophilic granulocyte series, grain counts increased with cell maturity, polymorphs being the most heavily labeled but all cells showed considerable heterogeneity in the degree of labeling. Combination of Scatchard analysis of binding with autoradiographic data indicated that mature granulocytes from murine bone marrow exhibited 50–500 G‐CSF receptors per cell.
Mononuclear macrophage (M‐CFC) colony formation in semisolid media in the absence of exogenous GM‐CSF
Authors: Zinzar Svetlana N. , Den Tiberius , Holland James F.
Published: February 2005

Human fetal bone marrow (FBM) cells were examined for the ability to form colonies in the absence of exogenous colony‐stimulating factor (CSF) in double layer agar, methylcellulose (MC), and in agar‐MC (agar underlayer, MC overlayer) culture systems. Without exogenous CSF, macrophage colonies (M‐CFC) were formed in a combined culture of agar and MC. Aggregates of 5–40 cells were observed on day 7. Gradually, large compact colonies which survived for 10–12 weeks of cultivation, were formed. They were composed of mononuclear monocytes and multinucleated cells. M‐CFC progenitors were nonadherent, but their progeny became adherent during differentiation within the colony. Colony formation was cell‐dose‐dependent. Depletion of monocytes increased the number of colonies in agar‐MC cultures and stimulated the development of some macrophage colonies in MC. Survival of monocyte progenitors was not dependent on CSF. Neither was their proliferation nor partial differentiation in agar‐MC cultures. CSF increased M‐CFC colony efficiency, however, if it was present when cultures were initiated. Addition of CSF to M‐CFC growing for 2–5 weeks in CSF‐deprived medium stimulated monocytes proliferation and transformation into macrophages. Epithelioid cells, and increase in the number of giant multinucleated cells, and granulocyte multiplication were also observed. The absolute dependence of macrophage colony formation on CSF described by others might be a result of inadequate culture conditions due to agar rather than an intrinsic physiological requirement.
Wheat germ agglutinin and concanavalin A inhibit the response of human fibroblasts to peptide growth factors by a post‐receptor mechanism
Authors: Kaplowitz Paul B.
Published: February 2005

The effect of plant lectins on amino acid uptake and DNA synthesis in cultured human skin fibroblasts stimulated by various peptide mitogens was studied. Wheat germ agglutinin (WGA), at a concentration of 5 μg/ml, which by itself had little effect on 3H‐aminoisobutyric acid (AIB) uptake, markedly inhibited stimulation of 3H‐AIB uptake by somatomedin‐C, insulin, epidermal growth factor (EGF) and platelet‐derived growth factor. This inhibition could not be overcome by increasing the concentration of peptide added. Neither WGA nor concanavalin A (Con A) significantly affected basal 3H‐thymidine incorporation. However both lectins, at concentrations of 5–20 μg/ml, decreased EGF‐and insulin‐stimulated DNA synthesis while succinyl Con A, a divalent lectin derivative, did not. The inhibitory effects of lectins on mitogenic stimulation were reversed by α‐methyl mannose (Con A) or N‐acetylglucosamine (WGA), and were not due to a reduction in the binding of growth factors to their receptors. It is concluded that certain lectins noncompetitively inhibit the response of human fibroblasts to multiple peptide mitogens at the post‐receptor level, possibly by interfering with lateral mobility and aggregation of mitogen‐receptor complexes.
DNA damage production in CHO cells at elevated temperatures
Authors: Warters Raymond L. , Brizgys Lucy M. , Axtell‐Bartlett Joann
Published: February 2005

Induction of DNA lesions in the nucleus of Chinese hamster ovary (CHO) cells was observed at hyperthermic temperatures using the alkaline filter elution and the alkaline sucrose gradient sedimentation methods. These lesions were observed principally at temperatures greater than 45°C with an activation energy of 140 kcal/mole. On alkaline sucrose gradients the cell genome was reduced to a 140 S or 2 × 108 dalton subunit of DNA independent of increasing exposure time at temperatures above 45°C. The large thermal activation energy and the limited DNA size reduction suggest the possible involvement of thermal denaturation of a nuclear polypeptide in the production of these nuclear lesions.
BrdU‐Hoechst flow cytometry reveals regulation of human lymphocyte growth by donor‐age–related growth fraction and transition rate
Authors: Kubbies M. , Schindler D. , Hoehn H. , Rabinovitch P. S.
Published: February 2005

An improved BrdU‐Hoechst flow assay was applied to cell kinetic studies of human lymphocyte cultures during a 24–96 hr interval after PHA stimulation. The assay shows that the duration of the initial lag phase and the proportions of noncycling cells increase as a function of donor age, whereas the rates of transition from each cell cycle compartment to the next decrease. Cell cycle arrest occurs in the first S and G2 phase after stimulation of lymphocytes from a 75‐year‐old donor but not from younger donors. The data are consistent with several models of cell cycle kinetics, so long as these models are modified to include a fraction of noncycling cells in each cell cycle compartment.
Stretch‐induced growth in chicken wing muscles: Role of soluble growth‐promoting factors
Authors: Summers Philip J. , Ashmore C. Robert , Lee Yu Bang , Ellis Stanley
Published: February 2005

The involvement of soluble growth‐promoting factors in stretch‐induced hypertrophy of the Patagilis muscle (PAT) in the chicken wing was investigated. Soluble extracts were prepared from young chicken PAT muscle made hypertrophic by passive stretch and from unstretched contralateral controls. Extracts were tested for their ability to stimulate cell proliferation and creatine phosphokinase (CPK) activity in primary monolayer cultures of chick embryo muscle cells.

Factors were present in muscle extracts which showed a dose‐dependent stimulation of cell proliferation and CPK activity in vitro. Passive stretch for 5 days produced a rapid hypertrophy of the PAT which was accompanied by a dramatic increase in the activity of the growth factor(s). Release of stretch resulted in an arrest of growth and an immediate fall in growth factor activity.

The difference in growth‐stimulating activity between control and stretched PAT extracts could be demonstrated in chicken transferrin‐sensitive chick myoblast cultures. Stretch thus induces an increase in a class‐specific growth factor, possibly Transferrin, in the PAT.

Stretched PAT extracts stimulated: (a) chick myoblast proliferation to a greater extent than an optimum concentration of chick embryo extract, and (b) CPK activity in vitro to a greater extent than excess Transferrin. Both control and stretched PAT extracts supported the growth of rat myoblasts. We conclude that PAT muscle extracts also contain unknown growth factor(s) which are different from Transferrin.
Characterization of erythropoietin‐like activity from mouse spleen cells
Authors: Fagg Barbara , Roitsch Carolyn A.
Published: February 2005

Supernatants from mouse spleen cell cultures contain a factor which acts in a similar manner to erythropoietin (Ep) to stimulate the formation of 2‐day erythroid (CFU‐E) colonies in vitro from bone marrow or fetal liver cells. Analysis of conditioned media by high performance liquid chromatography (HPLC) on anion exchange, reverse phase, molecular size exclusion, and hydroxyapatite columns demonstrated that the erythropoietin‐like activity (EpLA) has different biochemical characteristics to mouse Ep from anemic mouse serum. In addition, EpLA has a molecular weight (Mr), of 20,000 daltons determined by SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE), compared to 42,000 for mouse Ep. Partially purified EpLA was found to be active in vivo as well as in vitro. Highly purified preparations of γ‐interferon, Multilineage hemopoietic growth factor (Multi HGF), Interleukin‐2 (IL‐2), IL‐1, and colony stimulating factor 1 (CSF‐1) did not support CFU‐E colony formation. Thus, it was established that EpLA could not be attributed to other known components of spleen cell conditioned medium. Titration of mouse Ep and EpLA suggests that only a portion of the Ep‐responsive CFU‐E population in fetal liver is sensitive to EpLA.
Glucocorticoid stimulation of metabolism and glycerol‐3‐phosphate dehydrogenase activity in cultured heart cells
Authors: Freerksen Deborah L. , Hartzell Charles R.
Published: February 2005

The direct effects of the glucocorticoids hydrocortisone and corticosterone on myocardial metabolism were studied in cultured heart cells by assessing several parameters previously unreported. Hormone and growth factor concentrations were carefully controlled by using a serum‐free medium, which also allowed maintenance of cells in the absence of glucocorticoids. Heart cell beating rate, glucose uptake rate, and CO2 evolution from radioactively labeled glucose were increased by the addition of 0.03 μM corticosterone to the medium of cells maintained in culture for 11 days. There were no further changes in these parameters as steroid concentration was increased to 14.43 μM. The activity of NAD‐linked sn‐glycerol‐3‐phosphate dehydrogenase (EC 1.1.1.8) was increased by both corticosteroids and was dose dependent between 0.06 and 1.44 μM corticosterone. The difference between glycerol‐3‐phosphate dehydrogenase activity in cells maintained with hydrocortisone as compared to cells maintained without hydrocortisone increased with days in culture. The protein and DNA contents of dishes maintained with corticosteroid were depressed, demonstrating an inhibitory effect on cellular replication. Glucocorticoids have numerous direct effects on cardiac cell metabolism, and the nature of these effects suggests that secondary responses of the cell to chronic exposure are significant.
Interferon‐mediated inhibition of differentiation in a murine myoblast cell line
Authors: Multhauf Cecilia , Lough John
Published: February 2005

The effects of highly purified (> 5 × 107 IU/mg) murine β‐interferon (IFN) on a mouse myoblast line (MM14DZ) have been investigated to confirm and extend the previous observation that partially purified chicken interferon inhibits differentiation of cultured avian myoblasts (Lough et al., Biochem Biophys Res Commun 109:92, 1982). Cultures treated with 20–2,000 IU IFN/ml medium for 5 days exhibited dose‐dependent (1) inhibition of differentiation, as indicated by reduced myotube formation and creatine kinase (CK) activity and (2) increases in DNA content, suggesting that the inhibitory effect was accompanied by continued proliferation of myoblasts. Mock‐IFN had no such effects. Based on findings in other systems that IFN inhibits activity of ornithine decarboxylase (ODC), the polyamine products of which are required for myogenesis, the hypothesis that inhibition of differentiation was mediated by an effect of IFN on polyamine metabolism was tested. However, observations that (1) IFN‐treated myoblasts retained control levels of ODC activity and (2) exogenous polyamines did not prevent IFN‐inhibition did not indicate such a mechanism of action. On the other hand, treatment of control cultures with polyamines alone resulted in potentiation of myogenesis as revealed by precocious myotube formation and a marked increase in CK activity.