bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation and characterization of tunicamycin resistant mutants from Chinese Hamster ovary cells
Authors: Sudo Tadashi , Onodera Kazukiyo 
Published: February 2005

AbstractStable clones selected for resistance to tunicamycin (TM) have been isolated from Chinese Hamster Ovary (CHO) cells. The TMR phenotype is stable for more than nine months in the absence of the drug. The morphology of TMR mutant varies from epitheloid to abnormally elongate. The mutants do not display cross‐resistance for ConA but are slightly cross‐resistant to PHA. Biochemically labeled membrane proteins and glycoprotein of Vesicular stomatitis virus (VSV) grown in the TMR mutants revealed that the incorporation of radioactive glucosamine was markedly reduced in the mutants. The results indicate that TMR cells are a novel type of membrane mutant.
Nucleotide and pentose synthesis after serum‐stimulation of resting 3T6 fibroblasts
Authors: Smith Margaret L. , Buchanan John M. 
Published: February 2005

AbstractWe have investigated the de novo synthesis of intermediates of purine nucleotides in 3T6 fibroblasts and determined the manner by which the activity of this pathway is increased in resting cells by the addition of fresh serum. Within 30 minutes after stimulation, 3T6 cells began to synthesize increased amounts of purines by the de novo pathway as measured by increased amounts of formylglycinamide ribonucleotide, a representative intermediate of this pathway. Within 15 minutes after serum‐stimulation 3T6 cells exhibited a substantial increase in their capacity to synthesize ribose compounds, particularly in the form of 5‐phosphoribosylpyrophosphate (PRPP). The availability of PRPP appeared to be limiting for the synthesis of purine nucleotides in resting fibroblasts, but not necessarily in serum‐stimulated cells.The amount of the enzyme PRPP synthetase as measured in vitro remained constant for at least the first four hours. Therefore, a study was made of various compounds known to activate PRPP synthetase in vitro. No evidence was found that suggested involvement of concentrations of cyclic nucleotides or phosphate. Experiments with methylene blue, an artificial electron acceptor that stimulates the production of ribose 5‐phosphate by the hexose monophosphate shunt, indicated that one of the immediate consequences of the addition of serum is increased cycling of the pyridine nucleotide coenzymes, NADP+ and NADPH, and that the rapid increase in formation of ribose compounds and, consequently, purine nucleotides was caused as a result of modulation by this coenzyme. The relative ration of ATP:ADP:AMP as well as their concentrations remain constant in resting and serum‐stimulated cells under normal assay conditions. However, there was a substantial decrease in ATP concentrations with a corresponding increase in AMP concentration with methylene blue in the assay buffer. The production of AMP from ATP was 5‐fold greater in the serum‐stimulated than in the resting fibroblasts. The increased production of AMP is thus serum‐dependent and may reflect a basic enzymatic function of proliferative as compared to resting cells.
Colchicine stimulation of hyaluronate synthesis and secretion in bone organ culture
Authors: Severson Arlen R. 
Published: February 2005

AbstractParathyroid hormone (PTH) is known to have a number of effects on bone tissue in vitro, including the stimulation of calcium release and the synthesis and turnover of hyaluronate. PTH‐stimulated calcium release is inhibited by colchicine. Since hyaluronate may play a role in demineralization, calcium release into the media as a measure of bone resorption was correlated with the synthesis and secretion of 3H‐glucosamine‐labeled macromolecules. Newborn mice were labeled with 45Ca, and the calvaria removed and incubated in vitro in media containing 3H‐glucosamine. Addition of colchicine to the culture media inhibited the release of 45Ca into the media while stimulating the synthesis and secretion of 3H‐glucosamine labeled macromolecules. DEAE‐cellulose chromatography resolved the labeled macromolecular material into four peaks, of which the third peak containing hyaluronate demonstrated approximately twice the amount of radioactivity. PTH stimulation of calcium release was inhibited likewise by colchicine, while 3H‐glucosamine incorporation into labeled macromolecules was stimulated. Short term labeling studies emphasized the marked stimulatory effect that both PTH and colchicine have on the incorporation of 3H‐glucosamine into hyaluronate. PTH stimulated the incorporation of 35S‐sulfate, while colchicine markedly inhibited its incorporation into non‐dialyzable material. Both PTH and colchicine inhibited protein synthesis. Based on the observations, colchicine appears to stimulate the synthesis and secretion of hyaluronate and alter a number of metabolic pathways. Hyaluronate does not appear to be directly involved in the demineralization process.
The effect of protease inhibitors and decreased temperature on the degradation of different classes of proteins in cultured hepatocytes
Authors: Neff Nicola T. , Demartino George N. , Goldberg Alfred L. 
Published: February 2005

AbstractLeupeptin, chymostatin and antipain inhibited the degradation of long‐lived proteins in cultured rat hepatocytes by 20–30%, probably by inhibiting lysosomal proteases:(1) Leupeptin and chymostatin decreased to a similar extent the degradation of an exogenous protein 125I‐asialo fetuin, a process known to occur within lysosomes. (2) In extracts of cells treated with leupeptin, cathepsin B activity was inhibited by 35–50%. (3) Leupeptin, chymostatin and antipain inhibited proteolysis by homogenates of liver lysosomes but not by the supernatant fraction. These agents, however, do not appear to rapidly permeate the membrane of isolated lysosomes.Leupeptin, chymostatin and antipain did not inhibit the breakdown of short‐lived normal cell proteins, and ones containing amino acid analogs. Even when the amount of abnormal proteins was increased, such that it comprised a large fraction of cell protein, the degradation of these polypeptides was still very rapid and not affected by these inhibitors. The pathway for the degradation of short‐lived cell proteins thus appears distinct from that responsible for degradation of long‐lived cell proteins. In accord with this conclusion, reduction of the temperature of cultures inhibited the breakdown of long‐lived proteins to a much greater extent than it affected the breakdown of short‐lived ones.Treatment of cultured hepatocytes with glucagon, or deprivation for serum or amino acids stimulated the degradation of the more stable cell proteins but did not affect the breakdown of 125I‐asialo‐fetuin. Under these conditions leupeptin and chymostatin inhibited the breakdown of long‐lived cell proteins to the same extent as in control cultures. Thus, lysosomal enzymes seem to play an important role in protein breakdown both in fed hepatocytes and in cells where proteolysis is accelerated.
Transmembrane potential and ionic content of rat alveolar macrophages
Authors: Castranova V. , Bowman L. , Miles P. R. 
Published: February 2005

AbstractThe cell volume, cell water, intracellular ionic concentrations, and transmembrane potential of rat alveolar macrophages were determined. The measurements were made on cells which had been separated from the medium by centrifugation through dibutyl phthalate in order to greatly reduce the trapped extracellular space. The mean cell volume of the alveolar macrophages is 1,525 cubic microns and 72% of this volume is water. The intracellular fluid is high in Na+ (97 mM) and lower in K+ (50 mM) and the intracellular Cl− concentration is 64 mM. The transmembrane potential, as measured from the equilibrium distribution of tritiated triphenylmethyl phosphonium and by using the fluorescent probe, Di‐S‐C3(5), is approximately –37 millivolts. Neither Na+, K+, nor Cl− is distributed at equilibrium. However, the K+ permeability of alveolar macrophage membranes appears to be greater than Na+ permeability.
Inhibition of friend erythroleukemic cell differentiation by bromodeoxyuridine: Correlation with the amount of bromodeoxyuridine in dna
Authors: Ashman Charles R. , Davidson Richard L. 
Published: February 2005

AbstractThese studies were undertaken to examine the relationship between the inhibition by 5‐bromodeoxyuridine (BrdU) of erythroid differentiation in Friend erythroleukemia cells and the incorporation of BrdU into DNA. Experiments were carried out in which the incorporation of BrdU into DNA and the concentration of BrdU to which the cells were exposed were varied independently of each other. In addition, the ability of deoxycytidine (dC) to reverse the effects of BrdU on hemoglobin production and to reduce the amount of BrdU in DNA was analyzed. Under all the conditions tested, the effects of BrdU were correlated with the amount of Brd incorporated into nuclear DNA. These results differ from those of recent studies on the inhibition of pigmentation and the induction of mutations by BrdU in Syrian hamster melanoma cells. The results suggest that BrdU may be producing its biological effects by a variety of different mechanisms.
Enhanced rates of fluid pinocytosis during exponential growth and monolayer regeneration by cultured arterial endothelial cells
Authors: Davies Peter F. , Selden Sydney C. , Schwartz Stephen M. 
Published: February 2005

AbstractRates of fluid pinocytosis by bovine aortic endothelial cells were measured during various manipulations of growth status in vitro. Sparsely seeded cultures grew exponentially until a confluent monolayer was formed, at which time growth slowed. This change in growth rate coincided with a decline in the rate of pinocytosis to about one‐third that in the growing cultures. During the subsequent attainment of maximal cell density in the confluent monolayer, the pinocytic rate remained constant. There was close correlation between 3H‐thymidine labelling indices, as measured by autoradiography, and the rates of pinocytosis. Mechanical “wounding” of the confluent monolayer resulted in cell migration and proliferation. Twenty‐four hours after “wounding,” rates of pinocytosis per mg. cell protein were significantly enhanced. When regeneration of the monolayer was blocked by cytochalasin B, pinocytosis remained at the same rate as in the uninjured, confluent monolayer. These experiments support, and extend to endothelium, earlier observations that in growing cells pinocytosis proceeds at a higher rate than in non‐growing, quiescent cells. Furthermore, they raise the possibility that the transendothelial transport of macromolecules such as lipoproteins by receptor‐in‐dependent fluid pinocytosis in vivo may be altered by the growth status of the endothelium.
Sarcoma growth factor (SGF): Specific binding to epidermal growth factor (EGF) membrane receptors
Authors: De Larco Joseph E. , Todaro George J. 
Published: February 2005

AbstractCells transformed by murine sarcoma viruses (MSV) produce and release into their tissue culture media several polypeptide growth stimulating factors. One of these has been partially purified using Bio‐Gel P‐60 column chromatography followed by DEAE‐cellulose chromatography. This growth factor was assigned the name sarcoma growth factor (SGF), and is here shown to require the epidermal growth factor (EGF) receptor in order to function as a growth factor. DEAE‐cellulose chromatography yielded a product that was several‐fold purer than the material present in the Bio‐Gel P‐60 column pool II. The biologically active material from the DEAE‐cellulose column, when labeled with 125I, showed specific binding to EGF membrane receptors. The specific binding could be prevented with the addition of either unlabeled EGF or SGF. Both radiolabeled SGF and EGF will bind to live or fixed cells. We were able to bind 125I‐SGF as well as 125I‐EGF to fixed cells and elute the bound material from fixed receptors. The eluted SGF showed a greater than 25‐fold increase in specific binding. The biological activities of EGF and SGF could be bound to and eluted from fixed cells. A 3T3 clone lacking EGF receptors was unable to respond to either EGF or SGF, whereas it responded well to serum and several other purified growth factors. The SGF isolated using DEAE‐cellulose chromatography was unable to compete in a radioimmune assay using 125I‐EGF and antibody to purified mouse submaxillary gland EGF; it also was not precipitated by anti‐EGF antibody.From these studies it appears that the SGF produced and released by these MSV‐transformed cells combines with and requires the EGF receptor in order to exert its biological effects. The peptide, however, is antigenically distinct from mouse submaxillary gland EGF.
The regulation of haemopoiesis in long‐term bone marrow cultures: I. Role of L‐cell CSF
Authors: Dexter T. M. , Shadduck R. K. 
Published: February 2005

AbstractThe effects of L‐cell conditioned medium which contains granulocyte/macrophage colony stimulating factor (CSF); of highly purified L‐cell CSF; and the antiserum directed against L‐cell CSF, have been investigated in long‐term murine bone marrow cultures. Treatment of cultures with CSF containing conditioned medium led to a rapid decline in haemopoiesis. However, this inhibition of in vitro haemopoiesis is probably caused by materials other than CSF, since the addition of highly purified L‐cell CSF had no appreciable effect upon long‐term haemopoietic cell proliferation or differentiation. Furthermore, the inhibitory activity of L‐cell conditioned medium was not abrogated following neutralization of the CSF activity by CSF antiserum. The direct addition of CSF antiserum did not inhibit granulocyte or macrophage formation. These results suggest that long‐term cultures of murine marrow cells may show extensive interactions with stromal cells which are not influenced by exogenous stimulatory or inhibitory factors.
Quiescent cells but not cycling cells exhibit enhanced actin synthesis before they synthesize DNA
Authors: Riddle Veronica G. H. , Pardee Arthur B. 
Published: February 2005

AbstractMajor proteins synthesized by Swiss 3T3 cells at different stages of the cell cycle have been analyzed using double isotope labeling and one‐dimensional SDS‐polyacrylamide slab gels. The synthesis of actin was previously shown to be markedly enhanced a few hours after quiescent cells initiated growth following addition of serum. In contrast, the synthesis of actin remained at a constant rate, similar to that in quiescent cells, relative to synthesis of other proteins during the entire cell cycle. We conclude that enhanced actin synthesis is a process specific for the G0 to S transit, and may serve as a marker event during this interval. In contrast, three other proteins (90,000, 57,000, and 33,000 daltons) were synthesized throughout the cell cycle at higher rates than in G0 cells, and thus, are markers characteristic of cells traversing the cell cycle. A transient increase, such as seen for actin synthesis, by cells emerging from quiescence, may represent a process that these cells must perform before they can enter the G1 portion of the cell cycle. A transient event such as this need not be a periodic event that occurs during each cycle.
Na entry and Na‐K pump activity in murine, hamster, and human cells ‐ effect of monensin, serum, platelet extract, and viral transformation
Authors: Mendoza Stanley A. , Wigglesworth Noel M. , Pohjanpelto Pirkko , Rozengurt Enrique 
Published: February 2005

AbstractThe relationship between Na entry and the activity of the Na‐K pump has been investigated in a variety of cell types by testing the effect of the Naionophore monensin, mitogenic stimulation with serum and oncogenic transformation by SV40 and polyoma virus. We found that addition of monensin increases intracellular Na in quiescent cultures of murine, hamster, and human cells. In each case, the rise in intracellular Na by monensin is associated with an increase in the activity of the Na‐K pump, which was measured as ouabain‐inhibitable 86Rb uptake. The addition of serum to quiescent cultures stimulates 86Rb uptake in all cell types studied. Serum alone causes an increase in intracellular potassium with no consistent change in intracellular Na. In the presence of the Na‐K pump inhibitor ouabain, serum causes a marked increase in intracellular Na, with little change in intracellular K. This pattern is interpreted as indicating that the primary effect of serum is to increase Na entry into the cells. A low concentration of monensin (0.2 μg/ml) mimics the effect of serum on ion fluxes and content, which supports the conclusion that serum and monensin stimulate 86Rb uptake in the same manner, namely by increasing Na entry into the cells. In addition, a partially purified platelet extract stimulates Na entry and 86Rb uptake in quiescent 3T3 cells. Finally 3T3 cells transformed by SV40 or polyoma virus exhibit a higher rate of Na entry and of Na‐K pump activity than their untransformed 3T3 counterparts. All these results indicate that the rate of Na entry plays an important role in the regulation of the activity of the Na‐K pump and that an increase in Na and K movements is a rapid response elicited by serum in a variety of cell types.
The nucleus as the site of tRNA methylation
Authors: Colonna Alfredo , Kerr Sylvia J. 
Published: February 2005

AbstractMouse L‐cells were enucleated by exposure to cytochalasin B followed by centrifugation. The resulting karyoplasts, nuclei surrounded by a thin shell of cytoplasm and an outer cell membrane, and cytoplasts, the enucleated cell cytoplasm, were assayed for tRNA methyltransferase activity. The bulk of the enzyme activity was found to be localized in the nuclei. Analysis of the methylated nucleosides produced by the enzyme from the two sources showed that all the base‐specific enzyme activities which are found in whole cell extracts were present in the nuclear extracts.The cytoplast extracts retained a low but detectable enzyme activity, which was composed predominantly of only two base‐specific activities. This may represent tRNA methyltransferases of the mitochondria or may be cytoplasmic enzymes for late modification reactions.
Inhibitory effect of cyclic adenosine 2′,3′‐monophosphate on leucine incorporation by L5178Y cells
Authors: Fuhr Joseph E. , Stidham James D. 
Published: February 2005

AbstractThe specificity of the reported stimulation of protein synthesis affected by dibutyryl adenosine cyclic 3′,5′‐monophosphate in L5178Y cells was tested by assaying the effect of adenosine cyclic 2′,3′‐monophosphate. Protein synthesis was assayed as total acid precipitable 3H‐leucine. Leucine incorporation was inhibited by adenosine cyclic 2′,3′‐monophosphate. Alone, and in combination with actinomycin, 2′,3′‐cAMP inhibited leucine incorporation more rapidly and to a greater degree than did actinomycin alone. Since a similar potentiating effect was not observed with cordycepin, the hypothesis has been suggested that adenosine cyclic 2′,3′‐monophosphate inhibits protein synthesis by interfering with some post transcriptional event, perhaps modification of existing RNA. The results further suggest that 2,3 cyclic AMP may be antagonistic of the effects of 3,5 cyclic AMP, and as such may be useful as an inhibitor of that nucleotide's action.
Megakaryocytopoiesis in culture: Modulation by cholinergic mechanisms
Authors: Burstein Samuel A. , Adamson John W. , Harker Laurence A. 
Published: February 2005

AbstractTreatment of murine bone marrow cultures with the cholinergic agonist carbamylcholine enhanced megakaryocytic colony growth by as much as 65%. In contrast, adrenergic agonists had no such effect. Addition to cultures of dibutyryl cyclic GMP (db‐cGMP) also enhanced megakaryocytic colonies up to 50%, whereas dibutyryl cyclic AMP (db‐cAMP) had no effect. Sodium nitroprus‐side and sodium nitrite, putative guanyl cyclase activators, also enhanced colony numbers, as did imidazole, a postulated cGMP phosphodiesterase inhibitor. Preincubation of marrow for two hours with carbamylcholine resulted in both an increase in colony numbers (58%) and percent of progenitors in DNA synthesis (48%, compared to 14% for controls) as determined by tritiated thymidine suicide studies. Treatment of mice with the acetylcholinesterase inhibitor neostigmine resulted in an increase in CFU‐M/humerus (62%) and percent in DNA synthesis (45%). These data indicate that (1) cholinergic, but not adrenergic, agonists modulate megakaryocytopoiesis in culture; (2) this effect may be mediated by cyclic GMP; and (3) only a brief period of exposure of marrow cells to agonist results in enhancement of megakaryocytic colonies.
Senescence of cultured human diploid fibroblasts. Are mutations responsible?
Authors: Gupta Radhey S. 
Published: February 2005

AbstractTwo predictions of the error/mutation hypothesis of cellular sensecence (Orgel, '73) namely, (a) exponential accumulation of somatic mutations during the replicative lifespan and (b) shortening of culture lifespan upon treatment with mutagens have been examined experimentally in a strain of cultured human diploid fibroblasts. Our studies show that as cells traverse the replicative lifespan (from 10 to 75 mean population doublings (MPD); total lifespan ⋍ 95 MPD), no rapid and exponential increase occurs in the accumulation of mutations measured by the frequencies of Thgr (thioguanine resistance) and Dipr (diphtheria toxin resistance) mutants. Furthermore, repeated cycles of treatment (from 1‐ to 14‐times) of human fibroblasts with two mutagens, ethyl methane sulfonate (EMS) and N‐methyl‐N′nitro‐nitrosoguanidine, which led to a marked increase in the mutation frequency for the Dipr marker (⋍ 100‐fold), failed to shorten the lifespan of cultured fibroblasts. On the contrary, repeated mutagen treatment (12 times with EMS) prolonged the lifespan of one replicative culture (110 MPD versus 94–98 MPD). These results strongly indicate that mutations are unlikely to be the primary event in cellular senescence and suggest instead that senescence is probably controlled by one or more (specific) gene(s) whose expression can be modified by mutations.
Decay of hormone responsiveness in mouse melanoma cells in culture as a function of cell density
Authors: Fuller Bryan B. , Lebowitz Joyce 
Published: February 2005

AbstractCloudman S91 mouse melanoma cells lose their ability to demonstrate an MSH‐induced increase in tyrosinase activity as cell density increases. This loss in hormone responsiveness occurs before confluency is reached and cannot be reversed by exposure of cells to increasing concentrations of MSH. The failure of high‐density cultures to respond to MSH is apparently not the result of an inability of MSH to stimulate cAMP production, since either low‐ or high‐density cultures exposed to MSH demonstrate equivalent increases in intracellular levels of cAMP. Further, neither theophylline (1mM), dibutyryl cyclic AMP (10–4M), or prostaglandin E1 (10–6M) is effective in stimulating tyrosinase activity in melanoma cells cultured at densities exceeding 6 ± 104 cells/cm2. This finding suggests that the decay of hormone responsiveness occurs at a cellular site distal to cAMP production. The decrease in tyrosinase stimulation by MSH as cell density increases is also apparently not the result of an increase in activity of any soluble inhibitor of the enzyme, for cytosol preparations from high‐density cultures (105 cells/cm2) fail to inhibit tyrosinase activity in cell homogenates from low‐density cultures treated with MSH.