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Query: UNIPROT:P06889 (Mol)
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Nucleotide sequences of three novel rat long terminal repeats (LTR) of intracisternal A-particles (IAP) were determined and compared with two previously published solitary rat IAP LTRs from the genomic clone H12 (Furter et al. (1989) J. Biol. Chem. 264, 18276-18279) and from the upstream region of the oncomodulin (OM) gene (Banville and Boie (1989) J. Mol. Biol. 207, 481-490). These five LTRs have a length of 286 to 370 bp and show the major variability within the U3 region. The CCAAT and the TATA boxes, the AATAAA polyadenylation signals and the CA polyadenylation sites are well conserved in sequence and position in all five LTRs, whereas several putative transcriptional factor binding sites in the U3 domain show considerable heterogeneity. The transcriptional activities of three LTRs were tested in transient gene expression assays using the human growth hormone (hGH) reporter gene in chemically transformed T14c cells which produce considerable amounts of oncomodulin. Promoter strengths of the three investigated LTRs varied considerably.
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PMID:Sequence variations and promoter activities of long terminal repeats from rat intracisternal A-particles. 156 98

A set of genes is rapidly inducible when quiescent fibroblasts are stimulated by growth factors or by the activation of temperature-sensitive retroviral protein-tyrosine kinases. Most of these so-called immediate-early genes were cloned by differential cDNA hybridization. DNA sequence analysis identified many of them as putative members of the growth factor or of the transcription factor gene family, suggesting a role in signal transmission during the G0-to-G1 transition. In this study, we identified one of the genes that are rapidly inducible by the retroviral protein-tyrosine kinases v-Src and v-Fps of Rous sarcoma virus and Fujinami sarcoma virus, respectively, as the rhoB gene, a member of the ras gene superfamily whose products are GTP-binding proteins, rhoB is transiently activated at the transcriptional level by v-Fps and by epidermal growth factor. Its labile RNA is inducible in the presence of cycloheximide but not of actinomycin D. rhoB is strongly induced by epidermal growth factor and by platelet-derived growth factor both in subconfluent, serum-starved and in density-arrested Rat-2 fibroblasts. Fetal calf serum is a poor inducer, particularly in density-arrested cells, and phorbol esters do not increase rhoB expression at all. These data suggest that rhoB is inducible by protein-tyrosine kinases through a pathway not involving the activation of protein kinase C. Neither the closely related rhoC and rhoA genes nor the distantly related c-H-ras gene is rapidly inducible by mitogens. Thus, rhoB is the first known member of the small GTP-binding proteins among the immediate-early genes.
Mol Cell Biol 1991 Jul
PMID:The ras-related gene rhoB is an immediate-early gene inducible by v-Fps, epidermal growth factor, and platelet-derived growth factor in rat fibroblasts. 171 Jul 70

We have previously shown that there are multiple GTP-binding proteins (G proteins) with Mr values of about 20,000 in bovine brain membranes and identified one G protein with a Mr of 20,000 as the rho gene product. We have also shown that this rho gene product is ADP-ribosylated by an ADP-ribosyltransferase contaminated in botulinum toxin type C1. In the present studies, we have purified another G protein with a Mr of about 21,000 to near homogeneity from bovine brain membranes by several column chromatographies and identified it as the rhoA gene product. Further analysis of the amino acid sequence of the G protein, which we have purified and identified as the rho gene product previously, has revealed that this G protein is the rhoB gene product. The rhoA gene product binds maximally about 0.9 mol of [35S]guanosine 5'-(3-O-thio) triphosphate (GTP gamma S)/mol of protein with a K d value of about 20 nM. [35S]GTP gamma S-binding to the rhoA gene product is inhibited by pretreatment with N-ethylmaleimide. The rhoA gene product hydrolyzes GTP to liberate Pi with a turnover number of about 0.01 min-1. Moreover, the rhoA gene product is ADP-ribosylated by an ADP-ribosyltransferase contaminated in botulinum toxin type Cl. About 0.3 mol of ADP-ribose is maximally incorporated into 1 mol of the rhoA gene product. The ADP ribosylation of the rhoA gene product does not affect its GTP gamma S-binding or GTPase activity. These properties of the rhoA gene product are similar those of the rhoB gene product described previously. These results together with the earlier observations indicate that there are at least two rho gene products (rhoA, B) among three members of the rho gene family (rhoA, B, C) in bovine brain membranes and that both of them are ADP-ribosylated by an ADP-ribosyltransferase contaminated in botulinum toxin type C1.
Brain Res Mol Brain Res 1990 Jan
PMID:Purification and characterization from bovine brain membranes of a GTP-binding protein with a Mr of 21,000, ADP-ribosylated by an ADP-ribosyltransferase contaminated in botulinum toxin type C1--identification as the rhoA gene product. 215 99

To study the genetic expression and regulation of galactose-metabolizing enzymes, we mutagenized the mouse liver H2.35 cell line and selected for cell clones resistant to the toxic galactose analog, 2-deoxy-D-galactose (2-DOG). One cloned line, designated H12.10, was stably resistant to high levels of 2-DOG and was completely deficient in galactokinase activity. Galactokinase activity and growth sensitivity to 2-DOG could be restored by transfecting H12.10 cells with a plasmid containing the Escherichia coli galactokinase (galK) gene fused to a eucaryotic promoter; thus, the 2-DOG selection could be directed against transfected recombinant constructs in a liver cell line. We also found that H2.35 cells could not utilize galactose as a primary carbon source because of a deficiency in galactose-1-phosphate uridyltransferase; a variant line of H2.35 cells selected in galactose medium expressed higher levels of uridyltransferase activity. Finally, we found that in all mammalian cell lines tested, galactokinase expression was the same whether the medium contained glucose, galactose, or both sugars. These studies demonstrate differences between mammalian cells and yeast cells in the regulation of gal enzymes, and they define different schemes for obtaining altered expression of genes in the galactose metabolic pathway. The isogenic liver cell lines described here can also serve as model systems for studying galactosemias, which are inherited disorders of galactose metabolism in humans.
Mol Cell Biol 1990 Sep
PMID:Selection and analysis of galactose metabolic pathway variants of a mouse liver cell line. 216 34

Several microtubule-active drugs block cholinergically mediated catecholamine secretion from adrenal chromaffin cells without affecting secretion induced by other secretagogues. Interactions of these agents with nicotinic acetylcholine receptor-ion channel complexes from Torpedo californica electric organs were studied using radiolabeled probes for receptor and associated ion channel-binding sites. Colchicine, taxol, and the Vinca alkaloids had minimal affinity for cholinergic receptor-binding sites (nicotinic or muscarinic). The Vinca alkaloids (vinblastine, vincristine, vindesine) and colchicine inhibited [3H]perhydrohistrionicotoxin ([3H]H12-HTX) binding to the receptor-gated ion channel with IC50 values of 2-32 microM and 6 mM, respectively. The ability of the microtubule-active drugs to inhibit [3H]H12-HTX binding was increased by up to 5-fold in the presence of 1 microM carbamylcholine. The IC50 values for inhibition of [3H]H12-HTX binding by colchicine and three Vinca alkaloids were closely correlated with their abilities to inhibit acetylcholine-induced catecholamine secretion from cultured bovine adrenal chromaffin cells. As a consequence of its interaction (direct or indirect) with the ion channel, at least one Vinca alkaloid (vinblastine) stabilized a high agonist affinity conformation of the nicotinic receptor complex. beta-Lumicolchicine, an analog of colchicine devoid of microtubule activity, also blocked ion channel binding. On the other hand, taxol, a microtubule-stabilizing agent which also selectively blocks cholinergically mediated secretion, did not affect receptor or ion channel binding. The present results indicate that interactions with the nicotinic receptor-ion channel complex may underlie the actions of certain microtubule-active agents on catecholamine secretion by adrenal chromaffin cells.
Mol Pharmacol 1985 Jul
PMID:Interactions of microtubule-active agents with nicotinic acetylcholine receptors. Relationship to their inhibition of catecholamine secretion by adrenal chromaffin cells. 241 Jul 67

Neostigmine (Neo), pyridostigmine (Pyr), and physostigmine (Phy) at low concentrations inhibited acetylcholine (ACh) esterase, thereby indirectly potentiating ACh enhancement of [3H]perhydrohistrionicotoxin (H12-HTX) binding to the channel sites of the nicotinic ACh receptor of Torpedo membranes. However, at higher concentrations, they inhibited ACh action due to their direct binding to the ACh receptor. They displaced binding of [3H]ACh and 125I-alpha-bungarotoxin (alpha-BGT) to the receptor sites with the following order of decreasing potency: Neo greater than Phy greater than Pyr. Furthermore, Neo and Pyr potentiated [3H] H12-HTX binding to the receptor's channel sites. Preincubation of ACh receptors with any of the three carbamates reduced the rate of binding of 125I-alpha-BGT and increased the potency of carbamylcholine in inhibiting 125I-alpha-BGT binding, suggesting that the three carbamates act as partial agonists and potentiate receptor desensitization. Although none of the three carbamates inhibited [3H]H12-HTX binding to the receptor's closed channel conformation, only Phy was a potent inhibitor of [3H]H12-HTX binding to the carbamylcholine-activated conformation. The potency of Phy was not due to the absence of positive charge since Phy methiodide acted similarly. The data suggest that the major action of the three carbamates at nicotinic cholinergic synapses is inhibition of ACh-esterase. Their interactions with the nicotinic ACh receptor are with its "receptor" as well as allosteric "channel" sites, but they differ in their effects. Neo and Pyr act mainly as partial agonists, while Phy is mostly an inhibitor of the channel in the activated receptor conformation.
Mol Pharmacol 1985 Mar
PMID:Comparison of the actions of carbamate anticholinesterases on the nicotinic acetylcholine receptor. 397 72

The actions of the tertiary local anesthetic bupivacaine were studied on the nicotinic receptor-ionic channel complex (AChR) using electrophysiological and biochemical methods. Voltage clamp studies of the frog sartorius and cutaneous pectoris neuromuscular junction revealed a concentration-dependent depression of the decay time constant of the end-plate (tau EPC) and spontaneous miniature end-plate (tau MEPC) currents. The relationship of the reciprocal of either tau EPC or tau MEPC and bupivacaine concentration up to 100 microM was linear. Voltage dependence of EPC over the range +60 to -150 mV was reduced, whereas both EPC and MEPC decays were adequately described by a single exponential function at all concentrations tested. Peak MEPC and EPC amplitudes were also depressed in a concentration-dependent manner such that 100 microM bupivacaine reduced peak amplitude by about 50%. The current-voltage relationship remained linear under all conditions tested. Nerve-evoked responses were difficult to study at concentrations greater than 100 microM because of apparent blockade of nerve conduction. Extracellular recording of the MEPC afforded results similar to those obtained with EPCs. The tau MEPC could be reduced to less than 300 mu sec at a bupivacaine concentration of 400 microM. Fluctuation analysis showed that bupivacaine at concentrations of 10 and 25 microM did not change channel conductance but decreased single-channel lifetime to 76% and 39% of control values, respectively. Biochemical studies were performed on Torpedo californica membrane fragments using [3H]phencyclidine ([3H]PCP) and [3H]perhydrohistrionicotoxin ([3H]H12-HTX) as channel probes. Bupivacaine inhibited the binding of [3H]PCP and [3H]H12-HTX with inhibition constants (Ki) of 32 and 25 microM, respectively. The corresponding inhibition constants for bupivacaine methiodide were 1.8 and 3.2 microM. The preincubation of the membranes with carbamylcholine increased the affinity of bupivacaine for the ionic channel sites 5- to 8-fold and the affinity of bupivacaine methiodide 3- to 4-fold. Bupivacaine, however, had no affinity for the agonist recognition site as determined by [3H]ACh and [125I]alpha-bungarotoxin bindings. The electrophysiological and biochemical studies indicate that bupivacaine reacts primarily with the ionic channel of the nicotinic AChR. The results are consistent with a sequential model in which the drug interacts with the sites at the ionic channel of AChR in its open conformation, producing species with little or no conductance. From the present studies there is no evidence for an interaction of bupivacaine with the agonist binding site or closed states of AChR.
Mol Pharmacol 1984 Sep
PMID:Interactions of bupivacaine with ionic channels of the nicotinic receptor. Electrophysiological and biochemical studies. 609 Aug 84

In this study the effects of amantadine (1-adamantanamine) and its N-alkyl-substituted analogues [N-methyl- (NMA), N-ethyl- (NEA), N-propyl- (NPA), N-butyl- (NBA), and N,N-diethyl-amantidine (NNDEA)] were investigated on ionic channels of the electrically excitable membrane and of the nicotinic acetylcholine (ACh) receptors in frog sartorius muscles and on the binding of perhydrohistrionicotoxin (H12-HTX) to isolated membranes of the electric organ of the electric ray Torpedo. Amantadine and each analogue blocked the indirectly elicited twitch, but NPA, NBA, and NNDEA also potentiated the directly elicited twitch. The order of potency in inhibiting the indirect twitch was: NEA = NPA = NNDEA (10 microM) greater than NMA (15 microM) greater than NBA (40 microM) much greater than amantadine (130 microM). Neither amantadine nor its N-alkyl analogues affected miniature end-plate potential frequency or resting membrane potential but decreased miniature end-plate potential amplitude. Each compound prolonged the directly elicited action potential but did not alter delayed rectification. All of the compounds induced a concentration-dependent depression of the peak end-plate current (EPC) amplitude at negative membrane potentials and induced nonlinearity in the response at membrane potentials more negative than -40 mV. The order of potency in inhibiting the EPC (at -90 mV) was NNDEA (less than 0.5 microM) greater than NPA (less than 1.0 microM) greater than NBA (less than 2.0 microM) greater than NEA (19 microM) greater than NMA (42 microM) greater than amantadine (64 microM). Only NPA, NBA, and NNDEA depressed the peak EPC amplitude at positive membrane potentials as well. The shortening of the time constant of EPC decay by all compounds was concentration-dependent. At the higher concentrations examined, the slope of the relationship between the time constant of decay and membrane potential was reversed for all compounds. Only NPA induced a double-exponential decay of the EPC at positive membrane potentials. Neither amantadine nor its N-alkyl analogues inhibited the binding of [3H]ACh to its receptor in Torpedo electroplax but they inhibited the binding of [3H]H12-HTX binding to the ionic channel sites of the ACh receptor. The Ki for inhibition of [3H]H12-HTX binding was NEA = NNDEA (15 microM) greater than NMA (30 microM) greater than NPA = NBA (40 microM) greater than amantadine (60 microM). A gross correlation exists between their ability to block the indirect muscle twitch, miniature end-plate potential amplitude, peak EPC amplitude and the binding of [3H]H12-HTX. But, no correlation was found between these potencies and their antiviral activity. It is suggested that these compounds may interact with the ionic channel of the ACh receptor in its open and closed conformation.
Mol Pharmacol 1982 Jul
PMID:Structure-activity relationships of amantadine. I. Interaction of the N-alkyl analogues with the ionic channels of the nicotinic acetylcholine receptor and electrically excitable membrane. 612 6

Although substantial evidence supports a critical role for the activation of Raf-1 and mitogen-activated protein kinases (MAPKs) in oncogenic Ras-mediated transformation, recent evidence suggests that Ras may activate a second signaling pathway which involves the Ras-related proteins Rac1 and RhoA. Consequently, we used three complementary approaches to determine the contribution of Rac1 and RhoA function to oncogenic Ras-mediated transformation. First, whereas constitutively activated mutants of Rac1 and RhoA showed very weak transforming activity when transfected alone, their coexpression with a weakly transforming Raf-1 mutant caused a greater than 35-fold enhancement of transforming activity. Second, we observed that coexpression of dominant negative mutants of Rac1 and RhoA reduced oncogenic Ras transforming activity. Third, activated Rac1 and RhoA further enhanced oncogenic Ras-triggered morphologic transformation, as well as growth in soft agar and cell motility. Finally, we also observed that kinase-deficient MAPKs inhibited Ras transformation. Taken together, these data support the possibility that oncogenic Ras activation of Rac1 and RhoA, coupled with activation of the Raf/MAPK pathway, is required to trigger the full morphogenic and mitogenic consequences of oncogenic Ras transformation.
Mol Cell Biol 1995 Nov
PMID:Activation of Rac1, RhoA, and mitogen-activated protein kinases is required for Ras transformation. 756 96

The Ras-related protein Cdc42 plays a role in yeast cell budding and polarity. Two related proteins, Rac1 and RhoA, promote formation in mammalian cells of membrane ruffles and stress fibers, respectively, which contain actin microfilaments. We now show that microinjection of the related human Cdc42Hs into Swiss 3T3 fibroblasts induced the formation of peripheral actin microspikes, determined by staining with phalloidin. A proportion of these microspikes was found to be components of filopodia, as analyzed by time-lapse phase-contrast microscopy. The formation of filopodia was also found to be promoted by Cdc42Hs microinjection. This was followed by activation of Rac1-mediated membrane ruffling. Treatment with bradykinin also promoted formation of microspikes and filopodia as well as subsequent effects similar to that seen upon Cdc42Hs microinjection. These effects of bradykinin were specifically inhibited by prior microinjection of dominant negative Cdc42HsT17N, suggesting that bradykinin acts by activating cellular Cdc42Hs. Since filopodia have been ascribed an important sensory function in fibroblasts and are required for guidance of neuronal growth cones, these results indicate that Cdc42Hs plays an important role in determining mammalian cell morphology.
Mol Cell Biol 1995 Apr
PMID:The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts. 789 88


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