bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evidence for variation in the number of functional gene copies at the AmaRlocus in chinese hamster cell lines
Authors: Gupta Radhey S. , Chan David Y. H. , Siminovitch Louis 
Published: February 2005

AbstractThe hypothesis of functional hemizygosity has been examined for the α‐amanitin resistant (AmaR, a codominant marker) locus in a series of Chinese hamster cell lines. AmaRmutants were obtained from different cell lines, e.g., CHO, CHW, M3‐1 and CHO‐Kl, at similar frequencies. After fractionation of different RNA polymerase activities in the extracts by chromatographic procedures, the sensitivity of the mutant RNA polymerase II towards α‐amanitin was determined. While all of the RNA polymerase II activity in mutant CHO and CHO‐Kl lines became resistant to α‐amanitin inhibition, only about 50% of the activity is highly resistant in AmaRmutants of CHW and M3‐1 cell lines. The remaining activity in the latter cell lines shows α‐amanitin sensitivity similar to that seen with the wild‐type enzyme. This behaviour is similar to that observed with a 1:1 mixture of resistant and sensitive enzymes from CHO cells. These results, therefore, strongly indicate that while only one functional copy of the gene affected by α‐amanitin is present in CHO and CHO‐Kl cells, two copies of this gene are functional in the CHW and M3‐1 cell lines.
Differentiation‐related death of an established keratinocyte line in suspension culture
Authors: Morrissey James H. , Green Howard 
Published: February 2005

AbstractAn established keratinocyte line (XB), derived from a mouse teratoma, terminally differentiates in suspension culture in a manner similar to human epidermal keratinocytes. When surface‐grown XB cells are placed in suspension culture, they lose colony forming ability very rapidly; within three days the loss is virtually complete. Measurement of the ability of the suspended cells to synthesize protein and RNA show that they begin to lose both after 12 hours, the rate of uridine and glycine incorporation falling nearly to zero in about 36 hours. The cells then become insoluble in ionic detergents, owing to the formation of disulfide‐stabilized keratin filaments, and digest their nuclei.The total RNA content of the cells (a measure of ribosomes) begins to drop sharply about 12 hours after the cells are placed in suspension culture, and most RNA is eliminated by 24 hours. This process is independent of the presence of serum in the medium. DNA also begins to disappear from the cells, but this process is slower than ribosomal destruction and is strongly affected by the presence of serum. After seven days in the absence of serum, half the DNA still remains, and nearly all the nuclei are still visible, whereas during the same period in the presence of serum all visible nuclei and all DNA disappear.In contrast to the destructive process that takes place in the keratinocytes, 3T3 cells are much more stable in suspension culture. They show a reversible decline in their rate of amino acid incorporation, but no decline in their rate of uridine incorporation, and they undergo little loss in colony forming efficiency for several days. They retain most of their RNA and nuclei with full DNA content. The destructive process in suspended XB cells seems to be a model for the cell death that takes place in terminal differentiation of the keratinocyte.
Basis for differential cellular sensitivity to 8‐azaguanine and 6‐thioguanine
Authors: Van Diggelen Otto P. , Donahue Thomas F. , Shin Seung‐Il 
Published: February 2005

AbstractCellular resistance to the cytotoxic purine analogues 8‐azaguanine (AG) and 6‐thioguanine (TG) is usually mediated by a mutation leading to the loss or reduction in hypoxanthine phosphoribosyltransferase (HPRT) activity. However, stable AG‐resistant variants have often been shown to contain wild‐type levels of HPRT, while cellular resistance to TG is always accompanied by a profound deficiency in HPRT activity. Such AG‐resistant, HPRT‐positive cells are still sensitive to TG. To investigate the basis of this differential sensitivity, we examined the inhibition of the HPRT activity by AG and TG in whole cells, in cell‐free extracts, and with purified mouse HPRT. In addition, the relative incorporation and utilization of AG and TG by L929 cells were determined under a variety of culture conditions.Results show that, compared to TG, AG is generally a very poor substrate for HPRT. Incorporation of radioactive AG by HPRT‐positive cells was extremely sensitive to the free purine concentrations in the medium, so that under the usual culture conditions employing undialyzed serum, cellular uptake and utilization was minimal even when relatively high levels of AG were present. In contrast, the incorporation of radioactive TG was comparable to that of a natural substrate, hypoxanthine. These results indicate that the differential cellular sensitivity to AG and TG is due to the difference between these two guanine analogues as substrates of HPRT. Additional data indicate also that cellular resistance to TG is mediated exclusively by HPRT deficiency, but resistance to very high levels of AG may result through at least two other mechanisms not involving HPRT deficiency. These observations may help resolve some of the conflicting data in the literature, and demonstrate that TG is a better selective agent for the HPRT‐deficient phenotype.
Aminoacyl‐tRNA synthetase mutants degrade protein at a normal rate
Authors: Clark Jeffrey L. , Rabe Julie , Arfin Stuart M. 
Published: February 2005

AbstractThe stability of both rapidly and slowly degraded proteins in wild type CHO cells is similar to that in three ts aminoacyl‐tRNA synthetase mutants at both permissive and non‐permissive temperatures, although the degree of tRNA charging in the synthetase mutants differs considerably with temperature. These results indicate that the altered rate of protein breakdown seen under a variety of physiological conditions in eukaryotic systems is not mediated by uncharged tRNA.
A transient increase in amino acid transport modulated by insulin in differentiating muscle cells
Authors: Farfel Z. , Karlish S. , Prives J. 
Published: February 2005

AbstractDuring synchronous differentiation of embryonic chick muscle cells in culture, the Na‐dependent uptake of an amino acid analog, α‐amino isobutyric acid (AIB) undergoes an abrupt, transient increase. The increase in AIB uptake is concomitant with the rapid fusion of mononucleated myoblasts, and precedes the accumulation of muscle‐specific proteins. Subsequently, Nadependent AIB transport diminishes markedly during postfusional differentiation of myotubes. The rate of AIB uptake is increased by insulin both before and after myoblast fusion. This stimulation by insulin is restricted to the Nadependent component of total AIB uptake but is apparently not the result of insulin‐mediated increase in the trans‐membrane Na gradient.
Biochemical genetic analysis of pyrimidine biosynthesis in mammalian cells. II. Isolation and characterization of a mutant of chinese hamster ovary cells with defective dihydroorotate dehydrogenase (E.C. 1.3.3.1) activity
Authors: Stamato Thomas D. , Patterson David 
Published: February 2005

AbstractA mutant (A204) of Chinese hamster ovary cells (CHO‐K1), which is deficient in dihydroorotate (DHO) dehydrogenase (E.C. 1.3.3.1) activity, has been isolated by a replica plating procedure. The mutant does not show a requirement for exogenously added pyrimidines. Examination of intact cells shows that the mutant accumulates a large amount of carbamyl aspartate and is markedly but not totally deficient in biosynthesis of orotate from earlier precursors of pyrimidine biosynthesis, including aspartate and dihydroorotic acid, when compared to wild‐type cells. Analysis of cell‐free extracts of mutant and wild‐type cells shows that the mutant is deficient in DHO dehydrogenase activity, possessing ca. 5% of the wild‐type activity. This evidence leads to the conclusion that this mutant, A204, is in fact partially deficient in DHO dehydrogenase, and that in these cells it is this enzyme which carries out the fourth step of de novo pyrimidine biosynthesis.
RNA synthesis in growing and stationary cells of a culture of Scarlet rose. Disproportionate synthesis of ribosomal subunits in the stationary state
Authors: Ramagopal S. , Marcus A. 
Published: February 2005

AbstractRNA synthesis has been investigated in resting and growing cells of a culture of Scarlet rose. The rates of messenger RNA (mRNA) and ribosomal RNA (rRNA) synthesis are five‐ and ten‐fold higher, respectively, in the growing culture. In stationary phase cultures, newly synthesized 26S and 18S rRNA do not appear in the cytoplasm in equimolar amounts. Rather, the 26S/18S ratio of [3H]‐uridine labeled rRNA of stationary cells ranged from 0.9 to 1.3 while the ratio of the corresponding fraction from growing cells was 1.6 to 2.0. A similar result was obtained when cells were labeled with [3H‐CH3] methionine. Pulse chase experiments demonstrated that the nascent pre‐rRNA in resting cells could be chased into polysomes. These data are interpreted to indicate that a major part of the regulation of rRNA synthesis in stationary cells is at the level of the processing of the rRNA transcript.
Separation of cytoplasts and whole cells using density gradients of renografin
Authors: Lipsich Leah Ann , Lucas Joseph J. , Kates Joseph R. 
Published: February 2005

AbstractCytoplasts prepared from L929 or Hepa‐2 cells were separated from whole cells using density gradients of renografin. Using this technique, cytoplasts can be isolated from cell lines which cannot be routinely enucleated with an efficiency of 100%. The purified cytoplasts excluded the vital dye trypan blue and were utilized in nuclear transplantation experiments to reconstruct whole viable cells capable of division. In addition, the renografin gradient technique was used to separate the newly reconstructed cells from any contaminating “non‐renucleated” cytoplasts. This will permit immediate biochemical characterization of cytoplasmic‐nuclear hybrid cells without interference from contaminating cytoplasts.
Isolation and surface labeling of murine polymorphonuclear neutrophils
Authors: Watt Suzanne M. , Burgess Antony W. , Metcalf Donald 
Published: February 2005

AbstractMethods for the induction of an exudate of polymorphonuclear neutrophilic leukocytes (PMN) in the peritoneal cavity of C57BL, BALB/c, SJL and CBA mice were analysed. Peritoneal exudates in male mice were highly enriched for PMN (80‐90%) three hours after a single injection of calcium caseinate whereas eosionophils comprised less than 1% of the exudate population. Female mice were a less satisfactory source of PMN because the proportion of eosinophils in the exudate was variable. Purification of PMN from peritoneal exudate cells was performed on the basis of light scattering using a Becton‐Dickinson cell sorter or by density gradient centrifugation with graded polyvinylpyrroliodone‐coated silica particles (Percoll). Both techniques yielded approximately 97% pure PMN preparations. Electrophoretic analysis of the PMN proteins revealed an abundance of lactoferrin and actin, but several other proteins were also present in high concentrations. Proteolytic degradation of several high molecular weight proteins (>90,000) was prevented by the addition of phenylmethylsulphonyl fluoride (PMSF) and ethylene diamine tetracetic acid (EDTA). Surface iodination, using diphenyl, tetrachloroglycouril (IODO‐DEN), indicated that there were six tyrosine‐containing proteins present on the external cell membrane. The apparent molecular weights of these surface proteins ranged from 185,000 to 90,000 and the major 125I‐labeled protein had an apparent molecular weight of 90,000. Neither actin nor lactoferrin was labeled with 125I unless cell viability was lost during the iodination procedure. Standard conditions for labeling the cell surface only, required low iodide and IODO‐GEN concentrations. Biosynthetic labeling of PMN using 35S‐methionine increased the sensitivity of detection for most of the proteins, but some of the granule storage proteins (such as lactoferrin) were not effectively labeled within three hours.
Intracellular pH changes during the cell cycle in Tetrahymena
Authors: Gillies R. J. , Deamer D. W. 
Published: February 2005

AbstractThe equilibrium distribution of 5,5‐dimethyloxazoladine 2,4‐dione (DMO) between intra‐ and extracellular volume was used to estimate intracellular pH (pHi) in Tetrahymena pyriformis. In control experiments, DMO was found to equilibrate rapidly in response to a pH gradient. Under normal growth conditions, pHi was constant over a finite range of external pH, being maintained near pH 7.1 over the external pH range 5.25 to 7.3. This same range of external pH was also optimal for growth. pHi was monitored during the cell cycle of a synchronous population of T. pyriformis GL. The cells were synchronized either by starvation/refeeding or heat shock. Under both conditions, there were two alkaline shifts of approximately 0.4 pH units per cell cycle. These shifts in pH retained a constant remporal relationship to S phase and were not affected by changes in the time, duration, or magnitude of cytokinesis.
Erythropoietin responses and physical characterization of erythroid progenitor cells in rauscher virus infected BALB/c mice
Authors: Hasthorpe Suzanne , Bol Simon 
Published: February 2005

AbstractInfection of BALB/c mice with Rauscher leukemia virus (RLV) gives rise to pronounced erythrocytopoiesis manifesting in splenomegaly and is associated with progressive development of anemia. In the spleen erythroid colony forming units (CFU‐E) increase exponentially up to 800‐fold that of normal levels by the third week of infection. In vitro these CFU‐E are dependent on erythropoietin for colony formation, their erythropoietin requirements being higher than that of CFU‐E from normal mice. Numbers of CFU‐E in spleen and degree of splenomegaly in anemic RLV infected mice were also shown to be modified by red blood cell transfusion, but progression of the disease was not stopped. Erythroid burst forming units (BFU‐E) were also responsive to erythropoietin. However, a small proportion of cells also formed BFU‐E colonies at concentrations which did not support growth of normal marrow BFU‐E.When compared to normal, CFU‐E found in RLV‐infected spleen have similar velocity sedimentation rates. However, buoyant density separation of leukemic spleen cells indicated that CFU‐E were more homogeneous (modal density 1.0695 g/cm3) than CFU‐E from normal spleen. Analysis of physical properties of CFU‐E and the nonhemoglobinized erythroblast‐like cells, which accumulate in the spleen showed that they differed mainly in their distribution of cell diameter.Our findings show that erythroid progenitor cells in RLV infected mice are responsive to erythropoietin in vitro. Also in vivo erythropoiesis appears to be under control of erythropoietin but other factors which lead to progression of RLV disease apparently exist. Most proerythroblast‐like cells, which are characteristic of this disease, apparently lack the potential to form colonies and may be more mature than CFU‐E.
Epidermal growth factor and the control of proliferation of Balb 3T3 and benzo[a]pyrene‐transformed Balb 3T3 cells
Authors: Brown Kenneth D. , Holley Robert W. 
Published: February 2005

AbstractBenzo[a]pyrene‐transformed Balb 3T3 cells (BP3T3) exhibit “normal” growth controls at low concentrations of serum. Epidermal growth factor (EGF) stimulates DNA synthesis and cell division in both Balb 3T3 and BP3T3 cells at physiological concentrations. The growth response of BP3T3 cells to EGF is qualitatively the same as that of 3T3 cells, however, the transformed cells have a lower quantitative requirement. Both 3T3 and BP3T3 cells show a density‐dependent response to EGF, but the shift in the dose response curve for BP3T3 cells at high cell density is smaller than that seen for 3T3 cells. One cause of the restricted growth of 3T3 cells at high cell density compared with BP3T3 cells in the increased concentration of growth factor needed for stimulation of 3T3 cells at higher cell densities. A lower rate of depletion of other growth factory by BP3T3 cells may also explain the smaller effect of cell density on the EGF response of these cells.
Binding, internalization, and degradation of epidermal growth factor by Balb 3T3 and BP3T3 cells: Relationship to cell density and the stimulation of cell proliferation
Authors: Brown Kenneth D. , Yeh Yun‐Chi , Holley Robert W. 
Published: February 2005

AbstractEpidermal growth factor (EGF) stimulates the growth of both benzo[a]pyrene‐transformed Balb 3T3 cells (BP3T3) and untransformed Balb 3T3 cells. We describe here the binding, internalization, and degradation of [125I]‐EGF by BP3T3 cells and 3T3 cells. Binding of [125I]‐EGF reaches a maximum after 45 to 90 minutes incubation at 37°C. In both BP3T3 and 3T3 cells the extent of EGF binding required to stimulate DNA synthesis is density dependent; sparse cultures require a 15‐30% occupancy to elicit a maximal response whereas dense cultures require a 70‐85% occupancy. At physiological concentrations the total binding of [125I]‐EGF to 3T3 cells is higher than to BP3T3 cells, and this difference increases at higher cell densities. The rate of degradation of [125I]‐EGF is directly proportional to the total [125I]‐EGF binding in each cell type. This supports the hypothesis that one cause of the diminished serum requrement of BP3T3 cells is a reduced rate of utilization of serum growth factors.
Peptidase activity in Tetrahymena
Authors: Zdanowski Marek K. , Rasmussen Leif 
Published: February 2005

AbstractThis report strongly suggests that two compartments in Tetrahymena thermophila contain peptidase activity: the cytoplasm and the outer cell surface.Determinations of amino acid concentrations in the extracellular medium upon incubation of cells with peptides suggest that the surface‐bound peptidase activity hydrolyses di‐ and tri‐phenylalanine equally fast on a molar basis.Growth experiments designed to characterize the in vivo peptidase specificities showed that both T. thermophila and T. pyriformis can use L‐leucyl‐L‐leucine, but not L‐leucyl‐D‐leucine as a leucine donor. These results are independent of whether the cells form food vacuoles or not.
The tumor promoter 12‐0‐tetradecanoyl‐phorbol‐13‐acetate enhances the proliferative response of Balb/c‐3T3 cells to hormonal growth factors
Authors: Frantz Christopher N. , Stiles Charles D. , Scher Charles D. 
Published: February 2005

AbstractStimulation of Balb/c‐3T3 cell growth by TPA requires factors found in serum. We examined the interaction between TPA and serum growth factors in the stimulation of cell growth. The number of cells synthesizing DNA (incorporating 3H‐thymidine) within 24 to 30 hours after the addition of TPA and the growth factors to density‐inhibited Balb/c‐3T3 cultures in serum‐free medium was determined by autoradiography. With no additions or with TPA (30‐300 ng/ml) alone, only 3‐7% of cells synthesized DNA. However, TPA synergistically promoted DNA synthesis in combination with each of the defined serum growth fractions, platelet derived growth factor and platelet poor plasma. TPA also synergistically promoted DNA synthesis in combination with purified growth factors including fibroblast growth factor, insulin (10−6‐10−5 M), and epidermal growth factor. In all conditions, TPA enhancement of DNA synthesis also resulted in an increase in cell number. Because TPA synergistically enhanced the activity of each growth factor tested, it did not act identically to any of the growth factors.
Correlation between cell cycle duration and RNA content
Authors: Darzynkiewicz Zbigniew , Evenson Donald P. , Staiano‐Coico Lisa , Sharpless Thomas K. , Melamed Myron L. 
Published: February 2005

AbstractThe metachromatic fluorochrome acridine orange was used to differentially stain DNA and RNA in Chinese hamster ovary (CHO) cells and in mitogen‐stimulated human lymphocytes during their progression through the cell cycle. Green and red fluorescence of individual cells, representing cellular DNA and RNA, respectively, was measured by flow cytometry.CHO cells were synchronized by selective detachment at mitosis. Their rate of progression through G1 and subsequently through S phase correlated with the content of stainable RNA. The mean duration of the G1 phase was 5.2 hours for cells with high RNA content (highest 25 percentile population) and 8.1 hours for cells with low RNA (lowest 25 percentile). The duration of S phase was 5.9 and 7.5 hours for high‐ and low‐RNA, 25 percentile subpopulations, respectively.Lymphocytes synchronized at the G1/S boundary by hydroxyurea or 5‐fluorodeoxyuridine showed extremely high intecellular variation with respect to content of stainable RNA. After release from the block they traversed S phase at rates linearly proportional to the content of stainable RNA. The duration of S phase was five hours for cells with high RNA‐, six to nine hours for cells with moderate RNA‐ and up to 27 hours for cells with minimal RNA‐content.The data suggest that the rate of progression the cell cycle of individual cells within a population may be correlated with the number of ribosomes per cell.
A variant of S49 mouse lymphoma cells with enhanced secretion of cyclic AMP
Authors: Steinberg Robert A. , Steinberg M. Gwen , Van Daalen Wetters Theodoor 
Published: February 2005

AbstractA novel variant of S49 mouse lymphoma cells is described which is resistant to growth arrest and cytolysis by dibutyryl cyclic AMP but, in contrast to previously described variants, has normal cyclic AMP‐dependent protein kinase. The variant is also resistant to N6‐monobutyryl cAMP but is sensitive to killing by 8‐bromo cAMP and cholera toxin. Extracts of the variant appear to contain wild type levels of both O2′‐butyrylesterase and cyclic AMP phosphodiesterase activities. Accumulation of exogenous [3H]dibutyryl cyclic AMP is reduced in the variant suggesting a defect in either uptake or secretion of the analog or its metabolic products. Accumulation of cyclic AMP in variant cells after stimulation of adenylate cyclase with either isoproterenol or cholera toxin is also reduced compared with wild type cells, although cyclase activity of membranes prepared from the variant cells is normal. Extracellular accumulation of cyclic AMP after stimulation of variant cells with isoproterenol is greater than that found with wild type cells. It is concluded that the variant has an alteration in its cyclic AMP secretion mechanism resulting in more efficient extrusion of cyclic AMP than in wild type cells.
Trace element uptake by L‐cells as a function of trace elements in a synthetic growth medium
Authors: Zombola R. R. , Bearse R. C. , Kitos P. A. 
Published: February 2005

AbstractThe concentration of trace elements in L‐cells has been studied as a function of the trace metal content of the growth medium. Cells were cultured in synthetic media which contained varying trace amounts of the elements manganese, iron, cobalt, copper, zinc and molybdenum. The cellular concentration of the elements potassium, iron, copper and zinc were then determined. It was found that the cell accumulates trace metals at a different rate than they are made available. Deficiencies in zinc could be “induced” in the cell by increasing the concentration of iron, manganese and cobalt; cellular iron deficiencies were observed at larger medium concentrations of zinc, manganese, copper and cobalt. Trace metal uptake by the cell was seen to parallel the utilization by multicellular organisms.