5-alpha-reductase: 2012-09-23

2012年9月29日星期六

PPAR? prevents radiation-induced proinflammatory responses in microglia via transrepression of NF-?B and inhibition of the PKC?/MEK1/2/ERK1/2/AP-1 pathway.

PPAR? prevents radiation-induced proinflammatory responses in microglia via transrepression of NF-?B and inhibition of the PKC?/MEK1/2/ERK1/2/AP-1 pathway.

Free Radic Biol Med. 2012 May 1;52(9):1734-43

Authors: Schnegg CI, Kooshki M, Hsu FC, Sui G, Robbins ME

Abstract
Partial or whole-brain irradiation is often required to treat both primary and metastatic brain cancer. Radiation-induced normal tissue injury, including progressive cognitive impairment, however, can significantly affect the well-being of the approximately 200,000 patients who receive these treatments each year in the United States. Although the exact mechanisms underlying radiation-induced late effects remain unclear, oxidative stress and inflammation are thought to play a critical role. Microglia are key mediators of neuroinflammation. Peroxisomal proliferator-activated receptor (PPAR) ? has been shown to be a potent regulator of anti-inflammatory responses. Thus, we hypothesized that PPAR? activation would modulate the radiation-induced inflammatory response in microglia. Incubating BV-2 murine microglial cells with the PPAR? agonist L-165041 prevented the radiation-induced increase in: (i) intracellular reactive oxygen species generation, (ii) Cox-2 and MCP-1 expression, and (iii) IL-1? and TNF-? message levels. This occurred, in part, through PPAR?-mediated modulation of stress-activated kinases and proinflammatory transcription factors. PPAR? inhibited NF-?B via transrepression by physically interacting with the p65 subunit and prevented activation of the PKC?/MEK1/2/ERK1/2/AP-1 pathway by inhibiting the radiation-induced increase in intracellular reactive oxygen species generation. These data support the hypothesis that PPAR? activation can modulate radiation-induced oxidative stress and inflammatory responses in microglia.

PMID: 22387176 [PubMed - indexed for MEDLINE]

chir-258 dovitinib dna-pk

Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography.

Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography.

Mutat Res. 2012 Oct;751(2):158-246

Authors: Thompson LH

Abstract
The faithful maintenance of chromosome continuity in human cells during DNA replication and repair is critical for preventing the conversion of normal diploid cells to an oncogenic state. The evolution of higher eukaryotic cells endowed them with a large genetic investment in the molecular machinery that ensures chromosome stability. In mammalian and other vertebrate cells, the elimination of double-strand breaks with minimal nucleotide sequence change involves the spatiotemporal orchestration of a seemingly endless number of proteins ranging in their action from the nucleotide level to nucleosome organization and chromosome architecture. DNA DSBs trigger a myriad of post-translational modifications that alter catalytic activities and the specificity of protein interactions: phosphorylation, acetylation, methylation, ubiquitylation, and SUMOylation, followed by the reversal of these changes as repair is completed. "Superfluous" protein recruitment to damage sites, functional redundancy, and alternative pathways ensure that DSB repair is extremely efficient, both quantitatively and qualitatively. This review strives to integrate the information about the molecular mechanisms of DSB repair that has emerged over the last two decades with a focus on DSBs produced by the prototype agent ionizing radiation (IR). The exponential growth of molecular studies, heavily driven by RNA knockdown technology, now reveals an outline of how many key protein players in genome stability and cancer biology perform their interwoven tasks, e.g. ATM, ATR, DNA-PK, Chk1, Chk2, PARP1/2/3, 53BP1, BRCA1, BRCA2, BLM, RAD51, and the MRE11-RAD50-NBS1 complex. Thus, the nature of the intricate coordination of repair processes with cell cycle progression is becoming apparent. This review also links molecular abnormalities to cellular pathology as much a possible and provides a framework of temporal relationships.

PMID: 22743550 [PubMed - in process]

zm-447439 rad001 ecdysone

ZM447439, a novel promising aurora kinase inhibitor, provokes antiproliferative and proapoptotic effects alone and in combination with bio- and chemotherapeutic agents in gastroenteropancreatic neuroendocrine tumor cell lines.

ZM447439, a novel promising aurora kinase inhibitor, provokes antiproliferative and proapoptotic effects alone and in combination with bio- and chemotherapeutic agents in gastroenteropancreatic neuroendocrine tumor cell lines.

Neuroendocrinology. 2010;91(2):121-30

Authors: Georgieva I, Koychev D, Wang Y, Holstein J, Hopfenm�ller W, Zeitz M, Grabowski P

Abstract
Background: Therapeutic approaches to gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are still not satisfactory. A new direction in treatment options could be the novel aurora kinase inhibitor ZM447439, which was previously reported to interfere with the mitotic spindle integrity checkpoint and chromosome segregation, but does not interfere with other kinases when used up to 5 muM. Methods: We evaluated the antineoplastic effects of ZM447439 on growth and apoptosis of the GEP-NET cell lines BON, QGP-1 and MIP-101, representing the different malignant tumor types, using standard cell biological tests as crystal violet assays, caspase activation, DNA fragmentation and cell cycle analysis. Results: ZM447439 dose-dependently inhibited proliferation of all three cell lines with IC(50) values in the nanomolar to low micromolar range. Moreover, aurora kinase inhibition by ZM447439 potently induced apoptosis, which was accompanied by DNA fragmentation and caspase 3 and 7 activation. Furthermore, we observed cell cycle arrest at G(0)/G(1) phase as well as a block in G(2)/M transition. In addition, combined treatment with the chemotherapeutic agents streptozocin and cisplatin augmented significantly the antiproliferative effects of those agents. Conclusion: Aurora kinase inhibition by ZM447439 seems to be a promising new therapeutic approach in GEP-NETs, which should be evaluated in further clinical trials.

PMID: 19923785 [PubMed - indexed for MEDLINE]

dna-pk coxinhibitors c-met inhibitors

Low polydispersity (N-ethyl pyrrolidine methacrylamide-co-1-vinylimidazole) linear oligomers for gene therapy applications.

Low polydispersity (N-ethyl pyrrolidine methacrylamide-co-1-vinylimidazole) linear oligomers for gene therapy applications.

Eur J Pharm Biopharm. 2012 Aug 23;

Authors: Velasco D, R�thor� G, Newland B, Parra J, Elvira C, Pandit A, Rojo L, San Rom�n J

Abstract
Nonviral methods for gene delivery are becoming ever more prevalent along with the need to design new vectors that are highly effective, stable in biological fluids, inexpensive, and facile to produce. Here, we synthesize our previously reported monomer N-ethyl pyrrolidine methacrylamide (EPA) and evaluate its effectiveness in gene vector applications when copolymerized with 1-vinylimidazole (VI). A range of these novel linear cationic copolymers were synthesized via free radical polymerization with low molecular weights (oligomers) and low polydispersities showing two pK(a) values as the two co-monomers are cationic. DNA-polymer polyplexes had average sizes between 100 and 250nm and zeta-potentials between 10 and 25mV, and a strong dependence of composition on the size on the zeta-potential was observed. The cytotoxicity of the homopolymers, oligomers, and polyplexes toward human fibroblasts and 3T3 mouse fibroblasts was evaluated using the MTT and AlamarBlue? assays, proving that formulations could be made with toxicity as low as low molecular weight linear poly (dimethylaminoethyl methacrylate) (PDMAEMA). The transfection capability of the polyplexes measured using the G-luciferase marker gene far superseded PDMAEMA when evaluated in biological conditions. Furthermore, blood compatibility studies showed that these new oligomers exhibit no significant hemolysis or platelet activation above PBS controls. These new EPA based oligomers with low toxicity and ease of scalability show high transfection abilities in serum conditions, and blood compatibility showing its potential for systemic gene delivery applications.

PMID: 22952108 [PubMed - as supplied by publisher]

c-met inhibitors zm-447439 rad001

Conserved microRNA miR-8 controls body size in response to steroid signaling in Drosophila.

Conserved microRNA miR-8 controls body size in response to steroid signaling in Drosophila.

Genes Dev. 2012 Jul 1;26(13):1427-32

Authors: Jin H, Kim VN, Hyun S

Abstract
Body size determination is a process that is tightly linked with developmental maturation. Ecdysone, an insect maturation hormone, contributes to this process by antagonizing insulin signaling and thereby suppressing juvenile growth. Here, we report that the microRNA miR-8 and its target, u-shaped (USH), a conserved microRNA/target axis that regulates insulin signaling, are critical for ecdysone-induced body size determination in Drosophila. We found that the miR-8 level is reduced in response to ecdysone, while the USH level is up-regulated reciprocally, and that miR-8 is transcriptionally repressed by ecdysone's early response genes. Furthermore, modulating the miR-8 level correlatively changes the fly body size; either overexpression or deletion of miR-8 abrogates ecdysone-induced growth control. Consistently, perturbation of USH impedes ecdysone's effect on body growth. Thus, miR-8 acts as a molecular rheostat that tunes organismal growth in response to a developmental maturation signal.

PMID: 22751499 [PubMed - indexed for MEDLINE]

chir-258 dovitinib dna-pk

2012年9月28日星期五

Receptor binding kinetics and cellular responses of six N-formyl peptide agonists in human neutrophils.

Receptor binding kinetics and cellular responses of six N-formyl peptide agonists in human neutrophils.

Biochemistry. 2004 Jun 29;43(25):8204-16

Authors: Waller A, Sutton KL, Kinzer-Ursem TL, Absood A, Traynor JR, Linderman JJ, Omann GM

Abstract
The goal of this study was to elucidate the relationships between early ligand binding/receptor processing events and cellular responses for the N-formyl peptide receptor system on human neutrophils as a model of a GPCR system in a physiologically relevant context. Binding kinetics of N-formyl-methionyl-leucyl-phenylalanyl-phenylalanyl-lysine-fluorescein and N-formyl-valyl-leucyl-phenylalanyl-lysine-fluorescein to the N-formyl peptide receptor on human neutrophils were characterized and combined with previously published binding data for four other ligands. Binding was best fit by an interconverting two-receptor state model that included a low affinity receptor state that converted to a high affinity state. Response behaviors elicited at 37 degrees C by the six different agonists for the N-formyl peptide receptor were measured. Dose response curves for oxidant production, actin polymerization, and G-protein activation were obtained for each ligand; whereas all ligands showed equal efficacy for all three responses, the ED(50) values varied as much as 7000-fold. The level of agonism and rank order of potencies of ligands for actin and oxidant responses were the same as for the G-protein activation assay, suggesting that the differences in abilities of ligands to mediate responses were determined upstream of G-protein activation at the level of ligand-receptor interactions. The rate constants governing ligand binding and receptor affinity conversion were ligand-dependent. Analysis of the forward and reverse rate constants governing binding to the proposed signaling receptor state showed that it was of a similar energy for all six ligands, suggesting the hypothesis that ligand efficacy is dictated by the energy state of this ligand-receptor complex. However, the interconverting two-receptor state model was not sufficient to predict response potency, suggesting the presence of receptor states not discriminated by the binding data.

PMID: 15209517 [PubMed - indexed for MEDLINE]

find out more info GPCR Signaling G-protein Receptors

Yeast-based fluorescence reporter assay of G protein-coupled receptor signalling for flow cytometric screening: FAR1-disruption recovers loss of episomal plasmid caused by signalling in yeast.

Yeast-based fluorescence reporter assay of G protein-coupled receptor signalling for flow cytometric screening: FAR1-disruption recovers loss of episomal plasmid caused by signalling in yeast.

J Biochem. 2008 May;143(5):667-74

Authors: Ishii J, Tanaka T, Matsumura S, Tatematsu K, Kuroda S, Ogino C, Fukuda H, Kondo A

Abstract
Here, we describe a yeast-based fluorescence reporter assay for G protein-coupled receptor (GPCR) signalling using a flow cytometer (FCM). The enhanced green fluorescent protein (EGFP) gene was integrated into the FUS1 locus as a reporter gene. The engineered yeast was able to express the EGFP in response to ligand stimulation. Gene-disrupted yeast strains were constructed to evaluate the suitability of the yeast-based fluorescence screening system for heterologous GPCR. When receptor was expressed by episomal plasmid, the proportion of the signalling-activated cells in response to ligand stimulation decreased significantly. The GPCR-signalling-activated and non-activated cell clusters were individually isolated by analysing the fluorescence intensity at the single-cell level with FCM, and it was found that the plasmid retention rate decays markedly in the non-activated cell cluster. We attributed the loss of plasmid to G1 arrest in response to signalling, and successfully improved the plasmid retention rate by disrupting the FAR1 gene and avoiding cell cycle arrest. Our system will be a powerful tool for the quantitative and high-throughput GPCR screening of yeast-based combinatorial libraries using FCM.

PMID: 18281298 [PubMed - indexed for MEDLINE]

G-protein Receptors gpcr pathway NF-κB

Trichoderma G protein-coupled receptors: functional characterisation of a cAMP receptor-like protein from Trichoderma atroviride.

Trichoderma G protein-coupled receptors: functional characterisation of a cAMP receptor-like protein from Trichoderma atroviride.

Curr Genet. 2008 Dec;54(6):283-99

Authors: Brunner K, Omann M, Pucher ME, Delic M, Lehner SM, Domnanich P, Kratochwill K, Druzhinina I, Denk D, Zeilinger S

Abstract
Galpha subunits act to regulate vegetative growth, conidiation, and the mycoparasitic response in Trichoderma atroviride. To extend our knowledge on G protein signalling, we analysed G protein-coupled receptors (GPCRs). As the genome sequence of T. atroviride is not publicly available yet, we carried out an in silico exploration of the genome database of the close relative T. reesei. Twenty genes encoding putative GPCRs distributed over eight classes and additional 35 proteins similar to the Magnaporthe grisea PTH11 receptor were identified. Subsequently, four T. atroviride GPCR-encoding genes were isolated and affiliated to the cAMP receptor-like family by phylogenetic and topological analyses. All four genes showed lowest expression on glycerol and highest mRNA levels upon carbon starvation. Transcription of gpr3 and gpr4 responded to exogenously added cAMP and the shift from liquid to solid media. gpr3 mRNA levels also responded to the presence of fungal hyphae or cellulose membranes. Further characterisation of mutants bearing a gpr1-silencing construct revealed that Gpr1 is essential for vegetative growth, conidiation and conidial germination. Four genes encoding the first GPCRs described in Trichoderma were isolated and their expression characterized. At least one of these GPCRs is important for several cellular processes, supporting the fundamental role of G protein signalling in this fungus.

PMID: 18836726 [PubMed - indexed for MEDLINE]

find out more info GPCR Signaling G-protein Receptors

[Research progress of the biological characteristics of IkappaB kinase and its inhibitors].

[Research progress of the biological characteristics of IkappaB kinase and its inhibitors].

Yao Xue Xue Bao. 2011 Mar;46(3):253-60

Authors: Xue JY, Zhou B, Zhang DY, Wu XM

Abstract
The NF-kappaB pathway regulates the expression of over 150 target genes, e.g., cytokines, chemokines, leukocyte adhesion molecules and inducible effector enzymes. Consequently, it plays a crucial role in innate and adaptive immune responses, inflammatory response, stress responses, apoptosis and so on. IkappaB kinase (IKK) is the key of this pathway, and it owns a special structure which consists of catalytic subunit and regulatory subunit. Naturally, the activation of IKK needs the interaction of the two subunits and phosphorylation by its upstream kinases. Actually, there are two methods of activation of the NF-kappaB pathway, and both of the methods need the IKK complex. Given to the crucial role of IKK, researchers have isolated and synthesized amounts of IKK inhibitors, and these provide a great convenience to develop novel anti-inflammatory and anti-tumor drugs.

PMID: 21626777 [PubMed - indexed for MEDLINE]

NF-kB signaling pathway NF-kB pathway NF-kB signaling

Engineering the melanocortin-4 receptor to control G(s) signaling in vivo.

Engineering the melanocortin-4 receptor to control G(s) signaling in vivo.

Ann N Y Acad Sci. 2003 Jun;994:225-32

Authors: Srinivasan S, Vaisse C, Conklin BR

Abstract
G-protein-coupled receptors (GPCRs) are the largest known family of cell surface receptors, and they control many important physiological events, including sensory perception, chemotaxis, neurotransmission, and energy homeostasis. However, GPCR signaling can be difficult to study in vivo because of the multitude of GPCRs, the lack of specific synthetic agonists, and the fact that some GPCRs activate multiple signaling pathways. One method to circumvent these problems is to develop an engineered receptor that is unresponsive to its endogenous agonist, yet can be fully activated by synthetic, small-molecule drugs. Such a receptor, called a receptor activated solely by a synthetic ligand (RASSL), can be rapidly and reversibly activated by a small-molecule drug and would be a powerful tool to control G-protein signaling in vivo. Here we present the development of a G(s)-coupled RASSL based on the melanocortin-4 receptor (MC4R). MC4R couples exclusively to G(s) at physiologically relevant concentrations of its endogenous ligand, alpha-melanocyte-stimulating hormone (alpha-MSH). Data from human patients and structure-activity studies have shown that several mutations in MC4R cause a decreased affinity for alpha-MSH and can be exploited for RASSL development. Synthetic, small-molecule agonists of MC4R are now available and can be used to activate mutated receptors in vivo. We are engineering a series of mutations in MC4R to remove the peptide-binding site while retaining small-molecule binding and activation. The MC4R G(s) RASSL could be used to control many physiological responses associated with G(s) signaling such as heart rate, energy homeostasis, and cell proliferation.

PMID: 12851320 [PubMed - indexed for MEDLINE]

2012年9月27日星期四

Engineered G-protein Coupled Receptors are Powerful Tools to Investigate Biological Processes and Behaviors.

Engineered G-protein Coupled Receptors are Powerful Tools to Investigate Biological Processes and Behaviors.

Front Mol Neurosci. 2009;2:16

Authors: Nichols CD, Roth BL

Abstract
Understanding how discreet tissues and neuronal circuits function in relation to the whole organism to regulate physiological processes and behaviors is a fundamental goal of modern biological science. Powerful and important new tools in this discovery process are modified G-protein coupled receptors (GPCRs) known as 'Receptors Activated Solely by Synthetic Ligands (RASSLs),' and 'Designer Receptors Exclusively Activated by a Designer Drug (DREADDs).' Collectively, these are GPCRs modified either through rational design (RASSLs) or directed molecular evolution (DREADDs), that do not respond to native ligand, but functionally respond only to synthetic ligands. Importantly, the utility of these receptors is not limited to examination of the role of GPCR-coupled effector signal transduction pathways. Due to the near ubiquitous expression of GPCRs throughout an organism, this technology, combined with whole animal transgenics to selectively target expression, has the ability to regulate activity of discreet tissues and neuronal circuits through effector pathway modulation to study function and behavior throughout the organism. Advantages over other systems currently used to modify in vivo function include the ability to rapidly, selectively and reversibly manipulate defined signal transduction pathways both in short term and long term studies, and no need for specialized equipment due to convenient systemic treatment with activating ligand.

PMID: 19893765 [PubMed]

NF-kB pathway NF-kB signaling NF-kappaB signaling pathway

Modifying ligand-induced and constitutive signaling of the human 5-HT4 receptor.

Modifying ligand-induced and constitutive signaling of the human 5-HT4 receptor.

PLoS One. 2007;2(12):e1317

Authors: Chang WC, Ng JK, Nguyen T, Pellissier L, Claeysen S, Hsiao EC, Conklin BR

Abstract
G protein-coupled receptors (GPCRs) signal through a limited number of G-protein pathways and play crucial roles in many biological processes. Studies of their in vivo functions have been hampered by the molecular and functional diversity of GPCRs and the paucity of ligands with specific signaling effects. To better compare the effects of activating different G-protein signaling pathways through ligand-induced or constitutive signaling, we developed a new series of RASSLs (receptors activated solely by synthetic ligands) that activate different G-protein signaling pathways. These RASSLs are based on the human 5-HT(4b) receptor, a GPCR with high constitutive G(s) signaling and strong ligand-induced G-protein activation of the G(s) and G(s/q) pathways. The first receptor in this series, 5-HT(4)-D(100)A or Rs1 (RASSL serotonin 1), is not activated by its endogenous agonist, serotonin, but is selectively activated by the small synthetic molecules GR113808, GR125487, and RO110-0235. All agonists potently induced G(s) signaling, but only a few (e.g., zacopride) also induced signaling via the G(q) pathway. Zacopride-induced G(q) signaling was enhanced by replacing the C-terminus of Rs1 with the C-terminus of the human 5-HT(2C) receptor. Additional point mutations (D(66)A and D(66)N) blocked constitutive G(s) signaling and lowered ligand-induced G(q) signaling. Replacing the third intracellular loop of Rs1 with that of human 5-HT(1A) conferred ligand-mediated G(i) signaling. This G(i)-coupled RASSL, Rs1.3, exhibited no measurable signaling to the G(s) or G(q) pathway. These findings show that the signaling repertoire of Rs1 can be expanded and controlled by receptor engineering and drug selection.

PMID: 18338032 [PubMed - indexed for MEDLINE]

NF-kB signaling pathway NF-kB pathway NF-kB signaling

Myometrial tumor necrosis factor-? receptors increase with gestation and labor and modulate gene expression through mitogen-activated kinase and nuclear factor-?B.

Myometrial tumor necrosis factor-? receptors increase with gestation and labor and modulate gene expression through mitogen-activated kinase and nuclear factor-?B.

Reprod Sci. 2012 Jan;19(1):43-54

Authors: Alexander HA, Sooranna SR, Myatt L, Johnson MR

Abstract
Previously, we found that myometrial tumor necrosis factor-? (TNF-?) messenger RNA (mRNA) expression did not increase with preterm or term labor. To further investigate the role of TNF-? in human labor, we studied TNF-? receptor (TNFR1A and B) expression, regulation, and associated intracellular signaling pathways in human myometrial samples obtained both before and after the onset of labor and in primary cultures of uterine smooth muscle cells (USMCs). We found that the mRNA expression of both receptors increased with advancing gestation and labor and protein levels of TNFR1B were significantly higher in term laboring myometrial samples than in nonlabor controls. Tumor necrosis factor- treatment of USMCs activated all mitogen-activated protein kinase (MAPK) subtypes and nuclear factor ?-B (NF-?B). The TNF-? induced increases in the expression of TNFR1B and prostaglandin H synthase type 2 were reduced by inhibitors of NF-?B and MAPKs, respectively. The TNF-? induced increase in interleukin 8 (IL-8) appeared to be independent of MAPK and NF-?B pathway. These data suggest that the uterus may become more sensitive to the action of TNF-? with advancing gestation and labor and that TNF-? acts via MAPK and NF-?B to promote labor-associated gene expression.

PMID: 22228740 [PubMed - indexed for MEDLINE]

gpcr pathway NF-κB NF-kB signaling pathway

G(i)-coupled GPCR signaling controls the formation and organization of human pluripotent colonies.

G(i)-coupled GPCR signaling controls the formation and organization of human pluripotent colonies.

PLoS One. 2009;4(11):e7780

Authors: Nakamura K, Salomonis N, Tomoda K, Yamanaka S, Conklin BR

Abstract
BACKGROUND: Reprogramming adult human somatic cells to create human induced pluripotent stem (hiPS) cell colonies involves a dramatic morphological and organizational transition. These colonies are morphologically indistinguishable from those of pluripotent human embryonic stem (hES) cells. G protein-coupled receptors (GPCRs) are required in diverse developmental processes, but their role in pluripotent colony morphology and organization is unknown. We tested the hypothesis that G(i)-coupled GPCR signaling contributes to the characteristic morphology and organization of human pluripotent colonies.
METHODOLOGY/PRINCIPAL FINDINGS: Specific and irreversible inhibition of G(i)-coupled GPCR signaling by pertussis toxin markedly altered pluripotent colony morphology. Wild-type hES and hiPS cells formed monolayer colonies, but colonies treated with pertussis toxin retracted inward, adopting a dense, multi-layered conformation. The treated colonies were unable to reform after a scratch wound insult, whereas control colonies healed completely within 48 h. In contrast, activation of an alternative GPCR pathway, G(s)-coupled signaling, with cholera toxin did not affect colony morphology or the healing response. Pertussis toxin did not alter the proliferation, apoptosis or pluripotency of pluripotent stem cells.
CONCLUSIONS/SIGNIFICANCE: Experiments with pertussis toxin suggest that G(i) signaling plays a critical role in the morphology and organization of pluripotent colonies. These results may be explained by a G(i)-mediated density-sensing mechanism that propels the cells radially outward. GPCRs are a promising target for modulating the formation and organization of hiPS and hES cell colonies and may be important for understanding somatic cell reprogramming and for engineering pluripotent stem cells for therapeutic applications.

PMID: 19936228 [PubMed - indexed for MEDLINE]

NF-κB NF-kB signaling pathway NF-kB pathway

G protein-coupled receptors and their signaling pathways: classical therapeutical targets susceptible to novel therapeutic concepts.

G protein-coupled receptors and their signaling pathways: classical therapeutical targets susceptible to novel therapeutic concepts.

Curr Pharm Des. 2004;10(16):1937-58

Authors: Liebmann C

Abstract
In recent years, new strategies in cancer therapy have been developed targeting key signaling molecules in the receptor tyrosine kinase signal transduction pathway. In contrast, most therapeutical concepts to manipulate G protein-coupled receptors (GPCR)-mediated disorders are still limited to the use of receptor-specific agonists or antagonists. Visible progress in the understanding of GPCR signaling complexity, especially the detection of several families of highly target- and cell-specific regulator proteins of GPCRs, G proteins, and effector components may open new horizons to develop novel therapeutical concepts targeting GPCR signaling elements. Thus, this review will focus on different molecular levels that may be of particular interest in terms of new drug development such as: (i) GPCR subtypes, allosteric binding sites, dimerization and constitutive activity, the use of RAMPs (receptor-activity-modifying proteins) and RASSLs (receptor activated solely by synthetic ligands); (ii) AGS (activators of G protein signaling) and RGS (regulators of G protein signaling) proteins which modify G protein activity; (iii) the high diversity of isozymes involved in the generation, signal transmission, and degradation of second messenger molecules.

PMID: 15180530 [PubMed - indexed for MEDLINE]

NF-kB signaling NF-kappaB signaling pathway NF-kB signaling pathway

2012年9月26日星期三

Modifying ligand-induced and constitutive signaling of the human 5-HT4 receptor.

Modifying ligand-induced and constitutive signaling of the human 5-HT4 receptor.

PLoS One. 2007;2(12):e1317

Authors: Chang WC, Ng JK, Nguyen T, Pellissier L, Claeysen S, Hsiao EC, Conklin BR

PMID: 18338032 [PubMed - indexed for MEDLINE]

NF-kB pathway NF-kB signaling NF-kappaB signaling pathway

Statistical sequence analyses of G-protein-coupled receptors: structural and functional characteristics viewed with periodicities of entropy, hydrophobicity, and volume.

Statistical sequence analyses of G-protein-coupled receptors: structural and functional characteristics viewed with periodicities of entropy, hydrophobicity, and volume.

Proteins. 2004 Sep 1;56(4):650-60

Authors: Imai T, Fujita N

Abstract
G-protein-coupled receptors (GPCRs) play a crucial role in signal transduction and receive a wide variety of ligands. GPCRs are a major target in drug design, as nearly 50% of all contemporary medicines act on GPCRs. GPCRs are membrane proteins possessing a common structural feature, seven transmembrane helices. In order to design an effective drug to act on a GPCR, knowledge of the three-dimensional (3D) structure of the target GPCR is indispensable. However, as GPCRs are membrane bound, their 3D structures are difficult to obtain. Thus we conducted statistical sequence analyses to find information about 3D structure and ligand binding using the receptors' primary sequences. We present statistical sequence analyses of 270 human GPCRs with regard to entropy (Shannon entropy in sequence alignment), hydrophobicity and volume, which are associated with the alpha-helical periodicity of the accessibility to the surrounding lipid. We found periodicity such that the phase changes once in the middle of each transmembrane region, both in the entropy plot and in the hydrophobicity plot. The phase shift in the entropy plot reflects the variety of ligands and the generality of the mechanism of signal transduction. The two periodic regions in the hydrophobicity plot indicate the regions facing the hydrophobic lipid chain and the polar phospholipid headgroup. We also found a simple periodicity in the plot of volume deviation, which suggests conservation of the stable structural packing among the transmembrane helices.

PMID: 15281118 [PubMed - indexed for MEDLINE]

NF-kB signaling pathway NF-kB pathway NF-kB signaling

Quantitative and dynamic analyses of G protein-coupled receptor signaling in yeast using Fus1, enhanced green fluorescence protein (EGFP), and His3 fusion protein.

Quantitative and dynamic analyses of G protein-coupled receptor signaling in yeast using Fus1, enhanced green fluorescence protein (EGFP), and His3 fusion protein.

Biotechnol Prog. 2006 Jul-Aug;22(4):954-60

Authors: Ishii J, Matsumura S, Kimura S, Tatematsu K, Kuroda S, Fukuda H, Kondo A

Abstract
The mechanism of G protein-coupled receptor (GPCR) signaling in yeasts is similar to that in mammalian cells. Therefore, yeasts can be used in GPCR assays, and several ligand detection systems using a pheromone signaling pathway in yeasts have been developed by employing yeasts with disrupted chromosomal genes that code for proteins producing specific effects. In this study, the construction of yeast strains that can detect ligand binding mediated by interactions between the G protein and GPCR using either fluorescence or auxotrophic selectivity is demonstrated. The strain was constructed by integrating the fusion gene of pheromone-responsive protein (FUS1), enhanced green fluorescence protein (EGFP), and auxotrophic marker protein (HIS3) into the FUS1 locus. Moreover, the influence of gene disruptions on the yeast signal transduction cascade is closely investigated with respect to both quantitative and dynamic aspects to further develop a high-throughput screening system for the GPCR assay using yeasts. Yeast strains with a disrupted SST2 gene, which is a member of the RGS (regulator of G protein signaling) family, and a disrupted FAR1 gene, which mediates cell cycle arrest in response to a pheromone, were monitored by measuring their fluorescence and growth rate. This method will be applicable to other comprehensive GPCR ligand screening methods.

PMID: 16889369 [PubMed - indexed for MEDLINE]

NF-kB signaling NF-kappaB signaling pathway read more

Engineered G-protein Coupled Receptors are Powerful Tools to Investigate Biological Processes and Behaviors.

Engineered G-protein Coupled Receptors are Powerful Tools to Investigate Biological Processes and Behaviors.

Front Mol Neurosci. 2009;2:16

Authors: Nichols CD, Roth BL

PMID: 19893765 [PubMed]

NF-kappaB signaling pathway read more learn more

2012年9月25日星期二

Techniques: Recent developments in computer-aided engineering of GPCR ligands using the human adenosine A3 receptor as an example.

Techniques: Recent developments in computer-aided engineering of GPCR ligands using the human adenosine A3 receptor as an example.

Trends Pharmacol Sci. 2005 Jan;26(1):44-51

Authors: Moro S, Spalluto G, Jacobson KA

Abstract
G-protein-coupled receptors (GPCRs) represent the largest known family of signal-transducing molecules, and convey signals for light and many extracellular regulatory molecules. GPCRs are dysfunctional or dysregulated in several human diseases and are estimated to be the targets of >40% of the drugs used in clinical medicine today. The crystal structure of rhodopsin provides the first information on the three-dimensional structure of GPCRs, which now supports homology modeling studies and structure-based drug-design approaches. In this article, we review recent work on adenosine receptors, a family of GPCRs, and, in particular, on adenosine A(3) receptor antagonists. We focus on an iterative, bi-directional approach in which models are used to generate hypotheses that are tested by experimentation; the experimental findings are, in turn, used to refine the model. The success of this approach is due to the synergistic interaction between theory and experimentation.

PMID: 15629204 [PubMed - indexed for MEDLINE]

NF-kB signaling pathway NF-kB pathway NF-kB signaling

Chimeric yeast G-protein ? subunit harboring a 37-residue C-terminal gustducin-specific sequence is functional in Saccharomyces cerevisiae.

Chimeric yeast G-protein ? subunit harboring a 37-residue C-terminal gustducin-specific sequence is functional in Saccharomyces cerevisiae.

Biosci Biotechnol Biochem. 2012;76(3):512-6

Authors: Hara K, Inada Y, Ono T, Kuroda K, Yasuda-Kamatani Y, Ishiguro M, Tanaka T, Misaka T, Abe K, Ueda M

Abstract
Despite many recent studies of G-protein-coupled receptor (GPCR) structures, it is not yet well understood how these receptors activate G proteins. The GPCR assay using baker's yeast, Saccharomyces cerevisiae, is an effective experimental model for the characterization of GPCR-G? interactions. Here, using the yeast endogenous G? protein (Gpa1p) as template, we constructed various chimeric G? proteins with a region that is considered to be necessary for interaction with mammalian receptors. The signaling assay using the yeast pheromone receptor revealed that the chimeric G? protein harboring 37 gustducin-specific amino acid residues at its C-terminus (GPA1/gust37) maintained functionality in yeast. In contrast, GPA1/gust44, a variant routinely used in mammalian experimental systems, was not functional.

PMID: 22451393 [PubMed - indexed for MEDLINE]

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Lamin B1 loss is a senescence-associated biomarker.

Lamin B1 loss is a senescence-associated biomarker.

Mol Biol Cell. 2012 Jun;23(11):2066-75

Authors: Freund A, Laberge RM, Demaria M, Campisi J

Abstract
Cellular senescence is a potent tumor-suppressive mechanism that arrests cell proliferation and has been linked to aging. However, studies of senescence have been impeded by the lack of simple, exclusive biomarkers of the senescent state. Senescent cells develop characteristic morphological changes, which include enlarged and often irregular nuclei and chromatin reorganization. Because alterations to the nuclear lamina can affect both nuclear morphology and gene expression, we examined the nuclear lamina of senescent cells. We show here than lamin B1 is lost from primary human and murine cell strains when they are induced to senesce by DNA damage, replicative exhaustion, or oncogene expression. Lamin B1 loss did not depend on the p38 mitogen-activated protein kinase, nuclear factor-?B, ataxia telangiectasia-mutated kinase, or reactive oxygen species signaling pathways, which are positive regulators of senescent phenotypes. However, activation of either the p53 or pRB tumor suppressor pathway was sufficient to induce lamin B1 loss. Lamin B1 declined at the mRNA level via a decrease in mRNA stability rather than by the caspase-mediated degradation seen during apoptosis. Last, lamin B1 protein and mRNA declined in mouse tissue after senescence was induced by irradiation. Our findings suggest that lamin B1 loss can serve as biomarker of senescence both in culture and in vivo.

PMID: 22496421 [PubMed - indexed for MEDLINE]

G-protein Receptors gpcr pathway NF-κB

G(i)-coupled GPCR signaling controls the formation and organization of human pluripotent colonies.

G(i)-coupled GPCR signaling controls the formation and organization of human pluripotent colonies.

PLoS One. 2009;4(11):e7780

Authors: Nakamura K, Salomonis N, Tomoda K, Yamanaka S, Conklin BR

PMID: 19936228 [PubMed - indexed for MEDLINE]

NF-kB pathway NF-kB signaling NF-kappaB signaling pathway

NF-?B and Hypoxia: A Double-Edged Sword in Atherosclerosis.

NF-?B and Hypoxia: A Double-Edged Sword in Atherosclerosis.

Am J Pathol. 2012 Sep 20;

Authors: Sun X, Feinberg MW

Abstract
This Commentary highlights the article by Fang et al, which describes novel mouse models of chronic intermittent hypoxia (CIH)-induced atherosclerosis, revealing that loss of the NF-?B p50 subunit increased atherosclerosis in the presence of CIH.

PMID: 22999810 [PubMed - as supplied by publisher]

2012年9月24日星期一

Engineered GPCRs as tools to modulate signal transduction.

Engineered GPCRs as tools to modulate signal transduction.

Physiology (Bethesda). 2008 Dec;23:313-21

Authors: Pei Y, Rogan SC, Yan F, Roth BL

Abstract
Different families of G-protein-coupled receptors (GPCRs) have been engineered to provide exclusive control over the activation of these receptors and thus to understand better the consequences of their signaling in vitro and in vivo. These engineered receptors, named RASSLs (receptors activated solely by synthetic ligands) and DREADDs (designer receptors exclusively activated by designer drugs), are insensitive to their endogenous ligands but can be activated by synthetic drug-like compounds. Currently, the existing RASSLs and DREADDs cover the Gi, Gq, and Gs signaling pathways. These modified GPCRs can be utilized as ideal tools to study GPCR functions selectively in specific cellular populations.

PMID: 19074739 [PubMed - indexed for MEDLINE]

NF-kB signaling pathway NF-kB pathway NF-kB signaling

Understanding RAMPs through genetically engineered mouse models.

Understanding RAMPs through genetically engineered mouse models.

Adv Exp Med Biol. 2012;744:49-60

Authors: Kadmiel M, Fritz-Six KL, Caron KM

Abstract
The family of Receptor Activity Modifying Proteins (RAMPs) consists of three members, RAMP1, 2 and 3, which are each encoded by a separate gene and have diverse spatiotemporal expression patterns. Biochemical and pharmacological studies in cultured cells have shown that RAMPs can modulate several aspects of G receptor (GPCR) signaling, including receptor trafficking, ligand binding affinity, second messenger signaling and receptor desensitization. Moreover, these studies have shown that RAMPs can interact with several GPCRs other than the canonical calcitonin receptor-like receptor (CLR), with which they were first identified. Given these expanding roles for RAMPs, it becomes interesting to question how these biochemical and pharmacological properties bear significance in normal or disease physiology. To this end, several gene targeted knockout and transgenic models have been generated and characterized in recent years. Fortunately, they have each supported important roles for RAMPs during embryonic development and adulthood. This chapter provides a comprehensive overview of the most recent findings from gene targeted knockout mouse models and transgenic over-expression models, and gives special consideration to how comparative phenotyping approaches and conditional deletion strategies can be highly beneficial. In the future, these genetically engineered mouse models will provide both insights and tools for the exploitation of RAMP-based therapies for the treatment of human diseases.

PMID: 22434107 [PubMed - indexed for MEDLINE]

NF-kB signaling pathway NF-kB pathway NF-kB signaling

Quantitative and dynamic analyses of G protein-coupled receptor signaling in yeast using Fus1, enhanced green fluorescence protein (EGFP), and His3 fusion protein.

Quantitative and dynamic analyses of G protein-coupled receptor signaling in yeast using Fus1, enhanced green fluorescence protein (EGFP), and His3 fusion protein.

Biotechnol Prog. 2006 Jul-Aug;22(4):954-60

Authors: Ishii J, Matsumura S, Kimura S, Tatematsu K, Kuroda S, Fukuda H, Kondo A

PMID: 16889369 [PubMed - indexed for MEDLINE]

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Engineering the melanocortin-4 receptor to control G(s) signaling in vivo.

Engineering the melanocortin-4 receptor to control G(s) signaling in vivo.

Ann N Y Acad Sci. 2003 Jun;994:225-32

Authors: Srinivasan S, Vaisse C, Conklin BR

PMID: 12851320 [PubMed - indexed for MEDLINE]

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Directed molecular evolution of DREADDs: a generic approach to creating next-generation RASSLs.

Directed molecular evolution of DREADDs: a generic approach to creating next-generation RASSLs.

Nat Protoc. 2010 Mar;5(3):561-73

Authors: Dong S, Rogan SC, Roth BL

Abstract
G protein-coupled receptors (GPCRs) and their downstream signaling cascades contribute to most physiological processes and a variety of human diseases. Isolating the effects of GPCR activation in an in vivo experimental setting is challenging as exogenous ligands have off-target effects and endogenous ligands constantly modulate the activity of native receptors. Highly specific designer drug-designer receptor complexes are a valuable tool for elucidating the effects of activating particular receptors and signaling pathways within selected cell types in vivo. In this study, we describe a generic protocol for the directed molecular evolution of designer receptors exclusively activated by designer drugs (DREADDs). First, the yeast system is validated with the template receptor. Second, a mutant library is generated by error-prone PCR. Third, the library is screened by drug-dependent yeast growth assays. Mutants exhibiting the desired properties are selected for further rounds of mutagenesis or for characterization in mammalian systems. In total, these steps should take 6-8 weeks of experimentation and should result in the evolution of a receptor to be activated by the chosen ligand. This protocol should help improve the experimental targeting of select cell populations.

PMID: 20203671 [PubMed - indexed for MEDLINE]

NF-kB signaling pathway NF-kB pathway NF-kB signaling

2012年9月23日星期日

Fasudil Ameliorates disease Progression in Experimental Autoimmune Encephalomyelitis, Acting Possibly Through Antiinflammatory Effect.

Fasudil Ameliorates disease Progression in Experimental Autoimmune Encephalomyelitis, Acting Possibly Through Antiinflammatory Effect.

CNS Neurosci Ther. 2012 Sep 21;

Authors: Hou SW, Liu CY, Li YH, Yu JZ, Feng L, Liu YT, Guo MF, Xie Y, Meng J, Zhang HF, Xiao BG, Ma CG

Abstract
AIM: The purpose of this investigation was to further explore the mechanism(s) underlying the amelioration in EAE caused by Fasudil, particularly focusing on anti-inflammatory effect. METHODS: We induced a chronic-progressive experimental autoimmune encephalomyelitis (EAE) in B6 mice immunized with myelin oligodendrocyte glycoprotein(35-55) and performed Fasudil intervention in early and late stages of the disease. RESULTS: The administration of Fasudil (40�mg/kg, i.p) had a therapeutic effect in delaying the onset and ameliorating the severity of EAE, accompanied by the improvement in myelination and the decrease in inflammatory cells in spinal cords. Fasudil inhibited TLR-4, p-NF-kB/p65, and inflammatory cytokines (IL-1?, IL-6, and TNF-?) and enhanced IL-10 production in spinal cords. The ratio of arginase/iNOS was enhanced mainly in the spinal cords of EAE mice treated with Fasudil, reflecting a shift toward the M2 (antiinflammation) macrophage/microglia phenotype. The administration of Fasudil also induced the upregulation of CB2 receptor in spinal cords, but did not significantly trigger CB1 receptor. Levels of neurotrophic factors NGF, BDNF, and GDNF in the CNS were not altered by Fasudil. CONCLUSION: Fasudil ameliorates disease progression in EAE, acting possibly through antiinflammatory pathway.

PMID: 22994384 [PubMed - as supplied by publisher]

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Signaling through G protein coupled receptors.

Signaling through G protein coupled receptors.

Plant Signal Behav. 2009 Oct;4(10):942-7

Authors: Tuteja N

Abstract
Heterotrimeric G proteins (Galpha, Gbeta/Ggamma subunits) constitute one of the most important components of cell signaling cascade. G Protein Coupled Receptors (GPCRs) perceive many extracellular signals and transduce them to heterotrimeric G proteins, which further transduce these signals intracellular to appropriate downstream effectors and thereby play an important role in various signaling pathways. GPCRs exist as a superfamily of integral membrane protein receptors that contain seven transmembrane alpha-helical regions, which bind to a wide range of ligands. Upon activation by a ligand, the GPCR undergoes a conformational change and then activate the G proteins by promoting the exchange of GDP/GTP associated with the Galpha subunit. This leads to the dissociation of Gbeta/Ggamma dimer from Galpha. Both these moieties then become free to act upon their downstream effectors and thereby initiate unique intracellular signaling responses. After the signal propagation, the GTP of Galpha-GTP is hydrolyzed to GDP and Galpha becomes inactive (Galpha-GDP), which leads to its re-association with the Gbeta/Ggamma dimer to form the inactive heterotrimeric complex. The GPCR can also transduce the signal through G protein independent pathway. GPCRs also regulate cell cycle progression. Till to date thousands of GPCRs are known from animal kingdom with little homology among them, but only single GPCR has been identified in plant system. The Arabidopsis GPCR was reported to be cell cycle regulated and also involved in ABA and in stress signaling. Here I have described a general mechanism of signal transduction through GPCR/G proteins, structure of GPCRs, family of GPCRs and plant GPCR and its role.

PMID: 19826234 [PubMed - indexed for MEDLINE]

GPCR Signaling G-protein Receptors gpcr pathway

Engineering GPCR signaling pathways with RASSLs.

Engineering GPCR signaling pathways with RASSLs.

Nat Methods. 2008 Aug;5(8):673-8

Authors: Conklin BR, Hsiao EC, Claeysen S, Dumuis A, Srinivasan S, Forsayeth JR, Guettier JM, Chang WC, Pei Y, McCarthy KD, Nissenson RA, Wess J, Bockaert J, Roth BL

Abstract
We are creating families of designer G protein-coupled receptors (GPCRs) to allow for precise spatiotemporal control of GPCR signaling in vivo. These engineered GPCRs, called receptors activated solely by synthetic ligands (RASSLs), are unresponsive to endogenous ligands but can be activated by nanomolar concentrations of pharmacologically inert, drug-like small molecules. Currently, RASSLs exist for the three major GPCR signaling pathways (G(s), G(i) and G(q)). We review these advances here to facilitate the use of these powerful and diverse tools.

PMID: 18668035 [PubMed - indexed for MEDLINE]

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