共查询到20条相似文献,搜索用时 31 毫秒
1.
Cheng L Mahon GM Kostenko EV Whitehead IP 《The Journal of biological chemistry》2004,279(13):12786-12793
Dbs is a Rho-specific guanine nucleotide exchange factor that was identified in a screen for proteins whose expression causes deregulated growth in NIH 3T3 mouse fibroblasts. Although Rac1 has not been shown to be a substrate for Dbs in either in vitro or in vivo assays, the Rat ortholog of Dbs (Ost) has been shown to bind specifically to GTP.Rac1 in vitro. The dependence of the Rac1/Dbs interaction on GTP suggests that Dbs may in fact be an effector for Rac1. Here we show that the interaction between activated Rac1 and Dbs can be recapitulated in mammalian cells and that the Rac1 docking site resides within the pleckstrin homology domain of Dbs. This interaction is specific for Rac1 and is not observed between Rac1 and several other members of the Rho-specific guanine nucleotide exchange factor family. Co-expression of Dbs with activated Rac1 causes enhanced focus forming activity and elevated levels of GTP.RhoA in NIH 3T3 cells, indicating that Dbs is activated by the interaction. Consistent with this, activated Rac1 co-localizes with Dbs in NIH 3T3 cells, and natively expressed Rac1 relocalizes in response to Dbs expression. To summarize, we have characterized a surprisingly direct pleckstrin homology domain-mediated mechanism through which Rho GTPases can become functionally linked. 相似文献
2.
The regulation of adenylate cyclase by guanine nucleotides in Dictyostelium discoideum membranes 总被引:11,自引:0,他引:11
P J Van Haastert B E Snaar-Jagalska P M Janssens 《European journal of biochemistry》1987,162(2):251-258
Extracellular cAMP induces the activation of adenylate cyclase in Dictyostelium discoideum cells. Conditions for both stimulation and inhibition of adenylate cyclase by guanine nucleotides in membranes are reported. Stimulation and inhibition were induced by GTP and non-hydrolysable guanosine triphosphates. GDP and non-hydrolysable guanosine diphosphates were antagonists. Stimulation was maximally twofold, required a cytosolic factor and was observed only at temperatures below 10 degrees C. An agonist of the cAMP-receptor-activated basal and GTP-stimulated adenylate cyclase 1.3-fold. Adenylate cyclase in mutant N7 could not be activated by cAMP in vivo; in vitro adenylate cyclase was activated by guanine nucleotides in the presence of the cytosolic factor of wild-type but of not mutant cells. Preincubation of membranes under phosphorylation conditions has been shown to alter the interaction between cAMP receptor and G protein [Van Haastert (1986) J. Biol. Chem. in the press]. These phosphorylation conditions converted stimulation to inhibition of adenylate cyclase by guanine nucleotides. Inhibition was maximally 30% and was not affected by the cytosolic factor involved in stimulation. In membranes obtained from cells that were treated with pertussis toxin, adenylate cyclase stimulation by guanine nucleotides was as in control cells, whereas inhibition by guanine nucleotides was lost. When cells were desensitized by exposure to cAMP agonists for 15 min, and adenylate cyclase was measured in isolated membranes, stimulation by guanine nucleotides was lost while inhibition was retained. These results suggest that Dictyostelium discoideum adenylate cyclase may be regulated by Gs-like and Gi-like activities, and that the action of Gs but not Gi is lost during desensitization in vivo and by phosphorylation conditions in vitro. 相似文献
3.
We present the structure of the decanucleotide d(CGTATATACG) determined by single crystal X-ray diffraction at 1.58 A resolution. A netropsin drug is found in the minor groove with guanine stacked on a pyrrole ring of the drug, a feature described here for the first time. The stacked guanine is an extra-helical base coming from the end of a neighbour oligonucleotide. This observation may open the way to the development of minor groove binding drugs with a higher sequence selectivity. The oligonucleotide is in the B-conformation, but the terminal base-pairs are disrupted: the cytosine residues are disordered while the guanine residues penetrate into the minor groove of neighbouring duplexes. Four hydrated Ni ions with octahedral co-ordination are found associated with the N7 atoms of each guanine. The high affinity of these ions with guanine suggests that they may be used as probes for specific guanine residues. 相似文献
4.
Mammalian genes expressed in Drosophila: a transgenic model for the study of mechanisms of chemical mutagenesis and metabolism.
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Mammalian cytochrome P450s provide our first line of defence against the toxic effects of environmental chemicals. Ironically these enzymes also convert some compounds to their ultimate toxic or mutagenic species. Our knowledge of these mammalian enzymes and the role they play in chemical toxicity and mutagenesis has stemmed mostly from in vitro studies. In order to establish the role of specific enzymes in the toxicological response in vivo we have generated transgenic Drosophila which express mammalian cytochrome CYP2B1, which is a member of a large gene family encoding several important drug metabolising enzymes. The gene was fused to a Drosophila promoter which confers expression in the larval fat body. Using the Somatic Mutation And Recombination Test (SMART) we have demonstrated that transgenic larvae expressing the P450 are hypersensitive to the anticancer drug cyclophosphamide, a procarcinogenic substrate which is activated by the enzyme. This work demonstrates the potential of such transgenic Drosophila strains as an in vivo model for studying the role of specific mammalian drug metabolising enzymes in the pathways and metabolic cascades associated with the action of cytotoxic and carcinogenic chemicals, and also the chemical properties of specific classes of mutagen to be determined. 相似文献
5.
RhoGDI inhibits guanine nucleotide dissociation from post-translationally processed Rho and Rac proteins but its biochemical role in vivo is unknown. We show here that N-terminal effector site mutations in the Rac protein do not compromise its interaction with RhoGDI and that, whilst geranylgeranylation and -AAX proteolysis of the C-terminal CAAX motif of Rac1 and RhoA are required for efficient interaction with RhoGDI, methylesterification of the C-terminal cysteine residue is not required. In vitro, RhoGDI can form stable complexes with Rho and Rac proteins in both the GTP and GDP bound states. Furthermore the Rac-GTP--RhoGDI complex is resistent to the action of recombinant RhoGAP and recombinant BCR. Thus GDI, by complexing with Rac-GTP and preventing GAP stimulated GTP hydrolysis, may allow transit of the activated form of the Rac protein between physically separated activator and effector proteins in the cell. 相似文献
6.
7.
The nitroimidazole tested is structurally related to furazolidone in that it contains a 5-nitro-imidazole instead of 5-nitrofuran. The nitroimidazole was much less active in vitro than furazolidone against a spectrum of pathogenic organisms. However, it is active in vivo by oral administration against bacterial infections of mice and its low acute toxicity gives better therapeutic indices than are obtained with furazolidone. Plasma concentrations rise rapidly following oral administration of the nitroimidazole. Sufficient urinary excretion occurred to give significant activity in a mouse kidney infection incited by Staphylococcus aureus in which nitrofurantoin was inactive. 相似文献
8.
A. Kathrine Miller 《Applied microbiology》1971,22(3):480-481
Ronidazole, a nitroimidazole that has in vivo antiparasitic and antimycoplasmal activity, also has some in vivo antibacterial activity. 相似文献
9.
A new interaction between Abi.1 and βPIX involved in PDGF.activated actin cytoskeleton reorganisation 总被引:1,自引:0,他引:1
Members of the Rho family of GTPases are key regulators of the actin cytoskeleton. In particular, activated Racl stimulates membrane dorsal ruffle formation in response to platelet-derived growth factor (PDGF). Abl-interactor (Abi)- 1 and βP1X, a guanine nucleotide exchange factor for Racl, localise at these Rac1-induced actin structures and play important roles in the induction of membrane dorsal ruffling in response to PDGF in fibroblasts. Here, we demonstrate a novel interaction between Abi-1 and βPIX using the yeast two-hybrid system, in vitro pull-down assays, and in vivo co-immunoprecipitation experiments. In vitro, the C-terminal fragment of βPIX interacted with Abi-1, while in vivo the N-terminal fragment of βPIX interacted with Abi-1. The biological function of this interaction was investigated in mouse fibroblasts in response to PDGF stimulation. Abi-1 and βPIX co-localised in the cytoplasm and to membrane dorsal ruffles after PDGF treatment. We show that the co-expression of Abi-1 and truncated forms of βPIX in mouse fibroblasts blocked PDGF-induced membrane dorsal ruffles. Together, these results show that the interaction between Abi-1 and βPIX is involved in the formation of growth factor-induced membrane dorsal ruffles. 相似文献
10.
In vitro and in vivo assessment of herb drug interactions 总被引:3,自引:0,他引:3
Herbal products contain several chemicals that are metabolized by phase 1 and phase 2 pathways and also serve as substrates for certain transporters. Due to their interaction with these enzymes and transporters there is a potential for alteration in the activity of drug metabolizing enzymes and transporters in presence of herbal components. Induction and inhibition of drug metabolizing enzymes and transporters by herbal component has been documented in several in vitro studies. While these studies offer a system to determine the potential for a herbal component to alter the pharmacokinetics of a drug, they cannot always be used to predict the magnitude of any potential effect in vivo. In vivo studies are the ultimate way to determine the clinical importance of herb drug interactions. However, lack of content uniformity and lack of documentation of the bioavailability of herbal components makes even in vivo human studies difficult to interpret as the effect may be product specific. It appears that St. John's wort extract is probably one of the most important herbal product that increases the metabolism and decreases the efficacy of several drugs. Milk thistle on the other hand appears to have minimal effect on phase 1 pathways and limited data exists for phase 2 pathways and transporter activity in vivo. Further systematic studies are necessary to assess the significance of herb drug interactions. 相似文献
11.
Detecting cAMP-induced Epac activation by fluorescence resonance energy transfer: Epac as a novel cAMP indicator
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Ponsioen B Zhao J Riedl J Zwartkruis F van der Krogt G Zaccolo M Moolenaar WH Bos JL Jalink K 《EMBO reports》2004,5(12):1176-1180
Epac1 is a guanine nucleotide exchange factor for Rap1 that is activated by direct binding of cAMP. In vitro studies suggest that cAMP relieves the interaction between the regulatory and catalytic domains of Epac. Here, we monitor Epac1 activation in vivo by using a CFP-Epac-YFP fusion construct. When expressed in mammalian cells, CFP-Epac-YFP shows significant fluorescence resonance energy transfer (FRET). FRET rapidly decreases in response to the cAMP-raising agents, whereas it fully recovers after addition of cAMP-lowering agonists. Thus, by undergoing a cAMP-induced conformational change, CFP-Epac-YFP serves as a highly sensitive cAMP indicator in vivo. When compared with a protein kinase A (PKA)-based sensor, Epac-based cAMP probes show an extended dynamic range and a better signal-to-noise ratio; furthermore, as a single polypeptide, CFP-Epac-YFP does not suffer from the technical problems encountered with multisubunit PKA-based sensors. These properties make Epac-based FRET probes the preferred indicators for monitoring cAMP levels in vivo. 相似文献
12.
T Ishida K Kamiichi A Kuwahara M Doi M Inoue 《Biochemical and biophysical research communications》1986,136(1):294-299
The crystal structure of title complex has been analyzed by X-ray diffraction method as a model for elucidating the possible interaction between the phenylalanyl residue of proteins and the N7-protonated or methylated guanine base of nucleic acids. The guanine base is associated with the benzene ring of phenylalanine by stacking interaction, and further connected with the carboxyl group by the formation of a pair of hydrogen bonds. These two interaction modes are suggested to be responsible for the specific recognition of base sequence by protein. 相似文献
13.
Cyclic AMP signalling in Dictyostelium: G-proteins activate separate Ras pathways using specific RasGEFs 总被引:1,自引:0,他引:1
Kae H Kortholt A Rehmann H Insall RH Van Haastert PJ Spiegelman GB Weeks G 《EMBO reports》2007,8(5):477-482
In general, mammalian Ras guanine nucleotide exchange factors (RasGEFs) show little substrate specificity, although they are often thought to regulate specific pathways. Here, we provide in vitro and in vivo evidence that two RasGEFs can each act on specific Ras proteins. During Dictyostelium development, RasC and RasG are activated in response to cyclic AMP, with each regulating different downstream functions: RasG regulates chemotaxis and RasC is responsible for adenylyl cyclase activation. RasC activation was abolished in a gefA- mutant, whereas RasG activation was normal in this strain, indicating that RasGEFA activates RasC but not RasG. Conversely, RasC activation was normal in a gefR- mutant, whereas RasG activation was greatly reduced, indicating that RasGEFR activates RasG. These results were confirmed by the finding that RasGEFA and RasGEFR specifically released GDP from RasC and RasG, respectively, in vitro. This RasGEF target specificity provides a mechanism for one upstream signal to regulate two downstream processes using independent pathways. 相似文献
14.
Park B Nguyen NT Dutt P Merdek KD Bashar M Sterpetti P Tosolini A Testa JR Toksoz D 《The Journal of biological chemistry》2002,277(47):45361-45370
15.
C E Voogd 《Mutation research》1989,221(2):133-152
Azathioprine, an immunosuppressive drug, has been used for 25 years. Azathioprine is rapidly converted into a number of metabolites after absorption. Maximum blood levels in experimental animals (mice) were 11.3 micrograms/ml after a dosage of 33.3 mg/kg. Generally, levels of less than 1 microgram/ml are found. As azathioprine is ineffective in hypoxanthine guanine phosphoribosyltransferase (HPRT)-deficient patients, it will be clear that for immunosuppressive activity azathioprine must be metabolised. Regarding mutagenic activity, its mutagenicity for bacteria seems irrelevant for man because the nitroimidazole moiety can be reduced by bacteria but not or hardly at all by mammalian tissues. So 6-mercaptopurine (a metabolite of azathioprine) and its metabolites should be regarded as the active compounds. In vitro azathioprine can induce chromosome aberrations and other cytogenetic events at high, non-physiological doses. However, in view of the low blood levels it is unlikely that azathioprine can induce chromosome aberrations in kidney transplant patients. It is more probable that azathioprine inhibits the elimination of such aberrant cells through its immunosuppressive activity. It should be pointed out that in microbial mutagenicity systems also, azathioprine concentrations that are not reached in patients are needed to obtain an increased mutation rate. 相似文献
16.
Dynamics of GBF1, a Brefeldin A-sensitive Arf1 exchange factor at the Golgi 总被引:1,自引:0,他引:1
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Niu TK Pfeifer AC Lippincott-Schwartz J Jackson CL 《Molecular biology of the cell》2005,16(3):1213-1222
Trafficking through the Golgi apparatus requires members of the Arf family of GTPases, whose activation is regulated by guanine nucleotide exchange factors (GEFs). Once activated, Arf-GTP recruits effectors such as coat complexes and lipid-modifying enzymes to specific membrane sites, creating a domain competent for cargo concentration and transport. GBF1 is a peripherally associated Arf GEF involved in both endoplasmic reticulum-Golgi and intra-Golgi transport. The mechanism of GBF1 binding to membranes is unknown. As a first step to understanding the mechanism of membrane association, we constructed a yellow fluorescent protein-tagged version of GBF1 and performed fluorescence recovery after photobleaching analysis to determine its residence time on Golgi membranes. We find that GBF1 molecules are not stably associated with the Golgi but rather cycle rapidly on and off membranes. The drug brefeldin A (BFA), an uncompetitive inhibitor of the exchange reaction that binds to an Arf-GDP-Arf GEF complex, stabilizes GBF1 on Golgi membranes. Using an in vivo assay to monitor Arf1-GTP levels, we show that GBF1 exchange activity on Arf1 is inhibited by BFA in mammalian cells. These results suggest that an Arf1-GBF1-BFA complex is formed and has a longer residence time on Golgi membranes than GBF1 or Arf1 alone. 相似文献
17.
Evidence for two GTPases activated by thrombin in membranes of human platelets. 总被引:9,自引:6,他引:3
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Thrombin inhibits adenylate cyclase and stimulates GTP hydrolysis by high-affinity GTPase(s) in membranes of human platelets at almost identical concentrations. Both of these thrombin actions are similar to those observed with agonist-activated alpha 2-adrenoceptors coupling to the inhibitory guanine nucleotide-binding protein N1. However, stimulation of GTP hydrolysis caused by adrenaline (alpha 2-adrenoceptor agonist) and by thrombin at maximally effective concentrations was partially additive, whereas with regard to adenylate cyclase inhibition no additive response was observed. Furthermore, treatment of platelet membranes with pertussis toxin, which inactivates Ni and largely abolishes thrombin- and adrenaline-induced adenylate cyclase inhibition and adrenaline-induced GTPase stimulation, decreased the thrombin-induced stimulation of GTP hydrolysis by only about 30%. Additionally, the thiol reagent N-ethylmalemide (NEM) at rather low concentrations abolished thrombin- and adrenaline-induced stimulation of GTP hydrolysis was decreased by only 30-40% by treatment of platelet membranes with even high concentrations of NEM. Treatment with cholera toxin, which inhibits GTPase activity of the Ns (stimulatory guanine nucleotide-binding) protein, has no effect on thrombin-stimulated GTP hydrolysis. The data suggest that thrombin interaction with its receptor sites in platelet membranes leads to stimulation of two GTP-hydrolysing enzymes. One of these enzymes is apparently Ni and is also activated by agonist-activated alpha 2-adrenoceptors and is inactivated by pertussis toxin and NEM treatment. The other GTP-hydrolysing enzyme activated by thrombin may represent a guanine nucleotide-binding protein apparently involved in the coupling of thrombin receptors to the phosphoinositide phosphodiesterase. 相似文献
18.
Martínez SE Yuan L Lacza C Ransom H Mahon GM Whitehead IP Hake LE 《Molecular biology of the cell》2005,16(3):1152-1164
Polyadenylation-induced translation is an important regulatory mechanism during metazoan development. During Xenopus oocyte meiotic progression, polyadenylation-induced translation is regulated by CPEB, which is activated by phosphorylation. XGef, a guanine exchange factor, is a CPEB-interacting protein involved in the early steps of progesterone-stimulated oocyte maturation. We find that XGef influences early oocyte maturation by directly influencing CPEB function. XGef and CPEB interact during oogenesis and oocyte maturation and are present in a c-mos messenger ribonucleoprotein (mRNP). Both proteins also interact directly in vitro. XGef overexpression increases the level of CPEB phosphorylated early during oocyte maturation, and this directly correlates with increased Mos protein accumulation and acceleration of meiotic resumption. To exert this effect, XGef must retain guanine exchange activity and the interaction with CPEB. Overexpression of a guanine exchange deficient version of XGef, which interacts with CPEB, does not enhance early CPEB phosphorylation. Overexpression of a version of XGef that has significantly reduced interaction with CPEB, but retains guanine exchange activity, decreases early CPEB phosphorylation and delays oocyte maturation. Injection of XGef antibodies into oocytes blocks progesterone-induced oocyte maturation and early CPEB phosphorylation. These findings indicate that XGef is involved in early CPEB activation and implicate GTPase signaling in this process. 相似文献
19.
Identification of hypoxanthine and guanine as the co-repressors for the purine regulon genes of Escherichia coli 总被引:8,自引:0,他引:8
Addition of purine compounds to the growth medium of Escherichia coli and Salmonella typhimurium causes repressed synthesis of the purine biosynthetic enzymes. The repression is mediated through a regulatory protein, PurR. To identify the co-repressor(s) of PurR, two approaches were used: (i) mutations were introduced into purine salvage genes and the effects of different purines on pur gene expression were determined; (ii) purine compounds which dictate the binding of the PurR protein to its operator DNA were resolved by gel retardation. Both the in vivo and the in vitro data indicated that guanine and hypoxanthine are co-repressors. The toxic purine analogues 6-mercaptopurine and 6-thioguanine also activated the binding of PurR to its operator DNA. 相似文献
20.
Sequence-specific duplex DNA recognition by oligonucleotide-directed triple helix formation is a possible approach to in vivo gene inhibition. However, triple helix formation involving guanine-rich oligonucleotides is inhibited by physiological ions, particularly K+, most likely due to oligonucleotide aggregation via guanine quartets. Three oligodeoxynucleotide (ODN) derivatives were tested for their ability to resist guanine quartet-mediated aggregation, yet form stable triplexes. Electrophoretic mobility shift and dimethyl sulfate footprinting assays were used to analyze the formation of triplexes involving these oligonucleotide derivatives. In the absence of K+, all ODNs had similar binding affinities for the duplex target. Triplexes involving a 14mer ODN derivative containing 7-deazaxanthine substituted for three thymine bases or an 18mer ODN containing two additional thymines on both the 5' and 3' termini were abolished by 50 mM K+. Remarkably, triplexes involving an ODN derivative containing four 6-thioguanine bases substituted for guanine resisted K+ inhibition up to 200 mM. We hypothesize that the increased radius and decreased electronegativity of sulfur in the 6-position of guanine destabilize potential guanine quartets. These results improve the prospects for creating ODNS that might serve as specific and efficient gene repressors in vivo. 相似文献