bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin binding sites on rat liver nuclear membranes: Biochemical and immunofluorescent studies
Authors: Horvat Agnes
Published: February 2005

Preliminary investigations (Horvat et al., '75) indicated the nucleus of rat liver as a site for specific binding of insulin. In this report these observations are confirmed. Nuclei from rat liver were isolated in a highly purified state as verified by interference contrast and electron microscopy and by chemical analysis. Extensive scanning of the preparations did not reveal the presence of structures resembling plasma membranes. The nuclear envelope was isolated by a modification of the method of Kay et al. ('72). Electron micrographs showed the presence of nuclear “ghosts” and few other recognizable nuclear elements, but no plasma membranes (60–80 Å thick) were detected. The preparation was found to contain specific insulin binding activity. Specificity of the binding sites for insulin was demonstrated in competition studies with other polypeptide hormones and a synthetic insulin analog. Scatchard analysis of the binding data indicates the presence of a single class of high affinity receptors. In contrast to findings with plasma membranes the hormone‐receptor complex is very stable and the kinetics of the dissociation of bound [125I]‐insulin do not indicate negative cooperativity of the binding sites. Immunofluorescent labeling of intact, unfixed nuclei showed a specific fluorescent halo only around those nuclei that have been preincubated with insulin. All other controls were negative.
Methionine metabolism in BHK cells: Preliminary characterization of the physiological effects of cycloleucine, an inhibitor of S‐adenosylmethionine biosynthesis
Authors: Caboche Michel , Hatzfeld Jacques
Published: February 2005

Cycloleucine is in vitro a competitive inhibitor of methionine adenosyltransferase, an enzyme involved in S‐adenosylmethionine biosynthesis. The physiological effects of this drug on baby hamster kidney cells have been studied. When cells are grown in a medium containing 10 μM methionine, cycloleucine is an inhibitor of cell proliferation; high concentrations of methionine are able to withdraw this inhibition suggesting that cycloleucine toxicity is related to methionine metabolism. The drug does not primarily affect methionine uptake and its subsequent use for protein biosynthesis. Cycloleucine toxicity is correlated with a block of SAM biosynthesis and nucleic acids methylations. The actions of cycloleucine on progression in the cell cycle and DNA, RNA and protein biosynthesis are studied. The implications of these results are discussed.
Transforming viruses directly reduce the cellular growth requirement for a platelet derived growth factor
Authors: Scher Charles D. , Pledger W. J. , Martin Paul , Antoniades Harry , Stiles Charles D.
Published: February 2005

Early passage mouse embryo fibroblasts, mouse 3T3 cell lines, and early passage diploid human fibroblasts grew to higher cell densities in tissue culture medium supplemented with serum than in medium supplemented with defibrinogenated platelet‐poor plasma (PPP). Unlike the mouse cells, the human fibroblasts displayed this differential growth response only in the presence of hypophysiologic concentrations of calcium. The addition of heat‐treated extracts of human platelets to PPP‐supplemented medium stimulated the replication of both the normal mouse cells and early passage human embryo fibroblasts.

Human or mouse fibroblasts transformed by either retroviruses or by SV40, including SV40 infected “serum revertants” and “flat transformants,” grew to equal cell densities in medium supplemented with either serum or PPP. Infection of Balb/c‐3T3 cells with SV40 rapidly induced them to grow in PPP‐supplemented medium demonstrating that the ability of SV40‐transformed cell lines to proliferate in PPP‐supplemented medium does not arise from the cell culture selection procedures usually employed to obtain stable virus‐transformed cell lines. 3T3 cells infected but not transformed by retroviruses do not replicate in PPP‐supplemented medium demonstrating that reduction of the growth requirement for the platelet growth factor(s) by retroviruses is a transformation‐specific response. Cell cultures that did not proliferate well in PPP‐supplemented medium did not form tumors when inoculated into athymic nude mice. Many, although not all, of the lines which grew well in PPP medium were tumorigenic in nude mice. Together, these findings indicate that: (1) normal fibroblast‐like cells display a growth requirement for factor(s) present in serum but not found in PPP; (2) this serum specific growth factor is derived from platelets; (3) a primary response to viral transforming genes is a reduction in the growth requirement for these platelet‐derived factors; and (4) cells that have a reduced requirement for the platelet‐derived growth factor are often tumorigenic.
Plasminogen activator synthesis by cultured human embryonic lung cells: Characterization of the suppressive effect of corticosteroids
Authors: Rifkin Daniel B.
Published: February 2005

The synthesis of plasminogen activator (PA) by cultured human embryonic lung (HuEL) cells has been examined. The production of PA by these cells was found to be reversibly inhibited by physiological levels of glucocorticoids. The suppression of PA synthesis in HuEL cells was not accompanied by an inhibition of cell growth. Moreover, the glucocorticoid induced deinduction of plasminogen activator synthesis occurred in both growing and non‐growing cells. The inhibition of PA production by corticosteroids appeared to have a requirement for DNA‐dependent RNA synthesis since the inhibition of DNA‐dependent RNA synthesis at the time of exposure of cells to corticosteroids prevented the deinduction of PA.
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

The 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

An 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

Cellular 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

The 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

During 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

A 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

RNA 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

Cytoplasts 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

Methods 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

The 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

Infection 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

Benzo[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

Epidermal 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

This 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

Stimulation 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

The 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

A 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

The 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.
Isolation and characterization of tunicamycin resistant mutants from Chinese Hamster ovary cells
Authors: Sudo Tadashi , Onodera Kazukiyo
Published: February 2005

Stable 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

We 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

Parathyroid 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

Leupeptin, 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

The 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

These 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

Rates 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

Cells 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

The 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

Major 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

The 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

Mouse 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

The 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.