Wayne B. Anderson's research while affiliated with National Cancer Institute (USA) and other places
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Publications (89)
We have recently reported that the gestational age of the recipient determines the pattern and level of transgene expression following in utero retroviral gene transfer by direct injection into pre-immune fetal sheep (Molecular Therapy 11(2):284-293, 2005). These studies suggest that the developmental stage of each organ at the time of injection ma...
Amphotropic murine leukemia virus (A-MuLV) utilizes the PiT2 sodium-dependent phosphate transporter as its cell surface receptor to infect mammalian cells. The process of A-MuLV infection requires cleavage of the R peptide from the envelope protein. This occurs within virions thereby rendering them competent to fuse with target cells. Envelope prot...
Superoxide dismutases' (SODs) expression is altered in several diseases including Alzheimer, atherosclerosis, cancer and psoriasis. Previously, we reported a marked increase in Mn-SOD and Cu,Zn-SOD functional activity in human dermal psoriatic fibroblasts. As retinoic acid (RA) has been used in the treatment of psoriasis and a mechanism for its ben...
Recently we reported that simultaneous treatment of NIH 3T3 cells with the combination of phorbol myristate acetate (PMA) and hydrogen peroxide (H2O2) resulted in synergistic activation of Raf-1 kinase (Lee, M., Petrovics, G., and Anderson, W. B. (2003) Biochem. Biophys. Res. Commun. 311, 1026-1033). In this study we have demonstrated that PP2 (4-a...
We have previously demonstrated that a 33kDa C-terminal fragment of c-Raf-1 underwent a mobility shift in response to hydrogen peroxide (H(2)O(2)) and phorbol myristate acetate (PMA), respectively. In this study, we have demonstrated that H(2)O(2) induced the activation of N-terminal deletion mutant as well as full length Raf-1 kinase. The pharmaco...
Phorbol 12-myristate-13-acetate (PMA), a potent tumor promoter and activator of most protein kinase C (PKC) isotypes, was found to significantly inhibit the growth of low population density (1-5% confluency) NIH 3T3 cells. Higher cell population density (above 10% confluency) provided protection from this growth inhibitory effect of PMA. PMA-induce...
The syncytiotrophoblast is the major component of the human placenta, involved in feto-maternal exchanges and secretion of pregnancy-specific hormones. Multinucleated syncytiotrophoblast arises from fusion of mononuclear cytotrophoblast cells. In trisomy 21-affected placentas, we recently have shown that there is a defect in syncytiotrophoblast for...
Differences in oxidative damage, as measured by an increase in the carbonylation of macromolecules, were determined in situ with skin biopsies from psoriatic patients and controls. High levels of carbonyl residues were consistently detected in the dermis and never in the epidermis of sections of these skin biopsy samples. The dermis of psoriatic sk...
Amphotropic murine leukemia virus (A-MuLV) utilizes the Pit-2 sodium-dependent phosphate transporter as a cell surface receptor to infect mammalian cells. Previous studies established that infection of cells with A-MuLV resulted in the specific down-modulation of phosphate uptake mediated by Pit-2 and in resistance to superinfection with A-MuLV. To...
In fibroblasts, the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) stimulates phospholipase D (PLD)-mediated hydrolysis of both phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) by PKC-alpha-mediated nonphosphorylating and phosphorylating mechanisms. Here we have used NIH 3T3 fibroblasts overexpressing holo...
Since tumor promoter benzoyl peroxide (BPO) mimics phorbol esters in some aspects, its effects on protein kinase C (PKC) were previously studied. However, in those studies due to the presence of thiol agents in the PKC preparations, the sensitive reaction of BPO with redox-active cysteine residues in PKC was not observed. In this study, by excludin...
The membrane receptors for the gibbon ape leukemia retrovirus and the amphotropic murine retrovirus serve normal cellular
functions as sodium-dependent phosphate transporters (Pit-1 and Pit-2, respectively). Our earlier studies established that
activation of protein kinase C (PKC) by treatment of cells with phorbol 12-myristate 13-acetate (PMA) enh...
![FIG. 5. Effects of ROS on the saturation curves of specific [ 3 H]cAMP...](profile/Francoise-Raynaud/publication/13572309/figure/fig2/AS:601759420542982@1520482034358/Effects-of-ROS-on-the-saturation-curves-of-specific-3-HcAMP-binding-to-RI-from_Q320.jpg)
![FIG. 6. Effects of ROS on the saturation curves of specific [ 3 H]cAMP...](profile/Francoise-Raynaud/publication/13572309/figure/fig3/AS:601759420522510@1520482034416/Effects-of-ROS-on-the-saturation-curves-of-specific-3-HcAMP-binding-to-RII-from_Q320.jpg)
Oxygen free radicals may act as second messengers in signal transduction pathways and contribute to inflammatory diseases.
We studied the action in vitro of radiolytically generated hydroxyl radicals (⋅OH) and superoxide radicals (O⨪2) on the cAMP-dependent protein kinases, I and II (PKAI and -II, respectively). The effects of the gasses O2 and N2O...
In addition to acquired immunodeficiency syndrome (AIDS), persons infected with human immunodeficiency virus often develop cutaneous manifestations, including severe psoriasis. In previous studies, we have established that psoriatic fibroblasts and erythrocytes obtained from psoriatic patients exhibit decreased levels of cyclic adenosine monophosph...
Raf-1 protein serine/threonine kinase has been implicated in growth and damage-responsive signal transduction pathways. Several reports indicate an important role of Ras protein in the growth factor-induced activation of Raf-1. Here we investigated the possible involvement of Ras in ionizing radiation-induced activation of Raf-1. Irradiation of MDA...
Pleckstrin homology (PH) domains comprised of loosely conserved sequences of approximately 100 amino acid residues are a functional protein motif found in many signal-transducing and cytoskeletal proteins. We recently demonstrated that the PH domains of Tec family protein-tyrosine kinases Btk and Emt (equal to Itk and Tsk) interact with protein kin...
Pleckstrin homology (PH) domains comprised of loosely conserved sequences of ∼100 amino acid residues are a functional protein
motif found in many signal-transducing and cytoskeletal proteins. We recently demonstrated that the PH domains of Tec family
protein-tyrosine kinases Btk and Emt (equal to Itk and Tsk) interact with protein kinase C (PKC) a...
Previous studies have established that cyclic AMP-dependent protein kinase (PKA) activity, as well as 8-azido-[32P]-cAMP binding to the RI and RII regulatory subunits, are decreased in cells from psoriatic patients compared to cells from normal patients. Here we show that the exposure of normal human dermal fibroblasts in culture to hydrogen peroxi...
Antioxidant enzyme activities in fibroblasts and erythrocytes prepared from normal and psoriatic patients were measured and compared. The most significant differences were noted in superoxide dismutase (SOD) activities. A dramatic (5.2-fold) increase in Mn-SOD activity along with a lesser (1.8-fold) increase in CuZn-SOD activity was observed in fib...
Subcellular redistribution (translocation) was initiated by treatment of NIH 3T3 cells overexpressing different epitope-tagged fragments of PKC epsilon with PMA, and was analyzed by immunocytochemistry. The PMA-induced translocation of hole PKC epsilon, as well as fragments epsilon 2 (zinc finger domain + pseudosubstrate domain) and epsilon 7 (zinc...
Previously, we have reported a defect in the cAMP-dependent protein kinases (cAMP-PK) in psoriatic cells (i.e., a decrease in 8-azido-[32P]cAMP binding to the regulatory subunits and a decrease in phosphotransferase activity) which is rapidly reversed with retinoic acid (RA) treatment of these cells. This led us to examine a possible direct interac...
Monomethylethanolamine (1 mM) and dimethylethanolamine (1 mM) stimulated DNA synthesis 10- and 15-fold, respectively, in NIH 3T3 fibroblasts. In addition, simultaneous treatments with insulin (500 nM) and methylated ethanolamine analogues (1 mM or less) resulted in synergistic activation of DNA synthesis. The order of mitogenic potency of ethanolam...
Previously, we have reported a decrease in the binding of a cAMP analog to the regulatory subunits of cAMP-dependent protein kinase (cAMP-PK), as well as a decrease in cAMP-PK activities, in psoriatic cells. Retinoic acid (RA) treatment of these cells can induce an increase in cAMP-PK toward normal levels. To better define the effect of retinoic ac...
Activation of protein kinase C (PKC) by phorbol ester (PMA), or by diacylglycerol analogue (OAG) treatment of NIH 3T3 cells resulted in the rapid (within 2-5 min) stimulation (approx. 2-fold) of sodium-dependent phosphate (Pi) transport. Conversely, preincubation of these cells with forskolin and cholera toxin, or incubation with 8-bromo-cAMP, to a...
We have recently demonstrated a deficiency in the cyclic adenosine monophosphate (cAMP)-dependent protein kinases (PKA), the intracellular mediator of AMP, in psoriasis. This enzyme defect is expressed in fibroblasts and in red blood cells isolated from psoriatic patients. In these cells, the abnormality noted in cAMP binding to PKA correlates well...
In the conventional approach protein kinase activity and phorbol ester binding associated with protein kinase C (PKC) are measured by initially incubating samples in either test tubes or multiwell plates, followed by filtration of the terminated reaction mixture using either a manifold filtration device or a cell harvester. Here we report a method...
In fibroblasts, the mitogenic effects of sphingosine involves a rapid rise in the cellular content of phosphatidic acid (PtdOH) which may be due to the stimulation of phospholipase D, or inhibition of PtdOH phosphohydrolase, or both. Here, we demonstrate that in fibroblasts, 4-hydroxynonenal is a selective inhibitor of sphingosine-stimulated phosph...
Retinoylation (retinoic acid acylation) is a post-translational modification of proteins occurring in a variety of eukaryotic cell lines. There are at least 20 retinoylated proteins in the human myeloid leukemia cell line HL60 (N. Takahashi and T.R. Breitman (1990) J. Biol. Chem. 265, 19, 158-19, 162). Here we found that some retinoylated proteins...
The oxidant mitogen/tumor promoter, periodate, was used to selectively modify either the regulatory domain or the catalytic domain of protein kinase C (PKC) to induce oxidative activation or inactivation of PKC, respectively. Periodate, at micromolar concentrations, modified the regulatory domain of PKC as determined by the loss of ability to stimu...
Protein kinase C (PKC) activity was determined in different (membrane, nuclear and soluble) subcellular fractions prepared separately from the CA1 and CA3 subfields of Mongolian gerbils hippocampus at various time intervals following a single 5-min occlusion of the common carotid arteries. Soluble and nuclear PKC activities of the CA1 sector were f...
Evidence is presented that a differentiated mesodermal line (MES-1) from P19 EC cells express a high chemotactic response
to platelet-derived growth factor (PDGF) as assayed in a blind-well modified Boyden chamber. Compared to the NIH 3T3 fibroblasts
the chemotactic response of MES-1 is increased by 10-fold at 0.3 ng/ml of PDGF, 4-fold at 1.25 ng/m...
Addition of alcohols to NIH 3T3 fibroblasts, prelabeled with [2-14C]ethanolamine, resulted in increased degradation of [14C]phosphatidylethanolamine (PtdEtn). Long-chain alcohols, like octanol or nonanol, were more potent than methanol or ethanol. The main water-soluble product of alcohol-stimulated [14C]PtdEtn hydrolysis was [14C]ethanolamine. Add...
The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated the synthesis of sphingomyelin (CerPCho) from a [14C]choline-labelled phosphatidylcholine (PtdCho) pool in NIH 3T3 cells. Maximal stimulation (68%) of CerPCho synthesis, accompanied by an increase (38%) in its cellular content, required only 2 nM TPA. Higher concentrations of...
Treatment of PYS cells with the tumor promoter (TPA) has been previously shown to enhance calcium- and phospholipid-dependent protein kinase (PK.C) in the membranes and to decrease its activity in the cytosol. Evidence is presented that 0.1 microM TPA treatment of PYS cells causes an opposite effect on the cyclic AMP-dependent protein kinases (PK.A...
Retinoic acid-induced differentiation of F9 embryonal carcinoma cells to endoderm provokes the secretion of a protein factor that acts as both a chemoattractant and mitogen for smooth muscle cells. Undifferentiated F9 cells and PSA-5E (visceral endodermlike) cells produced little of this factor. However, PYS-2 (parietal endodermlike) and Dif 5 endo...
Exposure of protein kinase C to low concentrations of either N-chlorosuccinimide or H2O2 resulted in rapid and parallel loss of phosphotransferase activity and phorbol ester binding. This oxidative inactivation of protein kinase C also occurred in intact cells exposed to a low concentration of H2O2. With H2O2 treatment the rate of inactivation of p...
Teratocarcinoma cells in culture offer an in vitro system for studying certain aspects of embryonic differentiation. To gain some insight into regulatory systems that might be operative during early development, we have characterized the alterations that occur in the hormonal responsiveness of the membrane adenylate cyclase of different embryonal c...
We recently showed a deficiency of cyclic AMP (cAMP)-dependent protein kinases in psoriatic cells. In this work the effects of retinoids on cAMP-dependent protein kinases of fibroblasts from 7 normal subjects and 7 psoriatic patients were studied. The levels of RI and RII (two forms of the cAMP-dependent protein kinases) present in control and reti...
The physiologic regulation of protein kinase C activity appears to be modulated by its interaction with cellular membranes. Tumor promoter- and hormone-induced stabilization of protein kinase C to a membrane fraction in intact cells apparently reflects activation of the enzyme. Thus, measurement of changes in membrane-associated protein kinase C in...
Two different methods were used to study directly alpha-thrombin modulation of polyphosphoinositide breakdown in membranes prepared from Chinese hamster lung (CHL) fibroblasts. In the first one we labelled the lipid pool by incubating the intact cells with myo-[3H]inositol prior to membrane isolation; in the other we used exogenous [3H]PIP2 with ph...
Retinoic acid induces the differentiation of PCC4.aza 1R and Nulli-SCC1 embryonal carcinoma (EC) cells. In response to retinoic acid treatment, the levels of cyclic AMP (cAMP)-dependent protein kinases are enhanced in the plasma membrane within 17 hours and in the cytosol fractions of these cells within 2 to 3 days, as determined by phosphotransfer...
Phorbol esters induce a rapid phosphorylation of the antigenic epitope of the human IL-2 receptor identified by anti-Tac monoclonal antibody. The physiological activator of protein kinase C, diacylglycerol also stimulated the phosphorylation of the Tac epitope in intact activated human T lymphocytes. Stable derivatives of cyclic nucleotides had no...
Human teratocarcinoma cells in culture offer an in vitro system for studying certain aspects of embryonic differentiation. To gain some insight into regulatory systems that might be operative during early human development, we have characterized the alterations that occur in the hormonal responsiveness of human embryonal carcinoma cell adenylate cy...
The mammalian pineal gland is regulated by norepinephrine (NE) released into the pineal perivascular space from sympathetic nerve terminals; there is a 24-hour pattern of release, with high levels occurring at night. This rhythmic release of NE is controlled by a neural circuit which includes a circadian clock and passes through both central and pe...
The present study was carried out to determine if an insulin-like growth factor (IGF) type activity might be produced by embryonal carcinoma-derived cells. The cell line used to condition growth medium for the isolation of secreted growth factors was a newly established Dif 5 cell type. Dif 5 cells are a differentiated endoderm-like cell type deriv...
S-100-binding proteins, and calmodulin-binding proteins were isolated from S-100- and calmodulin-depleted bovine brain extract by Ca2+-dependent affinity chromatography using S-100- and calmodulin-coupled Sepharose columns respectively. The majority of the protein (80 to 90%) including calcineurin that bound to S-100 also bound to calmodulin and vi...
Interleukin-3 (IL-3) is a member of a family of growth and differentiation peptides, collectively referred to as colony-stimulating factors, which regulate haematopoiesis. IL-3 has been highly purified from medium conditioned by WEHI-3B cells, and recently the molecular cloning of complementary DNA for murine IL-3 has been reported. IL-3 seems to s...
Interleukin-2 (IL-2) is a regulatory peptide important for the growth and differentiation of antigen-specific T lymphocytes and large granular lymphocytes. Interaction of IL-2 with its specific receptor results in the promotion of S-phase progression as well as, in certain circumstances, the production and release of gamma-interferon (IFN-gamma). A...
The pineal gland has proven to be an excellent model for the study of adrenergic control systems. Noradrenaline, released from sympathetic nerve terminals in the pineal gland, regulates a large nocturnal increase in melatonin synthesis by stimulating the activity of arylalkylamine N-acetyltransferase (NAT, EC 2.3.1.87) 30-70-fold. An essential step...
Native calmodulin binds four calcium ions per molecule and exhibits strong Ca2+-dependent binding to phenyl-Sepharose. In contrast, calmodulin inactivated by oxidation of methionine residues or by deamidation binds fewer calcium ions (two per molecule) and shows relatively weak interaction with phenyl-Sepharose. Calmodulin inactivated by modificati...
We investigated changes in the activity and subcellular distribution of cyclic-AMP-dependent protein kinases (cAMP-PKs) in response to treatment with retinoic acid in three different embryonal carcinoma cell lines derived from the same teratoma 6050. After retinoic-acid treatment, F9 and PCC4 cells gave rise to parietal-like endoderm, while PC13 ce...
Calcium is involved in the regulation of numerous physiological and biochemical processes in response to cellular requirements and external stimuli. Cells can respond to certain hormones with a transient, localized increase in free intracellular Ca2+, and on this basis, Rasmussen initially proposed that Ca2+ might be regarded as a second messenger,...
To determine possible ectopic production of, and altered responsiveness to, specific hormones and growth factors which may be involved in mediating embryonic differentiation and development embryonal carcinoma cells in culture have been employed to serve as an in vitro system of embryogenesis. Exposure of F9 embryonal carcinoma cells to all-trans-r...
Exposure of F9 cells to all-trans-retinoic acid over a period of 6 days resulted in 4-fold induction of cell surface N-acetylglucosaminide beta (1----4)galactosyltransferase (GT) activity. The retinoic acid-induced GT activity was further enhanced by treatment of the cells with 8-bromo cyclic AMP. The ability of retinoic acid alone, or retinoic aci...
Treatment of F9 teratocarcinoma cells with all trans retinoic acid (RA) causes them to differentiate into two or three morphologically distinct cell types. Whereas the majority of these retinoid-derived cells exhibit properties resembling parietal endoderm, a small percentage of this differentiated cell population manifests properties distinct from...
A specific cellular protein of molecular weight of 53-55,000 (p53) has been shown to be induced in all SV40 transformed cells. A similar protein has also been shown to be present in embryonal carcinoma cells and in midgestation murine embryo primary cells, which are not infected by SV40. In embryo cell primaries the amount of the protein was shown...
Low concentrations of insulin (20 to 40 ng/ml) stimulate the growth of F9 embryonal carcinoma cells under defined, serum-free conditions. [125I] Insulin binding studies reveal the presence of high and low affinity receptor sites, and insulin does not compete for [125I] multiplication-stimulating activity (MSA) binding to F9 cells. The addition of a...
The present study was designed to assess the effectiveness of multiplication-stimulating activity (MSA), an insulin-like growth factor, in supporting the growth of F9 cells (an embryonal carcinoma line with properties similar to embryonic stem cells). Under serum-free growth conditions in a medium supplemented with transferrin and fibronectin, MSA...
Incubation of crude normal rat kidney membranes with activated cholera toxin in the presence of DTP, ATP and NAD results in a 10--20 fold stimulation of adenylate cyclase activity. Optimal choleragen activation f the cyclase is shown to be dependent upon the presence of a plasma membrane-associated reconstituting activity, which can be dissociated...
Calmodulin binds quantitatively to phenyl-Sepharose and octyl-Sepharose affinity columns in the presence of micromolar concentrations of Ca2+. In addition to EGTA, calmodulin also can be eluted from these affinity columns with low ionic strength buffer, non-ionic detergent (i.e., 1% Triton X-100), or ethylene glycol (50%), suggesting hydrophobic in...
Teratocarcinoma cells in culture offer an in vitro system for studying certain aspects of embryonic differentiation. To gain some insight into regulatory systems that might be operative during early development, we have characterized the alterations that occur in the hormonal responsiveness of the F9 embryonal carcinoma cell membrane adenylate cycl...
To determine if Chinese Hamster Ovary (CHO) cells in culture are able to metabolize steroids, CHO cells were incubated in defined medium with [14C]pregnenolone. As shown, [14C]pregnenolone is metabolized to progesterone and other delta 53 beta steroids; this steroidogenic response is appreciably enhanced upon exposure of the cells to 50 nM gonadotr...
Evidence is presented that brief trypsin treatment of intact normal rat kidney cells significantly increases the intracellular accumulation of cyclic AMP in response to added prostaglandin E1 (PGE1) and (−)-isoproterenol. Trypsin exhibits a concentration-dependent enhancement of hormone-stimulated accumulation of cyclic AMP in intact normal rat kid...
Hormonal responsiveness of the adenylate cyclase system of cultured F9 teratocarcinoma cells was investigated. Of numerous hormones tested only calcitonin, (−)isoproterenol, and prostaglandin E1, stimulate F9 adenylate cyclase activity. Of the active hormones, calcitonin is the most potent stimulator of cAMP formation. Treatment of intact F9 cells...
Treatment of intact normal rat kidney fibroblasts, or of purified NRK plasma membranes, with trypsin or papain markedly enhances adenylate cyclase activity [ATP pyrophosphatelyase (cyclizing) EC 4.6.1.1]. Limited proteolysis (25 μg/ml trypsin for 7 min) of confluent cells grown with unheated calf serum significantly increases cyclase activity, wher...
A rat kidney cortex lysosomal protease [Strewler, G. J., Manganiello, V. C. and Vaughan, M. (1977) J. Biol. Chem. 253, 390–394] which activates cyclic nucleotide phosphodiesterases has been shown to potentiate adenylate cyclase activity in normal rat kidney fibroblasts. The lysosomal protease increases GTP-, GTP plus prostaglandin E1-, and guanyl-5...
Normal rat kidney (NRK) cells growth arrested by picolinic acid and isoleucine deprivation exhibit an increased response to certain agents (i.e., prostaglandin E1, (−)-isoproterenol, and cholera toxin) which elevate intracellular cyclic AMP levels. The enhanced hormonal response is apparently due, at least in part, to increased adenylate cyclase ac...
The adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] of crude Chinese hamster ovary cell membranes was inhibited 30-40% by low concentrations (6-600 ng/ml) of calcium-dependent regulator (CDR). This inhibitory effect was lost at concentrations of CDR above 600 ng/ml. The adenylate cyclase activity of membranes prepared f...
The addition of the somatomedin-like growth factor Multiplication Stimulatory Activity (MSA) to intact chick embryo fibroblasts (CEF) in culture does not decrease the basal intracellular pool of cAMP. As a more sensitive means of determining a possible effect of MSA on cAMP metabolism we have assessed the ability of MSA to alter the accumulation of...
When Chinese Hamster Ovary (CHO) cells, incubated in serum-free medium, are exposed to gonadotropins a transient increase in the intracellular concentration of cyclic AMP is observed. Maximum accumulation of cyclic AMP is noted 30 minutes after addition of either human chorionic gonadotropin (hCG) or follicle stimulating hormone (FSH). Within one t...
THERE is currently wide interest in the biological role of glycolipids and glycoproteins of the cell membrane. As some transformed cells have reduced levels of the more complex gangliosides and neutral glycolipids (see refs 1–3 for reviews), it has been suggested that complex gangliosides and particularly ganglioside GM1 may have an important role...
NRK cells and many other cultured fibroblasts were found to contain the protease inhibitor, alpha 2 macroglobulin (alpha 2M). This alpha 2M is present as a result of uptake of alpha 2M from the calf serum in the culture medium. Some of this alpha 2M is released back into the medium. In radiolabeling experiments with 14C-amino acids, no radioactivit...
The effect of certain lipids on adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from fibroblasts in culture has been investigated. The unsaturated fatty acids, as well as lysolecithin, were found to act as potent inhibitors of fibroblast adenylate cyclase activity. Increasing the degree of unsaturation increases the ext...
The activity of the enzyme adenylate cyclase has been determined in normal rat kidney (NRK) cells and NRK cells transformed
by either the Kirsten sarcoma virus (Ki-SV) or the Moloney sarcoma virus (Mo-SV). The specific activity of the NRK enzyme
increases with increasing cell population density. Transformation apparently decreases this response, fo...
To elucidate further how cyclic adenosine 3':5' monophosphate (cyclic AMP) and the cyclic AMP receptor are able to stimulate gal mRNA synthesis an antiserum specific for cyclic AMP receptor and also cyclic GMP as inhibitors of cyclic AMP and cyclic AMP receptor action were utilized. When cyclic AMP, cyclic AMP receptor, DNA, and RNA polymerase are...
To investigate how cell population density influences the intracellular concentration of cyclic AMP we have measured adenylate cyclase and cyclic AMP phosphodiesterase activities and cyclic AMP levels at various stages of cell density in normal rat-kidney (NRK) cells, which exhibit contact-inhibition of growth, and in normal chick-embryo fibroblast...
An antiserum specific for the cyclic adenosine 3′,5′-monophosphate receptor from Escherichia coli has been employed to detect the presence of a similar protein in cellular extracts of a number of diverse organisms. In Ouchterlony double-diffusion experiments cellular extracts from Photobacterium fisheri, Aerobacter aerogenes, Proteus mirabilis, and...
Chick embryo fibroblasts (CEF) transformed by the Schmidt-Ruppin strain of Rous sarcoma virus (RSV-SR) have decreased adenylate cyclase activity. In cells infected by a temperature-sensitive mutant of this virus (RSV-SR-T5), enzyme activity is near normal when the cells are grown at the non-permissive temperature (41°C) but decreases at the permiss...
The activity of the enzyme adenylate cyclase, a component of the plasma membrane, has been determined in chick-embryo fibroblasts and in cells transformed by either Bryan high-titer strain of Rous sarcoma virus (RSV-BH) or a temperature-sensitive mutant of this virus (RSV-BH-Ta). Adenylate cyclase activity is reduced in cells transformed by the wil...
Cyclic AMP appears to regulate cell growth. Cyclic AMP levels are high in normal chicken embryo fibroblasts and drop to very low levels when the cells are transformed by the Bryan high-titer strain of Rous sarcoma virus. Cells infected with a temperature-sensitive mutant of the virus have normal levels of cyclic AMP at the nonpermissive (nontransfo...
The binding of cyclic adenosine 3′,5′-monophosphate (cyclic AMP) receptor (CRP) to DNA has been measured by a membrane filter assay and sucrose density gradient analysis. CRP binds to λpgal, λh80dlac ps, calf thymus, salmon sperm, and chicken blood DNA. CRP also binds to denatured chicken blood DNA and the separated strands of λpgal and λplac DNA....
Several analogues of cyclic adenosine 3′,5′-monophosphate (cyclic AMP) have been tested for their ability to inhibit the binding of cyclic AMP to cyclic AMP receptor (CRP) and to inhibit the cyclic AMP-CRP-dependent transcription of the gal operon. A number of the cyclic AMP derivatives are able to inhibit cyclic AMP binding to CRP, but of all the...
Optimal transcription of the lac operon in a defined purified in vitro system is dependent upon the concentrations of cyclic adenosine 3′,5′-monophosphate (cyclic AMP), the cyclic AMP-binding protein (CRP) and RNA polymerase. The concentration of CRP required for maximal lac transcription varies proportionately with the concentration of lac DNA use...
A procedure is described for the purification of cyclic adenosine 3',5'-monophosphate receptor protein (CRP) from Escherichia coli involving chromatography on DEAE-cellulose and phosphocellulose, (NH4)2SO4 precipitation, and filtration on Sephadex G-100. The preparation appears to be homogeneous as determined by sedimentation
equilibrium studies, i...
THE expression of the lac operon is controlled by a specific repressor which prevents transcription of the operon, as well as by cyclic AMP and a cyclic AMP receptor protein (CRP) which stimulate transcription1,2. The lac repressor is a protein and binds to lac DNA at the operator locus2. It could repress transcription by interfering with one of se...
In a transcription system containing λpgal8 DNA as template and Escherichia coli RNA polymerase, the addition of cyclic adenosine monophosphate receptor protein (CRP) and 3',5'-cyclic AMP causes a 15-fold increase in gal mRNA synthesis. Gal mRNA was measured by hybridizing the [³H]RNA product to the separated strands of λpgal8 DNA after removal of...
A cell free system has been developed in which only the components of the lactose operon are required for its transcription. Results obtained with this system suggest that the DNA of the operon contains a new regulator site, at which the cyclic AMP control system acts.
Citations
... However, the DNA used was a synthetic copolymer that bound the cAMP-CRP complex poorly and thereby resulted in an insensitive assay of poor precision. Other early work was based on an indirect in vitro system in which cIMP was reported to be unable to replace cAMP 22 . However, no details or data were given. ...
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... They are much closed to each other; a shift is equal to 5 bp and 22 bp for lac166 and gal178, respectively. A simultaneous binding of two RNAP molecules to both sites is impossible (Nisseley et al., 1971;Yu & Reznikoff, 1985;Choy & Adhya, 1993). On the other hand, RNAP can bind to DNA nonspecifically. ...
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... Datapoints obtained in presence of glucose are indicated with dashed lines (empty circles for raw datapoints, dashed black line for intracellular concentration). The dashed horizontal line (10 µM) indicates the cAMP-CRP binding affinity(24): above this level, cAMP binds CRP and pel expression is activated. ...
... Therefore our experimental evidence is entirely consistent with this particular motif-based prediction. 1 This consensus motif, together with our in vivo and in vitro data, suggests that PKC␣ is likely to directly phosphorylate Ser24. The finding that PMA-stimulated Ser24 phosphorylation is not affected by any of the non-PKC inhibitors (Fig. 5) further supports this notion, as does the findings that IRS1 and PKC␣ are constitutively associated in vivo (39,40) and that the PH domain itself is sufficient for this interaction (Fig. 6). ...
... We found that the A2M fragment decreased dramatically during the process of immortalization . Similarly, there was less binding and degradation of A2M in SV-40 transformed ®broblasts than in normal lung and skin ®broblasts (Mosher and Vaheri, 1980 ). Transformation of normal ®broblasts by Moloney sarcoma virus also decreased the uptake of A2M (Pastan et al., 1977 ). These results suggest that uptake of A2M into cells is linked to agerelated phenotypes that were lost during the process of immortalization. ...
... Cyclic adenosine monophosphate (cAMP), a second messenger, is one of the key regulators in mammalian cell proliferation and differentiation [14]. In the kinetoplastid family of parasites, cAMP plays an important role during cellular invasion and differentiation [15]. ...
... It is noteworthy that EGF by itself does not alter the basal cAMP level in hepatocytes. Similarly, EGF has been reported to decrease prostaglandin El-stimulated cAMP content of fibroblastic cells without affecting the basal unstimulated cAMP content (Anderson et al., 1979). Whether the effects of EGF in these two cell systems are the result of inhibition of adenylate cyclase or activation of cAMP phosphodiesterase activities remains to be elucidated. ...
... Chinese hamster ovary cells were grown to a density of 2-9 ϫ 10 6 cells/dish and showed that as cell number increased there was an increase in soluble CaM. Soluble CaM increased twofold, whereas the particulate fraction decreased by one-third (41). More soluble CaM is extracted from cancer cells than from noncancerous cells (122,138,145), whereas the particulate ApoCaM fraction is more abundant in noncancerous cells than cancerous ones (138). ...
Reference: Apocalmodulin
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... On the other hand, long chain fatty acids, e.g., stearic acid, oleic acid, linoleic acid, linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), are classified as penetration enhancers or membrane fluidizers due to their membrane-associated effects at cellular level [224][225][226]. The presence of double C bonds has been found to contribute to the cell membrane disrupting ability. ...
... We evaluated the PRDs for binding to the C1 domains of recombinant full-length human PKCα as described earlier. 13,14,44 We selected the PKCα isoform to allow maximum comparison with all the previous compounds in the HMI/PYR families, which were tested using PKCα. The compounds were used at a concentration range of 0.2−30 μM (raw data is available in the SI). ...
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