bluuebunny/crossref_metadata_2025_split huggingface dataset:
Topography of protein kinases and phosphoproteins in the plasma membrane of 3T3 cells
Authors: Boman Bruce M. , Zschunke Michael A. , Scott Robert E. 
Published: February 2005

AbstractThe plasma membrane of 3T3 cells contains at least two different endogenous cyclic AMP‐dependent protein kinase systems. One catalyzes the phosphorylation of endogenous protein substrates, i.e., PP24 and PP14, whereas the other catalyzes the phosphorylation of exogenous substrates. In this paper the topography of these cyclic AMP‐dependent phosphorylation systems is described. The results show that the kinases which phosphorylate only exogenous substrates are primarily localized to the outer plasma membrane surface whereas the endogenous cyclic AMP‐dependent protein kinase and its two endogenous substrates are localized to the cytoplasmic plasma membrane surface. The data also establish that neither the cytoplasmically orientated kinase nor its substrates has a transmembrane orientation even though factors acting on the outer plasma membrane can affect these proteins. This suggests that functional modulation of the cytoplasmically localized cyclic AMP‐dependent phosphorylation system can be mediated by a transmembrane regulatory mechanism. The importance of determining the topography of such plasma membrane phosphorylation systems is emphasized by recent studies which show that neoplastic transformation can be mediated at least in part by protein kinases and/or phosphoproteins which are localized on the cytoplasmic surface of the plasma membrane.
β‐Adrenergic stimulation inhibits calcium efflux and alters the morphology of cultured arterial muscle cells
Authors: Smith Jeffrey Bingham 
Published: February 2005

Abstractβ‐Adrenoreceptor stimulation with isoproterenol (IP) rapidly and reversibly rounded and arborized smooth muscle cells (SMC) cultured from rat aorta. The arborized SMC remained firmly attached to the substratum via a network of long, dendritelike processes. Arborization of the SMC correlated closely with increases in cellular cAMP produced by IP and a variety of other compounds. The intracellular calcium antagonist 8‐(N,N‐diethylamino)‐octyl‐3,4,5‐trimethoxybenzoate (TMB‐8) also rounded and arborized the SMC. Antitubulin compounds potently blocked arborization by IP, dibutyryl cAMP, and TMB‐8. The release of cell‐bound 45Ca2+ was followed in the presence and absence of IP. IP strikingly increased the amount of 45Ca2+ that remained cell bound between 20 and 120 min Propranolol and colchicine prevented IP from inhibiting the release of cell‐bound 45Ca2+. These results suggest that the modulation of Ca2+ transport is involved in the arborization of cultured SMC by cAMP.
Recovery of cyclic nucleotide regulation in protein‐kinase‐defective adrenal cells through somatic cell fusion
Authors: Schimmer Bernard P. , Horney Sandra J. , Williams Shirley A. , Aitchison William A. , Doherty Paul J. 
Published: February 2005

AbstractA mutant cell line (designated Kin‐8), isolated from the Y1 mouse adrenocortical tumor cell line on the basis of its resistance to growth‐inhibition by 8‐bromoadenosine 3′, 5′‐monophosphate (8BrcAMP), was resistant to the steroidogenic effects of the cyclic nucleotide analog and did not round up in the presence of 8BrcAMP as did responsive Y1 adrenal cells. In Kin‐8 cells, the mutation to cyclic nucleotide resistance was associated with a defective type 1 cAMP‐dependent protein kinase activity, suggesting an obligatory role for the enzyme in the regulation of these adrenal functions. In this study, the Kin‐8 mutant was fused with a rat glioma cell line (C6) in order to analyze the genetic behavior of the protein kinase mutation in somatic cell hybrids. The growth of C6 glial cells was inhibited by 8BrcAMP and its cAMP‐dependent protein kinase responded normally to cAMP. In addition, C6 cells had no capacity for steroidogenesis nor did they round up when treated with 8BrcAMP. In Kin‐8 × C6 hybrids, the protein kinase mutation seemed to behave recessively. The growth of hybrid cells was inhibited by 8BrcAMP and the protein kinase responded to cAMP over a normal range. Kin‐8 × C6 hybrids, when treated with 8BrcAMP, exhibited steroidogenic activities which were greater than the activity measured in either fusion partner and which had lower ED50 values for 8BrcAMP. In addition, Kin‐8 × C6 hybrids rounded up in the presence of 8BrcAMP, a morphologic change unlike that seen with either fusion partner. The effect of 8BrcAMP on the steroidogenic activity and morphology of Kin‐8 × C6 hybrids was reminiscent of the effects of the cyclic nucleotide on cAMP‐responsive, parental Y1 adrenal cells. These results suggest that cell fusion provided a normal cAMP‐dependent protein kinase for Kin‐8 cells and led to the recovery of a cAMP‐responsive adrenal phenotype. These results provide additional evidence in support of an obligatory role for cAMP‐dependent protein kinase in the regulation of adrenal steroridogenesis, cell division, and cell shape.
Effects of cystic fibrosis serum on calcium influx and secretion using isolated tracheal epithelial cells
Authors: Wooten M. W. , Rudick M. J. , Rudick V. L. , Kramer R. I. 
Published: February 2005

AbstractHamster trachea epithelial (HTE) cells were shown to respond to 20% Cystic fibrosis serum (CFS) by secreting twice as much protein as in the presence of 20% normal human serum (NHS). Seum from obligate heterozygotes (HHS) produced an intermediate effect. A peak of Ca2+ entry into the HTE cells occurred about 30 min after exposure to 20% CFS, followed by a slow decline to basal levels. In contrast, 20% NHS did not cause an influx of Ca2+ and HHS produced an influx to about half that of CFS. Increasing concetrations (5‐30%) of pooled NHS had no effect on HTE cell Ca2+ uptake or secretion, but pooled CFS and HHS caused progressive increases in Ca2+ influx and protein secretion from 10 to 25% sera. The CFS‐induced Ca2+ influx and secretion were about twice those of HHS throughout the range of serum concentrations tested, suggesting the presence of a modulatory influence in HHS. When EGTA was used to chelate extracellular Ca2+ in the presence of CFS, Ca2+ influx was prevented and there was no stimulation of secretion. lonophore A23187 allowed Ca2+ entry into HTE cells in the presence or absence ofserum and a heightened level of secretory activity followed. The time course of Ca2+ influx under the influence of CFS was shown to correspond to the efflux of Na+ from the cells. Also verapamil, a Ca2+ channel blocking agent, inhibited CFS‐induced Ca2+ influx by 50% at 10−5M and prevented secretion. Thus, it appears that CFS, but not NHS, contains an agent which stimulates Ca2+ uptake into HTE cells by means of a Ca2+ channel and/or Na+‐Ca2+ exchange mechanism, and that increased intracellular Ca2+ levels then trigger secretion. The intermediate HTE cellr esponse to HHS suggests that half of the CFS stimulatory agent is present as would be expected in a gene dose effect, lending support for a agentic basis for CF.
Modulation of Ca2+‐mediated K+‐gating of erythrocyte ghosts by external Ca‐EGTA
Authors: Benjamin A. M. , Quastel D. M. J. 
Published: February 2005

AbstractUsing 86RB+ as a marker for K+ permeability, we find that extracellular Ca‐EGTA influences the rate of 86Rb+ efflux from erythrocyte ghosts preloaded with 86Rb+ and “buffered” Ca2+. At an internal free Ca2+, where the rate of 86Rb+ efflux is minimal and uninfluenced by either external EGTA or external Ca2+, external Ca‐EGTA at 0.2–0.5 mM can raise the flux rate to as high as can be attained by raising internal Ca2+, in the presence of an excess externally either of Ca2+ or of EGTA. Higher concentrations of Ca‐EGTA (up to 1–2 mM) diminish the flux rate. External Ca‐EDTA or Mg‐EDTA can substitute for Ca‐EGTA in enhancing and suppressing flux rate. The peak rate is insensitive to external free Ca2+ but depends on internal Ca2+; internal Mg‐EDTA does not substitute for internal Ca‐EGTA. Thus, the erythrocyte membrane is asymmetric with respect to its interaction with Ca2+ and Ca‐EGTA. Also, 22Na+ does not substitute for 86Rb+. The peak rate of 86Rb+ flux produced by external Ca‐EGTA is diminished by chlorpromazine (0.1 mM) and augmented by 1‐propranolol (25 μM), in the same way as the rate produced by increasing internal Ca2+. The results suggest that external Ca‐EGTA enhances the affinity of internal Ca2+ for its receptor(s) which operate the K+‐gate at the inner surface of the membrane. At external concentrations of Ca‐EGTA above 1–2 mM, 86Rb+ flux rate again rises with increase of Ca‐EGTA. This phenomenon does not depend upon internal Ca2+, is not affected by chlorpromazine or by 1‐propranolol, and is associated with an enhanced permeability to 22Na+, inulin, and haemoglobin.
The in vitro cell culture age and cell density of articular chondrocytes alter sulfated‐proteoglycan biosynthesis
Authors: Malemud Charles J. , Papay Robert S. 
Published: February 2005

AbstractThe effect of cell culture age and concomitant changes in cell density on the biosynthesis of sulfated‐proteoglycan by rabbit articular chondrocytes in secondary monolayer culture was studied. Low density (LD, 2 d), middle density (MD, 5‐7 d), and high density (HD, 12‐15 d) cultures demonstrated changes in cellular morphology and rates of DNA synthesis. DNA synthesis was highest at LD to MD densities, but HD cultures continued to incorporate [3H]‐thymidine. LD cultures incorporated 35SO4 into sulfated‐proteoglycans at a higher rate than MD or LD cultures. The qualitative nature of the sulfated‐proteoglycans synthesized at the different culture ages were analyzed by assessing the distribution of incorporated 35SO4 in associative and dissociative CsCI density gradients and by elution profiles on Sepharose CL‐2B. Chondrocytes deposited into the extracellular matrix (cell‐associated fraction) 35SO4‐labeled proteoglycan aggregate. More aggregated proteoglycan was found in the MD and HD cultures than at LD. A 35SO4‐labeled aggregated proteoglycan of smaller hydrodynamic size than that found in the cell‐associated fraction was secreted into the culture medium at each culture age. The proteoglycan monomer (A1D1) of young and older cultures had similar hydrodynamic sizes at all cell culture ages and cell densities. The glycosaminoglycan chains of A1D1 were hydrodynamically larger in the younger LD cultures than in the older HD cultures and consisted of only chondroitin 6 and 4 sulfate chains. A small amount of chondroitin 4,6 sulfate was detected, but no keratan sulfate was measured. The A1D2 fractions of young LD cultures contained measurable amounts of dermatan sulfate; no dermatan sulfate was found in older MD or HD cultures. These studies indicated that chondrocytes at LD synthesized a proteoglycan monomer with many of the characteristics of young immature articular cartilage of rabbits. These results also indicated that rapidly dividing chondrocytes were capable of synthesizing proteoglycans which form aggregates with hyaluronic acid. Culture age and cell density appears primarily to modulate the synthesis of glycosaminoglycan types and chain length. Whether or not these glycosaminoglycans are found on the same or different core proteins remains to be determined.
β adrenergic receptor repopulation of C6 glioma cells after irreversible blockade and down regulation
Authors: Homburger V. , Pantaloni C. , Lucas M. , Gozlan H. , Bockaert J. 
Published: February 2005

AbstractC6 glioma cells possess β adrenergic receptors coupled with adenylate cyclase which can be irreversibly blocked by bromoacetylaminomethylpindolol (Br‐AAM‐pindolol), a β adrenergic antagonist. With 1 μM Br‐AAM‐pindolol, more than 80% of β adrenergic receptors, labeled by (3H)‐dihydroalprenolol ((3H)‐DHA), were blocked. After this blockade, new β adrenergic receptors were synthesized only during cell division. However, at cell confluency when the cell number was constant, turnover of β adrenergic receptors was barely detectable. Cycloheximide (1 μg/ml) inhibited cell growth as well as reappearance of β adrenergic receptors. A 90% loss of β adrenergic receptors in C6 glioma cells was obtained after down‐regulation for 15 h with 10 μM isoproterenol, a β adrenergic agonist. After removal of the agonist, recovery of β‐adrenergic‐sensitive adenylate cyclase was complete within 2 to 3 days, whereas β adrenergic receptors reached 90% of control value within 6 days. The half‐life of the receptor recovery was 2 to 3 days. Pretreatment of C6 glioma cells by Br‐AAM‐pindolol and subsequent cell exposure to isoproterenol indicated that down regulation and recovery of unblocked β adrenergic receptors did occur; however isoproterenol did not accelerate the biosynthesis of β adrenergic receptors. The recovery of both biological response and β adrenergic receptor occupancy was restored both in the presence or absence of cycloheximide (1 μg/ml), a concentration which blocked 90% of protein synthesis. Our results suggest that reappearance of β adrenergic receptors in C6 glioma cells, following isoproterenol‐induced down regulation, was not due to synthesis of new receptors but to recycling of the β adrenergic receptors.
Regulation of angiotensin I‐converting enzyme activity in serially cultivated bovine endothelial cells
Authors: Rosen Eliot M. , Noveral James P. , Mueller Stephen N. , Levine Elliot M. 
Published: February 2005

AbstractAngiotensin I‐converting enzyme (ACE) activity was measured in lysates of cloned and uncloned cultures of bovine fetal aortic endothelial cells. The expression of ACE activity in these cells was complex, and influenced by subcultivation, cell density, serum, cumulative population doublings, and clonal heterogeneity. The ACE specific activity at any point in the in vitro lifespan was determined, at least in part, by interaction of these culture variables. After subcultivation to subconfluent densities, cellular ACE specific activity decreased markedly and did not reach detectable levels until cells attained confluent densities. The use of different suppliers' lots of serum in the growth medium resulted in different cellular ACE specific activities. The ACE specific activity decreased as cultures were serially subcultivated, but remained detectable throughout the lifespan, suggesting a linkage between the proliferative history of an endothelial cell and its remaining capacity to express ACE. Increased ACE activity was observed when cells at the end of their lifespan were cultured at high densities. Cloned strains behaved similarly to the uncloned parent culture, except that they exhibited a wide range of ACE specific activities.
Modulation of beta‐adrenergic response in rat brain astrocytes by serum and hormones
Authors: Wu Doris K. , Morrison Richard S. , de Vellis Jean 
Published: February 2005

AbstractPurified astrocyte cultures from neonatal rat cerebrum respond to isoproterenol, a beta‐adrenergic agonist, with a transient rise in cAMP production. This astroglial property was regulated by serum, a chemically defined medium (serum‐free medium plus hydrocortisone, putrescine, prostaglandin F2α, insulin, and fibroblast growth factor) and epidermal growth factor. Compared to astrocytes grown in serum‐supplemented medium, astrocytes grown in the chemically defined medium were nonresponsiveto isoproterenol stimulation, and this difference did not appear to be due to selection of a subpopulation of cells by either medium. The data suggest that a decreased beta‐adrenergic receptor number and an increased degradation of cAMP may account for the reduced response to beta‐adrenergic stimulation. The nonresponsive state of astrocytes in the defined medium was reversible when the medium was replaced with serum‐supplemented medium. An active substance(s) in serum was responsible for restoring the responsiveness of astrocytes. Each of the five components of the defined medium had little effect by itself; however, together they acted synergistically to desensitize astrocytes to beta‐adrenergic stimulation. On the other hand, epidermal growth factor, a potent mitogen for astrocytes, was very competent by itself in reducing the cAMP response of astrocytes to beta‐adrenergic stimulation. Thus purified astrocytes grown in the chemically defined medium appear to be a good model for the study of hormonal interactions and of serum factors which may modulate the beta‐adrenergic response.
Cultured mouse marrow stromal cell lines. II. Distinct subtypes differing in morphology, collagen types, myelopoietic factors, and leukemic cell growth modulating activities
Authors: Zipori Dov , Duksin Dan , Tamir Merana , Argaman Anat , Toledo Jeki , Malik Zvi 
Published: February 2005

AbstractA series of stromal cell lines were studied for their growth properties, electron microscopic morphology, cytochemical profile, collagen types, production of myelopoietic factors, and modulation of leukemic cell growth. Three cell types were identified in addition to the previously described macrophages (14M and 14M1) and preadipocytes (14F). MBA‐1 cells were found to be fibroblasts by their ability to synthesize collagen types I and III, while the cell line MBA‐13 shared properties in common with both fibroblasts and endothelial cells (collagen types I, III, IV, V). The third cell type, represented by the stromal cell line MBA‐2, produced mainly collagen types IV and V and exhibited junctional complexes between adjacent cells. All of the cell lines tested produced and secreted a macrophage‐colony‐stimulating factor, CSF‐1. MBA‐2 and to a lesser extent, MBA‐13, produced an additional activity resistant to anti‐CSF‐1 antiserum. Trypsin extraction of outer surface components from two clones of the MBA‐2 cell line (MBA‐2.1 and MBA‐2.4) yielded high molecular weight factor(s) that specifically inhibited the growth of a plasmacytoma cell line (MPC‐11). Such inhibitory activity was not detected in other stromal cell lines. It is possible that this variability in the nature of stromal cell lines represents corresponding diversity of cell types comprising the hematopoietic microenvironment in vivo.
Regulation of thymidylate synthase enzyme synthesis in 5‐fluorodeoxyuridine‐resistant mouse fibroblasts during the transition from the resting to growing state
Authors: Jenh Chung‐Her , Rao Lakshmi G. , Johnson Lee F. 
Published: February 2005

AbstractThymidylate synthase (TS) activity is very low in resting mouse 3T6 fibroblasts but increases sharply in growth‐stimulated cells at about the same time the cells enter S phase. To study the mechanism responsible for the increase in TS level, we isolated a 5‐fluorodeoxyuridine (5‐FdUrd)‐resistant cell line (LU3‐7) that overproduces TS and its mRNA about 50–100‐fold. In this paper we show that the LU3‐7 cells were able to rest in the GO state of the cell cycle when maintained in medium containing 0.5% serum. When the serum concentration was increased to 10% the resting cells reentered the cell cycle and began DNA replication about 12 hr later. TS activity remained at the resting level until DNA replication began, then increased at later times. The increase was not affected when the cells were stimulated in the presence of DNA synthesis inhibitors. The rate of synthesis of TS (as determined in a pulselabeling experiment) remained at the resting level for the first 10 hr following stimulation, then increased 8–9‐fold by 25 hr following serum stimulation. The half‐life of TS in growing LU3‐7 cells was measured in a pulse‐chase experiment and found to be greater than 24 hr. Therefore the increase in TS activity was primarily due to an increase in the rate of synthesis of the enzyme. Since TS gene expression appears to be regulated in a similar manner in LU3–7 cells and in the parental 3T6 cells, the LU3–7 cells should be a good model system for detailed analysis of the mechanism for regulating TS gene expression in mammalian cells.
Subpopulations of fibroblasts from mouse skeletal muscle defined by clonal variation for 5′ nucleotidase expression
Authors: Turker Mitchell S. , Ogburn Charles E. , Martin George M. 
Published: February 2005

AbstractA primary cloning technique has been employed for the isolation of nine spontaneously transformed cell lines from mouse skeletal muscle. Four of these lines were isolated after selection for partial resistance to the purine (adenine) analog 2′6′diaminopurine and five were isolated from non‐selected control dishes. Four of the nonselected lines and three of the selected lines demonstrated a fibroblastoid morphology in vitro. The other two cell lines (one from each group) were epithelioid. Two of the three selected fibroblastoid lines were found to contain significant quantities of the enzyme 5′nucleotidase (EC3.1.3.5), whereas the four nonselected fibroblastlike lines, one selected fibroblastlike line, and the two epithelioid lines did not. In the two cell lines expressing 5′nucleotidase activity, this expression was stable in the absence of selective pressure. Histochemical staining of mouse skeletal muscle for 5′nucleotidase activity demonstrated positive staining in the cells of small blood vessels and in a subset of the connective tissue cells. The bulk of the skeletal muscle tissue, however, had no detectable 5′nucleotidase activity. We propose that the two cultivatable types of fibroblastoid cell lines represent distinct classes of fibroblastlike cells in vivo, reflecting alternative states of stable cellular differentiation involving 5′nucleotidase expression.
Proliferation and differentiation in culture of mast cell progenitors derived from mast cell‐deficient mice of genotype W/Wv
Authors: Suda Toshio , Suda Junko , Spicer Samuel S. , Ogawa Makio 
Published: February 2005

AbstractMice of genotype W/Wv have less than 1% of normal mast cells in the skin, stomach, and cecum. In order to further clarify the mechanism of this deficiency, we studied committed mast cell progenitors and multipotent progenitors, which are capable of mast cell differentiation in clonal culture. The relative concentration of mast cell progenitors in the bone marrow, spleen, and peripheral blood of W/Wv mice was similar to that of +/+ mice. However, the cellularity of the marrows of W/Wv mice was 54% of that of their normal littermates. Identification of mast cells was established by metachromatic staining with toluidine blue, transmission electron microscopy, and demonstration of membrane receptors for immunoglobulin E. The time course of colony formation and the morphology of W/Wv mast cell colonies in culture was identical to that of normal littermates. The percentages of mast cells in individual multi‐lineage colonies were extremely variable. The histamine content of mast cells derived from W/Wv mice was similar to that of mast cells from +/+ mice. These studies demonstrated the normal capacity for differentiation and proliferation in culture of mast cell progenitors from W/Wv mice.
Linear relationship of phlorizin‐binding capacity and hexose uptake during differentiation in a clone of LLC‐PK1 cells
Authors: Amsler Kurt , Cook John S. 
Published: February 2005

AbstractWith a clone of (Cl 4) of LLC‐PK cells, which develop a high capacity for Na+‐dependent hexose uptake over time (days) in culture, we show that increasing uptake capacity is paralleled by an increase in the number of phlorizin‐binding sites in the population. The linear relationship between binding and hexose transport is the same whether the cells differentiate spontaneously or are induced by either methylisobutylxanthine or hexamethylene bisacet‐amide. The constancy of the relationship suggests that the primary factor in transport development is the number of transporters in the cells rather than other possible factors like a change in membrane potential or decreased efflux. The Kd for phlorizin binding is .08 ± .04 μM, and corresponds to Ki of 0.10 μM for transport inhibition. The turnover number of the transporter is estimated to be 170 ± 40 molecules per second of α‐methyl glucoside.