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Basal Activity (basal + activity)
Selected AbstractsInterplay of constitutively released nucleotides, nucleotide metabolism, and activity of P2Y receptorsDRUG DEVELOPMENT RESEARCH, Issue 2-3 2001Eduardo R. Lazarowski Abstract At least six mammalian P2Y receptors exist that are specifically activated by ATP, UTP, ADP or UDP. Although the existence of ectoenzymes that rapidly metabolize extracellular nucleotides is well established, the relative flux of ATP and UTP through their extracellular metabolic products remains undefined. In addition, the existence of basal nucleotide release and the contribution of resting levels of ATP and UTP to P2 receptor activation are poorly understood. In the absence of exogenous agonists, an apyrase-sensitive inositol phosphate accumulation was observed in resting 16HBE14o, human bronchial epithelial cells endogenously expressing P2Y receptors and in 1321N1 human astrocytoma cells expressing a recombinant P2Y2 receptor. To test whether nucleotide release may account for basal P2 receptor activities, the rates of extracellular accumulation and metabolism of endogenous ATP were examined with resting 16HBE14o,, C6 rat glioma, and 1321N1 cell cultures. Although extracellular ATP concentrations (1-5 nM) remained unchanged for up to 12 h, [,32P] ATP included in the medium (as a radiotracer) was completely degraded within 120 min, indicating that ATP release balanced ATP hydrolysis. The calculated basal rates of ATP release ranged from 20 to 200 fmol/min per million cells. HPLC analysis during steady state revealed that the gamma-phosphate of ATP was reversibly transferred to species further identified as UTP and GTP, implicating ecto-nucleoside diphosphokinase (NDPK)-catalyzed phosphorylation of endogenous UDP and GDP. At steady state, the final 32P-products of [,32P]ATP metabolism were 32P-orthophosphoric acid and a species further purified and identified as 32P-pyrophosphate. Constitutive nucleotide release balanced by the concerted activities of ecto-ATPase, ecto-ATP pyrophosphatase, and ecto-NDPK may determine the resting levels of extracellular nucleotides and therefore, the basal activity of P2 receptors. Drug Dev. Res. 53:66,71, 2001. © 2001 Wiley-Liss, Inc. [source] Fatty acid incorporation in endothelial cells and effects on endothelial nitric oxide synthaseEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 9 2007S. Couloubaly Abstract Background The nature of fatty acids provided by the diet as well as plasma lipid metabolism can modify the composition and properties of plasma membrane and thus the activity of membrane proteins. In humans, as well as in experimental models, diabetes is associated with both an alteration in serum lipid profile and a documented endothelial dysfunction. This in vitro study investigated on an immortalized human endothelial cell line (EA.hy 926) the specific effects of several free fatty acids (FFAs) on the composition of cellular membranes and the regulation of endothelial nitric oxide synthase (eNOS). Materials and methods 0·1% of lipid deprived serum was added to the incubation medium with 25 mm glucose in order to study the effects of individual fatty acids: myristic acid, palmitic acid, stearic acid, oleic acid or linoleic acid at 100 µm bound with albumin. The effects of the FFAs on the endothelial nitric oxide synthase were investigated on mRNA level by quantitative PCR, on protein level and Ser1177 phosphorylation by Western blot and on enzymatic activity on living cells using radiolabelled arginine. Results Free linoleic acid increased the membrane content in n-6 fatty acids (mainly C18: n-6 and its metabolites) with a decrease in saturated and monounsaturated fatty acids. These conditions decreased the basal eNOS activity and reduced the phosphorylation of eNOS-Ser1177 due to activation by histamine. Free palmitic acid enriched the membranes with 16 : 0 with a slight decrease in monounsaturated fatty acids. These conditions increased eNOS activation without increasing Ser1177 phosphorylation upon histamine activation. The addition of the other FFAs also resulted in modifications of membrane composition, which did not to affect eNOS-Ser1177 phosphorylation. Conclusion Among the fatty acids used, only modification of the membrane composition due to linoleic acid supply disturbed the basal enzymatic activity and Ser1177 phosphorylation of eNOS in a way that limited the role of histamine activation. Linoleic acid might involve the dysfunction of both eNOS basal activity and its phosphorylation status and may then contribute to an impaired vasodilatation in vivo. [source] Effects of nicotine in the dopaminergic system of mice lacking the alpha4 subunit of neuronal nicotinic acetylcholine receptorsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2003L. M. Marubio Abstract The mesostriatal dopaminergic system influences locomotor activity and the reinforcing properties of many drugs of abuse including nicotine. Here we investigate the role of the ,4 nicotinic acetylcholine receptor (nAChR) subunit in mediating the effects of nicotine in the mesolimbic dopamine system in mice lacking the ,4 subunit. We show that there are two distinct populations of receptors in the substantia nigra and striatum by using autoradiographic labelling with 125I ,-conotoxin MII. These receptors are comprised of the ,4, ,2 and ,6 nAChR subunits and non-,4, ,2, and ,6 nAChR subunits. Non-,4 subunit-containing nAChRs are located on dopaminergic neurons, are functional and respond to nicotine as demonstrated by patch clamp recordings. In vivo microdialysis performed in awake, freely moving mice reveal that mutant mice have basal striatal dopamine levels which are twice as high as those observed in wild-type mice. Despite the fact that both wild-type and ,4 null mutant mice show a similar increase in dopamine release in response to intrastriatal KCl perfusion, a nicotine-elicited increase in dopamine levels is not observed in mutant mice. Locomotor activity experiments show that there is no difference between wild-type and mutant mice in basal activity in both habituated and non-habituated environments. Interestingly, mutant mice sustain an increase in cocaine-elicited locomotor activity longer than wild-type mice. In addition, mutant mice recover from depressant locomotor activity in response to nicotine at a faster rate. Our results indicate that ,4-containing nAChRs exert a tonic control on striatal basal dopamine release, which is mediated by a heterogeneous population of nAChRs. [source] Properties of ecdysteroid receptors from diverse insect species in a heterologous cell culture system , a basis for screening novel insecticidal candidatesFEBS JOURNAL, Issue 11 2009Joshua M. Beatty Insect development is driven by the action of ecdysteroids on morphogenetic processes. The classic ecdysteroid receptor is a protein heterodimer composed of two nuclear receptors, the ecdysone receptor (EcR) and Ultraspiracle (USP), the insect ortholog of retinoid X receptor. The functional properties of EcR and USP vary among insect species, and provide a basis for identifying novel and species-specific insecticidal candidates that disrupt this receptor's normal activity. A heterologous mammalian cell culture assay was used to assess the transcriptional activity of the heterodimeric ecdysteroid receptor from species representing two major insect orders: the fruit fly, Drosophila melanogaster (Diptera), and the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera). Several nonsteroidal agonists evoked a strong response with the L. decemlineata heterodimer that was consistent with biochemical and in vivo evidence, whereas the D. melanogaster receptor's response was comparatively modest. Conversely, the phytoecdysteroid muristerone A was more potent with the D. melanogaster heterodimer. The additional presence of juvenile hormone III potentiated the inductive activity of muristerone A in the receptors from both species, but juvenile hormone III was unable to potentiate the inductive activity of the diacylhydrazine methoxyfenozide (RH2485) in the receptor of either species. The effects of USP on ecdysteroid-regulated transcriptional activity also varied between the two species. When it was tested with D. melanogaster EcR isoforms, basal activity was lower and ligand-dependent activity was higher with L. decemlineata USP than with D. melanogaster USP. Generally, the species-based differences validate the use of the cell culture assay screen for novel agonists and potentiators as species-targeted insecticidal candidates. [source] Repressive domain of unliganded human estrogen receptor , associates with Hsc70GENES TO CELLS, Issue 12 2005Satoko Ogawa Estrogen receptor (ER) is a hormone-inducible transcription factor as a member of the nuclear receptor gene superfamily. Unliganded ER is transcriptionally silent and capable of DNA binding; however, it is unable to suppress the basal activity of the target gene promoters, unlike non-steroid hormone receptors that associate with corepressors in the absence of their cognate ligands. To study the molecular basis of how unliganded human ER, is maintained silent in gene regulation upon the target gene promoters, we biochemically searched interactants for hER,, and identified heat shock protein 70 (Hsc70). Hsc70 appeared to associate with the N-terminal hormone binding E domain, that also turned out a transcriptionally repressive domain. Competitive association of Hsc70 with a best known coactivator p300 was observed. Thus, these findings suggest that Hsc70 associates with unliganded hER,, and thereby deters hER, from recruiting transcriptional coregulators, presumably as a component of chaperone complexes. [source] Nonstructural 3/4A protease of hepatitis C virus activates epithelial growth factor,induced signal transduction by cleavage of the T-cell protein tyrosine phosphatase,HEPATOLOGY, Issue 6 2009Erwin Daniel Brenndörfer The hepatitis C virus (HCV) is a worldwide major cause of chronic liver disease with a high tendency to establish a persistent infection. To permit persistent replication of viral genomes through the cellular translation machinery without affecting host cell viability, viruses must have developed mechanisms to control cellular cascades required for sufficient viral replication, on the one hand, and to adapt viral replication to the cellular requirements on the other hand. The present study aimed to further elucidate mechanisms by which HCV targets growth factor signaling of the host cell and their implications for viral replication. The study describes a novel mechanism by which HCV influences the activation of the epithelial growth factor receptor/Akt pathway through a nonstructural (NS)3/4A-dependent down-regulation of the ubiquitously expressed tyrosine phosphatase T cell protein tyrosine phosphatase (TC-PTP). NS3/4A is demonstrated to cleave TC-PTP protease-dependently in vitro at two cleavage sites. The in vivo relevance of this finding is supported by the fact that down-regulation of TC-PTP protein expression could also be demonstrated in HCV-infected individuals and in transgenic mice with intrahepatic expression of NS3/4A. Conclusion: This down-regulation of TC-PTP results in an enhancement of epithelial growth factor (EGF)-induced signal transduction and increases basal activity of Akt, which is demonstrated to be essential for the maintenance of sufficient viral replication. Hence, therapeutic targeting of NS3/4A may not only disturb viral replication by blocking the processing of the viral polyprotein but also exerts unforeseen indirect antiviral effects, further diminishing viral replication. (HEPATOLOGY 2009;49:1810,1820.) [source] RANKL Treatment Releases the Negative Regulation of the Poly(ADP-Ribose) Polymerase-1 on Tcirg1 Gene Expression During Osteoclastogenesis,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 11 2006Guillaume E Beranger Abstract The Tcirg1 gene encodes the osteoclast-specific a3 isoform of the V-ATPase a subunit. Using the mouse osteoclastic model RAW264.7 cells, we studied Tcirg1 gene expression, and we identified PARP-1 as a transcriptional repressor negatively regulated by RANKL during osteoclastogenesis. Introduction: The TCIRG1 gene encodes the a3 isoform of the V-ATPase a subunit, and mutations at this locus account for ,60% of infantile malignant osteopetrosis cases. Using RAW264.7 cells as an osteoclastic differentiation model, we undertook a transcriptional study of the mouse Tcirg1 gene focused on the 4-kb region upstream of the transcription starting point. Materials and Methods: The promoter activity of serial-deletion fragments of the Tcirg1 gene promoter was monitored throughout the RAW264.7 cell differentiation process. We next performed EMSA, UV cross-linking, affinity purification, mass spectrometry analysis, gel supershift, and siRNA transfection experiments to identify the factor(s) interacting with the promoter. Results: The ,3946/+113 region of the mouse Tcirg1 gene displayed a high basal promoter activity, which was enhanced by RANKL treatment of RAW264.7 cells. Constructs deleted up to ,1589 retained this response to RANKL. A deletion up to ,1402 induced a 3-fold enhancement of the basal activity, whereas RANKL response was not affected. EMSA experiments led us to identify within the ,1589/,1402 region, a 10-nucleotide sequence, which bound a nuclear protein present in nondifferentiated RAW264.7 cells. This interaction was lost using nuclear extracts derived from RANKL-treated cells. Affinity purification followed by mass spectrometry analysis and gel supershift assay allowed the identification of poly(ADP-ribose) polymerase-1 (PARP-1) as this transcriptional repressor, whereas Western blot experiments revealed the cleavage of the DNA-binding domain of PARP-1 on RANKL treatment. Finally, both PARP-1 depletion after siRNA transfection and RAW264.7 cell treatment by an inhibitor of PARP-1 activity induced an increase of a3 mRNA expression. Conclusions: We provide evidence that the basal transcription activity of the Tcirg1 gene is negatively regulated by the binding of PARP-1 protein to its promoter region in mouse pre-osteoclast. On RANKL treatment, PARP-1 protein is cleaved and loses its repression effect, allowing an increase of Tcirg1 gene expression that is critical for osteoclast function. [source] Inhibition of constitutive activity of nuclear transcription factor kappaB sensitizes doxorubicin-resistant cells to apoptosisJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2009Charitha Gangadharan Abstract Doxorubicin is one of the most effective agents used in the treatment of various tumors. Its use is restricted by the development of resistance to apoptosis, the mechanism of which is not fully understood. Nuclear transcription factor kappaB (NF-,B) has been shown both to block apoptosis and to promote cell proliferation, and hence has been considered as an important target for anticancer drug development. We found that in wild type and Dox-revertant MCF-7 cells, Doxorubicin induced NF-,B was transient and Dox-resistant cells showed high basal activity of NF-,B and expression of genes dependent on it. Moreover, in resistant cells Doxorubicin was unable to induce apoptosis as detected by assays for reactive oxygen intermediates generation, lipid peroxidation, cytotoxicity, PARP degradation and Bcl-2 expression. High basal expressions of multi-drug resistant protein and transglutaminase were found in Dox-resistant cells and inhibition of NF-,B decreased those amounts and also sensitized these cells by Doxorubicin. These observations collectively suggest that high NF-,B activity confers resistance to Doxorubicin and its inhibition potentiates apoptosis. This study indicates that NF-,B plays an important role in chemoresistance and establishes the fact that inhibition of NF-,B will be a novel approach in chemotherapy. J. Cell. Biochem. 107: 203,213, 2009. © 2009 Wiley-Liss, Inc. [source] Co-localization of PARP-1 and lamin B in the nuclear architecture: A halo-fluorescence- and confocal-microscopy studyJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2005Melita Vidakovi Abstract A functional interaction between poly(ADP-ribose) polymerase-1 (PARP-1) and lamin B has recently been proposed by nuclear fractionation, crosslinking, and immunoprecipitation experiments. Here we use fluorescence microscopy to verify and extend these findings. We analyze nuclear halo preparations by fluorescence in situ immuno staining (FISIS), which shares attributes with traditional nuclear fractionation techniques, and by confocal laser scanning microscopy (CLSM). The results agree in that a major part of the enzyme co-localizes with lamin B under physiological conditions, where PARP-1 only has basal activity. After DNA damage and the associated activation of PARP-1, and during the subsequent entry into apoptosis, dramatic changes occur: a gradual release of the enzyme from the lamina, accompanied by its accumulation in nucleoli. Our observations are in line with biochemical evidence for lamin B-PARP-1 interactions under physiological conditions and suggest ways by which these interactions are modified to support PARP-functions in damage and its fate in apoptosis. © 2005 Wiley-Liss, Inc. [source] Role of protein kinase C-dependent A-kinase anchoring proteins in lysophosphatidic acid-induced cAMP signaling in human diploid fibroblastsAGING CELL, Issue 6 2006Ji-Heon Rhim Summary Previously, we reported that lysophosphatidic acid (LPA)-induced adenosine 3,,5,-cyclic monophosphate (cAMP) production by human diploid fibroblasts depends on the age of the fibroblasts. In this study, we examined the role of A-kinase anchoring proteins (AKAP) in the regulation of LPA-stimulated cAMP production in senescent fibroblasts. We found that levels of protein kinase C (PKC)-dependent AKAPs, such as Gravin and AKAP79, were elevated in senescent cells. Co-immunoprecipitation experiments revealed that Gravin and AKAP79 do not associate with adenylyl cyclase type 2 (AC2) but bind to AC4/6, which interacts with calcium-dependent PKCs ,/, both in young and senescent fibroblasts. When the expression of Gravin and AKAP79 was blocked by small interference RNA transfection, the basal level of cAMP was greatly reduced and the cAMP status after LPA treatment was also reversed. Protein kinase A showed a similar pattern in terms of its basal activity and LPA-dependent modulation. These data suggest that Gravin and to a lesser extent, AKAP79, may play important roles in maintaining the basal AC activity and in coupling the AC systems to inhibitory signals such as Gi, in young cells, and to stimulatory signals such as PKCs in senescent cells. This study also demonstrates that Gravin is especially important for the long-term activation of PKC by LPA in senescent cells. We conclude that LPA-dependent increased level of cAMP in senescent human diploid fibroblasts is associated with increases in Gravin levels resulting in its increased binding with and activation of calcium-dependent PKC ,/, and AC4/6. [source] Regulation of Tyrosine Hydroxylase Activity and Phosphorylation at Ser19 and Ser40 via Activation of Glutamate NMDA Receptors in Rat StriatumJOURNAL OF NEUROCHEMISTRY, Issue 6 2000Niklas Lindgren Abstract: The activity of tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of dopamine, is stimulated by phosphorylation. In this study, we examined the effects of activation of NMDA receptors on the state of phosphorylation and activity of tyrosine hydroxylase in rat striatal slices. NMDA produced a time-and concentration-dependent increase in the levels of phospho-Ser19 -tyrosine hydroxylase in nigrostriatal nerve terminals. This increase was not associated with any changes in the basal activity of tyrosine hydroxylase, measured as DOPA accumulation. Forskolin, an activator of adenylyl cyclase, stimulated tyrosine hydroxylase phosphorylation at Ser40 and caused a significant increase in DOPA accumulation. NMDA reduced forskolin-mediated increases in both Ser40 phosphorylation and DOPA accumulation. In addition, NMDA reduced the increase in phospho-Ser40 -tyrosine hydroxylase produced by okadaic acid, an inhibitor of protein phosphatase 1 and 2A, but not by a cyclic AMP analogue, 8-bromo-cyclic AMP. These results indicate that, in the striatum, glutamate decreases tyrosine hydroxylase phosphorylation at Ser40 via activation of NMDA receptors by reducing cyclic AMP production. They also provide a mechanism for the demonstrated ability of NMDA to decrease tyrosine hydroxylase activity and dopamine synthesis. [source] Influence of progesterone on myometrial contractility in pregnant mice treated with lipopolysaccharideJOURNAL OF OBSTETRICS AND GYNAECOLOGY RESEARCH (ELECTRONIC), Issue 6 2007Hiroshi Anbe Abstract Aim:, To evaluate the effect of progesterone on interleukin (IL)-6, prostaglandin (PG) E2 and nitric oxide (NO) metabolite (NOx) production and contractile activity by NO in pregnant mice treated with lipopolysaccharide (LPS). Methods:, Pregnant C57BL mice on day 14 of gestation were killed 6 h after i.p. injection of LPS (400 ,g/kg) or vehicle. Progesterone (2 mg) was subcutaneously injected 2 h before LPS treatment. Uterine rings were equilibrated in Krebs-Henseleit solution (37°C) bubbled with 20% O2 and 5% CO2 (pH 7.4) for sampling and isometric tension recording. IL-6, PGE2 and NOx productions were measured from the bathing solution. Changes in spontaneous contractile activity in response to cumulative concentrations of l -arginine, diethylamine/nitric oxide (DEA/NO, the NO donor), and 8-bromo-cGMP (8-br-cGMP) were compared. Integral contractile activity over 10 min after each concentration was calculated and expressed as percentage change from basal activity. Statistical analyses were performed using one-way anova followed by Dunnett's test (significance was defined as P < 0.05). Results:, Interleukin-6 (34.7 ± 6.0 pg/g tissue), PGE2 (66.8 ± 6.7 pg/g tissue) and NOx (51.0 ± 5.4 pmol/2 mL/g wet tissue) production were significantly stimulated by LPS treatment (138.2 ± 23.2, 147.0 ± 29.0, 98.6 ± 16.2, respectively; P < 0.05). l -arginine, DEA/NO and 8-br-cGMP concentration-dependently inhibited spontaneous contractions in uterine rings both in LPS-treated and -untreated animals. Treatment with LPS significantly attenuated the maximal inhibition induced by l -arginine, DEA/NO and 8-br-cGMP in uterine rings from pregnant mice. Progesterone significantly decreased the levels of IL-6 production (74.9 ± 12.1, P < 0.05), but not PGE2 and NOx production, and contractile responses by l -arginine, DEA/NO and 8-br-cGMP. Conclusions:, The administration of LPS is associated with increases in IL-6, PGE2 and NO, and these increases may or may not have a role to play in LPS-induced preterm labor. Progesterone reduced the LPS-induced increase in IL-6 production and this may be one of the ways that progesterone reduces the risk of preterm labor. [source] Peroxisome Proliferator-Activated Receptors (PPAR) and the Mitochondrial Aldehyde Dehydrogenase (ALDH2) Promoter In Vitro and In VivoALCOHOLISM, Issue 7 2001David W. Crabb Background : The aldehyde dehydrogenase 2 (ALDH2) promoter contains a nuclear receptor response element (NRRE) that represents an overlapping direct repeat-1 (DR-1) and -5 (DR-5) element. Because DR-1 elements are preferred binding sites for peroxisome proliferator-activated receptors (PPARs), we tested the hypothesis that PPARs regulate ALDH2 expression. Methods: We examined the ability of PPAR isoforms to bind to the ALDH2 NRRE in electrophoretic mobility shift assays, their ability to activate the transcription of promoter-reporter constructs containing this NRRE, the effect of PPAR ligands on ALDH2 expression in liver, and the role of the PPAR, on the expression of ALDH2 by using PPAR,-null mice. Results: In vitro translated PPARs bound the ALDH NRRE with high affinity. Mutation of the NRRE indicated that binding was mediated by the DR-1 element. Cotransfection of PPAR expression plasmids showed that PPAR, had no effect on expression of heterologous promoter constructs containing the NRRE. PPAR, slightly induced expression, whereas PPAR, repressed basal activity of the promoter and blocked induction by hepatocyte nuclear factor 4. Treatment of rats with the PPAR ligand clofibrate repressed expression of ALDH2 in rats fed either stock rodent chow or a low-protein diet. Consistent with the transfection data, expression of ALDH2 protein was not different in PPAR,-null mice. Treatment of the mice with the PPAR, agonist WY14643 slightly decreased the level of ALDH2 protein in both wild-type and PPAR,-null mice, suggesting that the effect of WY14643 was not mediated by the receptor. Conclusions: These data indicate that ALDH2 is not part of the battery of lipid metabolizing enzymes and proteins regulated by PPAR, [source] Potential Use of Biomarkers in Zooplankton as Early Warning Signals of Ecotoxicological Risk in the Marine Food ChainMARINE ECOLOGY, Issue 2002Roberta Minutoli Abstract. Zooplankton is an essential component of the marine and brackish food chains. The ecotoxicological risk of zooplanktonic communities, estimated by the modern methodological approach of biomarkers, can be used as an early warning signal of ecosystem health. The aim of this project is to estimate the potential use of several biomarkers (esterases, mixed function oxidases, porphyrins) in zooplanktonic organisms. Studies were carried out with different zooplanktonic crustaceans: the copepods Acartia margalefi and Acartia latisetosa collected in Ganzirri Lake (Messina); the mysid Siriella clausi collected in Faro Lake (Messina); the mysids Diamysis bahirensis, Siriella armata and Mysidopsis gibbosa collected in Stagnone di Marsala (Palermo); the Antarctic euphausiids Euphausia crystallorophias and Euphausia superba; the am-phipod Streetsia challengeri and the euphausiid Meganycthiphanes norvegica collected after a shore-stranding along Messina's Ionian coast. Moreover, experiments were carried out with the benthic decapods Eriphia verrucosa and Pachygrapsus marmoratus from a rocky shore of Messina's Ionian coast. Acetylcholinesterase activity (AChE) was determined in homogenates of whole organisms. The key result of this project concerns the different AChE activity basal values of different crustacean species. Particular attention should be paid to the difference in basal activity found between the Antarctic and the Mediterranean species. [source] The HOG MAP kinase pathway is required for the induction of methylglyoxal-responsive genes and determines methylglyoxal resistance in Saccharomyces cerevisiaeMOLECULAR MICROBIOLOGY, Issue 1 2005Jaime Aguilera Summary A sudden overaccumulation of methylglyoxal (MG) induces, in Saccharomyces cerevisiae, the expression of MG-protective genes, including GPD1, GLO1 and GRE3. The response is partially dependent on the transcriptional factors Msn2p/Msn4p, but unrelated with the general stress response mechanism. Here, we show that the high-osmolarity glycerol (HOG)-pathway controls the genetic response to MG and determines the yeast growth capacity upon MG exposure. Strains lacking the MAPK Hog1p, the upstream component Ssk1p or the HOG-dependent nuclear factor Msn1p, showed a reduction in the mRNA accumulation of MG-responsive genes after MG addition. Moreover, hyperactivation of Hog1p by deletion of protein phosphatase PTP2 enhanced the response, while blocking the pathway by deletion of the MAPKK PBS2 had a negative effect. In addition, the activity of Hog1p affected the basal level of GPD1 mRNA under non-inducing conditions. These effects had a great influence on MG resistance, as hog1, and other HOG-pathway mutants with impaired MG-specific expression displayed MG sensitivity, whereas those with enhanced expression exhibited MG resistance as compared with the wild-type. However, MG does not trigger the overphosphorylation of Hog1p or its nuclear import in the parental strain. Moreover, dual phosphorylation of Hog1p appears to be dispensable in the triggering of the transcriptional response, although a phosphorylable form of Hog1p is fundamental for the transcriptional activity. Overall, our results suggest that the basal activity of the HOG-pathway serves to amplify the expression of MG-responsive genes under non-inducing and inducing conditions, ensuring cell protection against this toxic glycolytic by-product. [source] Inhibition of NF-,B activation with designed ankyrin-repeat proteins targeting the ubiquitin-binding/oligomerization domain of NEMOPROTEIN SCIENCE, Issue 9 2007Emanuel Wyler Abstract The link between the NF-,B signal transduction pathway and cancer is now well established. Inhibiting this pathway is therefore a promising approach in the treatment of certain cancers through a pro-apoptotic effect in malignant cells. Owing to its central role in the pathway, the I,B kinase (IKK) complex is a privileged target for designing inhibitors. Previously, we showed that oligomerization of NEMO is necessary for IKK activation and defined a minimal oligomerization domain (CC2-LZ) for NEMO, and we developed NEMO peptides inhibiting NF-,B activation at the level of the IKK complex. To improve the low-affinity inhibitors, we used ribosome display to select small and stable proteins with high affinity against the individual CC2-LZ because the entire NEMO protein is poorly soluble. Several binders with affinities in the low nanomolar range were obtained. When expressed in human cells, some of the selected molecules, despite their partial degradation, inhibited TNF-,-mediated NF-,B activation while having no effect on the basal activity. Controls with a naive library member or null plasmid had no effect. Furthermore, we could show that this NF-,B inhibition occurs through a specific interaction between the binders and the endogenous NEMO, resulting in decreased IKK activation. These results indicate that in vitro selections with the NEMO subdomain alone as a target may be sufficient to lead to interesting compounds that are able to inhibit NF-,B activation. [source] Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by G,iGDP and G,,THE JOURNAL OF PHYSIOLOGY, Issue 14 2009Moran Rubinstein G protein activated K+ channels (GIRK, Kir3) are switched on by direct binding of G,, following activation of Gi/o proteins via G protein-coupled receptors (GPCRs). Although G,i subunits do not activate GIRKs, they interact with the channels and regulate the gating pattern of the neuronal heterotetrameric GIRK1/2 channel (composed of GIRK1 and GIRK2 subunits) expressed in Xenopus oocytes. Coexpressed G,i3 decreases the basal activity (Ibasal) and increases the extent of activation by purified or coexpressed G,,. Here we show that this regulation is exerted by the ,inactive' GDP-bound G,i3GDP and involves the formation of G,i3,, heterotrimers, by a mechanism distinct from mere sequestration of G,,,away' from the channel. The regulation of basal and G,,-evoked current was produced by the ,constitutively inactive' mutant of G,i3, G,i3G203A, which strongly binds G,,, but not by the ,constitutively active' mutant, G,i3Q204L, or by G,,-scavenging proteins. Furthermore, regulation by G,i3G203A was unique to the GIRK1 subunit; it was not observed in homomeric GIRK2 channels. In vitro protein interaction experiments showed that purified G,, enhanced the binding of G,i3GDP to the cytosolic domain of GIRK1, but not GIRK2. Homomeric GIRK2 channels behaved as a ,classical' G,, effector, showing low Ibasal and strong G,,-dependent activation. Expression of G,i3G203A did not affect either Ibasal or G,,-induced activation. In contrast, homomeric GIRK1* (a pore mutant able to form functional homomeric channels) exhibited large Ibasal and was poorly activated by G,,. Expression of G,i3GDP reduced Ibasal and restored the ability of G,, to activate GIRK1*, like in GIRK1/2. Transferring the unique distal segment of the C terminus of GIRK1 to GIRK2 rendered the latter functionally similar to GIRK1*. These results demonstrate that GIRK1 containing channels are regulated by both G,i3GDP and G,,, while GIRK2 is a G,,-effector insensitive to G,i3GDP. [source] Selective expression of connective tissue growth factor in fibroblasts in vivo promotes systemic tissue fibrosisARTHRITIS & RHEUMATISM, Issue 5 2010Sonali Sonnylal Objective Connective tissue growth factor (CTGF) is a cysteine-rich secreted matricellular protein involved in wound healing and tissue repair. Enhanced and prolonged expression of CTGF has been associated with tissue fibrosis in humans. However, questions remain as to whether CTGF expression alone is sufficient to drive fibrosis. This study was undertaken to investigate whether CTGF alone is sufficient to cause fibrosis in intact animals and whether its effects are mediated through activation of transforming growth factor , (TGF,) signaling or through distinct signal transduction pathways. Methods We generated mice overexpressing CTGF in fibroblasts under the control of the fibroblast-specific collagen ,2(I) promoter enhancer. Tissues such as skin, lung, and kidney were harvested for histologic analysis. Mouse embryonic fibroblasts were prepared from embryos (14.5 days postcoitum) for biochemical analysis. Results Mice overexpressing CTGF in fibroblasts were susceptible to accelerated tissue fibrosis affecting the skin, lung, kidney, and vasculature, most notably the small arteries. We identified a marked expansion of the myofibroblast cell population in the dermis. RNA analysis of transgenic dermal fibroblasts revealed elevated expression of key matrix genes, consistent with a fibrogenic response. CTGF induced phosphorylation of p38, ERK-1/2, JNK, and Akt, but not Smad3, in transgenic mouse fibroblasts compared with wild-type mouse fibroblasts. Transfection experiments showed significantly increased basal activity of the CTGF and serum response element promoters, and enhanced induction of the CTGF promoter in the presence of TGF,. Conclusion These results demonstrate that selective expression of CTGF in fibroblasts alone causes tissue fibrosis in vivo through specific signaling pathways, integrating cues from the extracellular matrix into signal transduction pathways to orchestrate pivotal biologic responses relevant to tissue repair and fibrosis. [source] Hypoxia and glucocorticoid signaling converge to regulate macrophage migration inhibitory factor gene expressionARTHRITIS & RHEUMATISM, Issue 8 2009Laura M. Elsby Objective Macrophage migration inhibitory factor (MIF) is a proinflammatory mediator involved in the pathogenesis of rheumatoid arthritis. This study was undertaken to identify the MIF promoter elements responsible for regulating gene expression. Methods Luciferase reporter gene assays were used to identify the MIF promoter sequence responsible for basal activity. Bioinformatic analysis was used to predict transcription factor binding sites, and electrophoretic mobility shift assay (EMSA) was used to demonstrate transcription factor binding. Chromatin immunoprecipitation (ChIP) was used to demonstrate transcription factor loading on the MIF promoter. Results We identified the minimal promoter sequence required for basal MIF promoter activity that was also capable of conferring glucocorticoid-dependent inhibition in a T lymphocyte model cell line. Deletion studies and EMSA revealed 2 elements in the MIF promoter that were responsible for basal promoter activity. The 5, element binds CREB/activating transcription factor 1, and the 3, element is a functional hypoxia-responsive element binding hypoxia-inducible factor 1,. Further studies demonstrated that the cis elements are both required for glucocorticoid-dependent inhibition. ChIP demonstrated glucocorticoid-dependent recruitment of glucocorticoid receptor , to the MIF promoter in lymphocytes within 1 hour of treatment and a concomitant decrease in acetylated histone H3. Conclusion Our findings indicate that hypoxia and glucocorticoid signaling converge on a single element regulating MIF; this regulatory unit is a potential interacting node for microenvironment sensing of oxygen tension and glucocorticoid action in foci of inflammation. [source] MOUSE STRAIN-SPECIFIC DIFFERENCES IN CARDIAC METABOLIC ENZYME ACTIVITIES OBSERVED IN A MODEL OF ISOPROTERENOL-INDUCED CARDIAC HYPERTROPHYCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 1-2 2007Michael D Faulx SUMMARY 1Alterations in myocardial energy metabolism accompany pressure overload-induced hypertrophy. We previously described a novel model of catecholamine-induced hypertrophy in which A/J mice exhibit more robust cardiac hypertrophy than B6 mice. Accordingly, we assessed the influence of mouse strain on the activities of key myocardial metabolic enzymes and whether there are strain-related metabolic adaptations to short-term, high-dose isoproterenol (ISO) administration. 2Thirty-nine male mice (19 A/J mice, 20 B6 mice), aged 12,15 weeks, were randomly assigned to receive either ISO (100 mg/kg, s.c.) or vehicle (sterile water) daily for 5 days. On Day 6, all hearts were excised, weighed, freeze clamped and assayed for pyruvate dehydrogenase (PDH), medium chain acyl-CoA dehydrogenase, carnitine palmitoyl transferase I and citrate synthase activities. Plasma fatty acids (FA) were also measured. 3The ISO-treated A/J mice demonstrated greater percentage increases in gravimetric heart weight/bodyweight ratio than ISO-treated B6 mice (24 vs 3%, respectively; P < 0.001). All enzyme activities were significantly greater in vehicle-treated B6 mice than in A/J mice, illustrating a greater capacity for aerobic metabolism in B6 mice. Administration of ISO reduced PDHa (active form) activity in B6 mice by 47% (P < 0.001), with no significant change seen in A/J mice. Free FA levels were not significantly different between groups; thus, the differences in PDHa were not due to changes in FA. 4The basal activity of myocardial metabolic enzymes is greater in B6 mice than in A/J mice and ISO alters myocardial PDH activity in a mouse strain-dependent manner. Compared with A/J mice, B6 mice demonstrate less ISO-induced cardiac hypertrophy, but greater activity of key enzymes regulating FA and carbohydrate oxidation, which may protect against the development of hypertrophy. The metabolic adaptations associated with ISO-induced hypertrophy differ from those reported with pressure overload hypertrophy. [source] | ||||||||||||||||