5-alpha-reductase: 2012-09-09

2012年9月15日星期六

Structure and function analyses of the purified GPCR human vomeronasal type 1 receptor 1.

Structure and function analyses of the purified GPCR human vomeronasal type 1 receptor 1.

Sci Rep. 2011;1:172

Authors: Corin K, Baaske P, Geissler S, Wienken CJ, Duhr S, Braun D, Zhang S

Abstract
The vomeronasal system is one of several fine-tuned scent-detecting signaling systems in mammals. However, despite significant efforts, how these receptors detect scent remains an enigma. One reason is the lack of sufficient purified receptors to perform detailed biochemical, biophysical and structural analyses. Here we report the ability to express and purify milligrams of purified, functional human vomeronasal receptor hVN1R1. Circular dichroism showed that purified hVN1R1 had an alpha-helical structure, similar to that of other GPCRs. Microscale thermophoresis showed that hVN1R1 bound its known ligand myrtenal with an EC(50) approximately 1 �M. This expression system can enable structural and functional analyses towards understanding how mammalian scent detection works.

PMID: 22355687 [PubMed]

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5-Oxo-eicosatetraenoic acid-induced chemotaxis: identification of a responsible receptor hGPCR48 and negative regulation by G protein G(12/13).

5-Oxo-eicosatetraenoic acid-induced chemotaxis: identification of a responsible receptor hGPCR48 and negative regulation by G protein G(12/13).

J Biochem. 2006 Mar;139(3):543-9

Authors: Koike D, Obinata H, Yamamoto A, Takeda S, Komori H, Nara F, Izumi T, Haga T

Abstract
While screening genes encoding G protein-coupled receptors (GPCRs) in the human genome, we and other groups have identified a GPCR named hGPCR48 as a high affinity receptor for 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), which is arachidonic acid metabolite and an endogenous chemoattractant for granulocytes. Using Chinese hamster ovary (CHO) cells stably expressing hGPCR48, we show here that activation of the receptor causes the chemotaxis of the cells toward 5-oxo-ETE. We also show that the chemotaxis of human granulocytes toward 5-oxo-ETE is inhibited by pretreatment with anti-hGPCR48 antibodies, indicating that hGPCR48 is an endogenous receptor responsible for chemotaxis of granulocytes toward 5-oxo-ETE. In addition, we show that the chemotaxis of CHO cells expressing hGPCR48 is suppressed by pretreatment with pertussis toxin, and enhanced by overexpression of the carboxy terminal peptides of Galpha (12/13) subunits or a regulator of the G protein signaling domain of p115RhoGEF, both of which are known to suppress G(12/13)-dependent signaling pathways. These results indicate that hGPCR48 couples with G(i/o) and G(12/13) proteins, which then initiate or attenuate the chemotaxis of the cells toward 5-oxo-ETE, respectively.

PMID: 16567419 [PubMed - indexed for MEDLINE]

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Classification of G-protein coupled receptors at four levels.

Classification of G-protein coupled receptors at four levels.

Protein Eng Des Sel. 2006 Nov;19(11):511-6

Authors: Gao QB, Wang ZZ

Abstract
G-protein coupled receptors (GPCRs) are transmembrane proteins which via G-proteins initiate some of the important signaling pathways in a cell and are involved in various physiological processes. Thus, computational prediction and classification of GPCRs can supply significant information for the development of novel drugs in pharmaceutical industry. In this paper, a nearest neighbor method has been introduced to discriminate GPCRs from non-GPCRs and subsequently classify GPCRs at four levels on the basis of amino acid composition and dipeptide composition of proteins. Its performance is evaluated on a non-redundant dataset consisted of 1406 GPCRs for six families and 1406 globular proteins using the jackknife test. The present method based on amino acid composition achieved an overall accuracy of 96.4% and Matthew's correlation coefficient (MCC) of 0.930 for correctly picking out the GPCRs from globular proteins. The overall accuracy and MCC were further enhanced to 99.8% and 0.996 by dipeptide composition-based method. On the other hand, the present method has successfully classified 1406 GPCRs into six families with an overall accuracy of 89.6 and 98.8% using amino acid composition and dipeptide composition, respectively. For the subfamily prediction of 1181 GPCRs of rhodopsin-like family, the present method achieved an overall accuracy of 76.7 and 94.5% based on the amino acid composition and dipeptide composition, respectively. Finally, GPCRs belonging to the amine subfamily and olfactory subfamily of rhodopsin-like family were further analyzed at the type level. The overall accuracy of dipeptide composition-based method for the classification of amine type and olfactory type of GPCRs reached 94.5 and 86.9%, respectively, while the overall accuracy of amino acid composition-based method was very low for both subfamilies. In comparison with existing methods in the literature, the present method also displayed great competitiveness. These results demonstrate the effectiveness of our method on identifying and classifying GPCRs correctly. GPCRsIdentifier, a corresponding stand-alone executable program for GPCR identification and classification was also developed, which can be acquired freely on request from the authors for academic purposes.

PMID: 17032692 [PubMed - indexed for MEDLINE]

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

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 pathway NF-kB signaling NF-kappaB signaling pathway

Modification of the beta 2-adrenergic receptor to engineer a receptor-effector complex for gene therapy.

Modification of the beta 2-adrenergic receptor to engineer a receptor-effector complex for gene therapy.

J Biol Chem. 2001 Aug 24;276(34):31596-601

Authors: Small KM, Brown KM, Forbes SL, Liggett SB

Abstract
Depressed G-protein-coupled receptor (GPCR) signaling has been implicated as a component of the pathophysiology of a number of complex diseases including heart failure and asthma, and augmentation or restoration of signaling by various means has been shown to improve organ function. Because some properties of native GPCRs are disadvantageous for ectopic therapeutic expression, we utilized the beta(2)-adrenergic receptor (beta(2)AR) as a scaffold to construct a highly modified therapeutic receptor-effector complex (TREC) suitable for gene therapy. Altogether, 19 modifications were made to the receptor. The ligand-binding site was re-engineered in TM-3 so that a beta-hydroxylmethyl side chain acts as a proton donor for the binding of a novel ligand. In addition, sites critical for agonist-promoted down-regulation in the amino terminus and for phosphorylation by GPCR kinases, and protein kinases A and C, in the third intracellular loop and the carboxyl terminus of the receptor were altered. These modifications of the receptor resulted in depressed agonist-stimulated adenylyl cyclase activity (26.8 +/- 2.1 versus 41.4 +/- 8 pmol/min/mg for wild-type beta(2)AR). This was fully restored by fusing the carboxyl terminus of the modified receptor to G alpha(s) (43.3 +/- 2.7 pmol/min/mg). The fully modified fused receptor was not activated by beta-agonists but rather by a nonbiogenic amine agonist that itself failed to activate the wild-type beta(2)AR. This two-way selectivity thus provides targeted activation based on physiologic status. Furthermore, the TREC did not display tachyphylaxis to prolonged agonist exposure (desensitization was 1 +/- 5% versus 55 +/- 4% for wild-type beta(2)AR). Thus, despite extensive alterations in regions of conformational lability, the beta(2)AR can be tailored to have optimal signaling characteristics for gene therapy. As a general paradigm, TRECs for enhancement of other G-protein signaling appear to be feasible for modification of other pathologic states.

PMID: 11402033 [PubMed - indexed for MEDLINE]

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2012年9月14日星期五

Molecular mechanisms of ligand binding, signaling, and regulation within the superfamily of G-protein-coupled receptors: molecular modeling and mutagenesis approaches to receptor structure and function.

Molecular mechanisms of ligand binding, signaling, and regulation within the superfamily of G-protein-coupled receptors: molecular modeling and mutagenesis approaches to receptor structure and function.

Pharmacol Ther. 2004 Jul;103(1):21-80

Authors: Kristiansen K

Abstract
The superfamily of G-protein-coupled receptors (GPCRs) could be subclassified into 7 families (A, B, large N-terminal family B-7 transmembrane helix, C, Frizzled/Smoothened, taste 2, and vomeronasal 1 receptors) among mammalian species. Cloning and functional studies of GPCRs have revealed that the superfamily of GPCRs comprises receptors for chemically diverse native ligands including (1) endogenous compounds like amines, peptides, and Wnt proteins (i.e., secreted proteins activating Frizzled receptors); (2) endogenous cell surface adhesion molecules; and (3) photons and exogenous compounds like odorants. The combined use of site-directed mutagenesis and molecular modeling approaches have provided detailed insight into molecular mechanisms of ligand binding, receptor folding, receptor activation, G-protein coupling, and regulation of GPCRs. The vast majority of family A, B, C, vomeronasal 1, and taste 2 receptors are able to transduce signals into cells through G-protein coupling. However, G-protein-independent signaling mechanisms have also been reported for many GPCRs. Specific interaction motifs in the intracellular parts of these receptors allow them to interact with scaffold proteins. Protein engineering techniques have provided information on molecular mechanisms of GPCR-accessory protein, GPCR-GPCR, and GPCR-scaffold protein interactions. Site-directed mutagenesis and molecular dynamics simulations have revealed that the inactive state conformations are stabilized by specific interhelical and intrahelical salt bridge interactions and hydrophobic-type interactions. Constitutively activating mutations or agonist binding disrupts such constraining interactions leading to receptor conformations that associates with and activate G-proteins.

PMID: 15251227 [PubMed - indexed for MEDLINE]

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Autophagy-dependent NF?B regulation.

Autophagy-dependent NF?B regulation.

Cell Cycle. 2012 Feb 1;11(3):436-7

Authors: Cianfanelli V, Cecconi F

PMID: 22262191 [PubMed - indexed for MEDLINE]

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

Targeted acetylation of NF-kappaB/RelA and histones by epigenetic drugs reduces post-ischemic brain injury in mice with an extended therapeutic window.

Targeted acetylation of NF-kappaB/RelA and histones by epigenetic drugs reduces post-ischemic brain injury in mice with an extended therapeutic window.

Neurobiol Dis. 2012 Aug 30;

Authors: Lanzillotta A, Pignataro G, Branca C, Cuomo O, Sarnico I, Benarese M, Annunziato L, Spano P, Pizzi M

Abstract
Nuclear factor-kappaB (NF-?B) p50/RelA is a key molecule with a dual effect in the progression of ischemic stroke. In harmful ischemia, but not in preconditioning insult, neurotoxic activation of p50/RelA is characterized by RelA-specific acetylation at Lys310 (K310) and deacetylation at other Lys residues. The derangement of RelA acetylation is associated with activation of Bim promoter. OBJECTIVE: With the aim of producing neuroprotection by correcting altered acetylation of RelA in brain ischemia, we combined the pharmacological inhibition of histone deacetylase (HDAC) 1-3, known to reduce global RelA acetylation, and the activation of sirtuin 1, endowed with a specific deacetylase activity on the K310 residue of RelA. To afford this aim, we tested the clinically used HDAC 1-3 inhibitor entinostat (MS-275) and the sirtuin 1 activator resveratrol. METHODS: We used the mouse model of transient middle cerebral artery occlusion (MCAO) and primary cortical neurons exposed to oxygen glucose deprivation (OGD). RESULTS: The combined use of MS-275 and resveratrol, by restoring normal RelA acetylation, elicited a synergistic neuroprotection in neurons exposed to OGD. This effect correlated with MS-275 capability to increase total RelA acetylation and resveratrol capability to reduce RelA K310 acetylation through the activation of an AMP-activated protein kinase-sirtuin 1 pathway. The synergistic treatment reproduced the acetylation state of RelA peculiar of preconditioning ischemia. Neurons exposed to the combined drugs totally recovered the optimal histone H3 acetylation. Neuroprotection was reproduced in mice subjected to MCAO and treated with MS-275 (20?g/kg and 200?g/kg) or resveratrol (6800?g/kg) individually. However, the administration of lowest doses of MS-275 (2?g/kg) and resveratrol (68?g/kg) synergistically reduced infarct volume and neurological deficits. Importantly, the treatment was effective even when administered 7h after the stroke onset. Chromatin immunoprecipitation analysis of cortices harvested from treated mice showed that the RelA binding and histone acetylation increased at the Bcl-x(L) promoter and decreased at the Bim promoter. CONCLUSION: Our study reveals that epigenetic therapy shaping acetylation of both RelA and histones may be a promising strategy to limit post-ischemic injury with an extended therapeutic window.

PMID: 22971966 [PubMed - as supplied by publisher]

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

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]

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Theiler's murine encephalomyelitis virus induced phenotype switch of microglia in vitro.

Theiler's murine encephalomyelitis virus induced phenotype switch of microglia in vitro.

J Neuroimmunol. 2012 Sep 8;

Authors: Gerhauser I, Hansmann F, Puff C, Kumnok J, Schaudien D, Wewetzer K, Baumg�rtner W

Abstract
The present in vitro study aimed to define the involvement of astrocytes and microglia in the initial inflammatory response of Theiler's murine encephalomyelitis (TME), a virus-induced mouse model of multiple sclerosis, and whether intralesional microglia exert pro- (M1) or anti-inflammatory (M2) effects following TME virus (TMEV) infection. Therefore astrocytes and microglia were purified from neonatal murine brains and inoculated either with TMEV or mock-solution. Gene expression of IL-1, IL-2, IL-10, IL-12, TNF, TNF receptors (TNFR1, TNFR2), TGF?1, IFN? and transcription factors NF-?B (p50, p65) and AP-1 (c-jun, c-fos) were quantified using RT-qPCR at 6, 48, and 240h post infection (hpi). In addition, IL-1, IL-10, IL-12, TNF and TGF?1 mRNA transcripts were investigated at 168 hpi in TMEV- and mock-infected SJL/J mice. Overall in vitro astrocytes showed a significant higher amount of viral RNA compared to microglia. In addition, TMEV-infected astrocytes showed higher numbers of IL-1, IL-12 and TNF transcripts at 48 hpi. In microglia high IL-10 and low IL-12 mRNA levels were detected at 48 hpi, while the opposite was the case at 240 hpi. In addition, TNF mRNA was increased in microglia at 240 hpi. In addition, the observed up-regulation of IL-1, IL-12 and IL-10 in the early phase of TME in vivo substantiates the relevance of these cytokines during the disease induction. Summarized data indicate that TMEV infection of microglia induces a switch from the anti-inflammatory (M2) during the early phase to the pro-inflammatory (M1) phenotype in the later phase of the infection. The simultaneous expression of TNF and its receptors by both cell types might generate autocrine feedback loops possibly associated with pro-inflammatory actions of astrocytes via TNFR1.

PMID: 22967352 [PubMed - as supplied by publisher]

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

2012年9月13日星期四

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]

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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

PMID: 19074739 [PubMed - indexed for MEDLINE]

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

Plant signaling in stress: G-protein coupled receptors, heterotrimeric G-proteins and signal coupling via phospholipases.

Plant signaling in stress: G-protein coupled receptors, heterotrimeric G-proteins and signal coupling via phospholipases.

Plant Signal Behav. 2008 Feb;3(2):79-86

Authors: Tuteja N, Sopory SK

Abstract
Plant growth and development are coordinalely controlled by several internal factors and environmental signals. To sense these environmental signals, the higher plants have evolved a complex signaling network, which may also cross talk with each other. Plants can respond to the signals as individual cells and as whole organisms. Various receptors including phytochromes, G-proteins coupled receptors (GPCR), kinase and hormone receptors play important role in signal transduction but very few have been characterized in plant system. The heterotrimeric G-proteins mediate the coupling of signal transduction from activated GPCR to appropriate downstream effectors and thereby play an important role in signaling. In this review we have focused on some of the recent work on G-proteins and two of the effectors, PLC and PLD, which have been shown to interact with Galpha subunit and also discussed their role in abiotic stress tolerance.

PMID: 19516978 [PubMed - in process]

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Osteoblast expression of an engineered Gs-coupled receptor dramatically increases bone mass.

Osteoblast expression of an engineered Gs-coupled receptor dramatically increases bone mass.

Proc Natl Acad Sci U S A. 2008 Jan 29;105(4):1209-14

Authors: Hsiao EC, Boudignon BM, Chang WC, Bencsik M, Peng J, Nguyen TD, Manalac C, Halloran BP, Conklin BR, Nissenson RA

Abstract
Osteoblasts are essential for maintaining bone mass, avoiding osteoporosis, and repairing injured bone. Activation of osteoblast G protein-coupled receptors (GPCRs), such as the parathyroid hormone receptor, can increase bone mass; however, the anabolic mechanisms are poorly understood. Here we use "Rs1," an engineered GPCR with constitutive G(s) signaling, to evaluate the temporal and skeletal effects of G(s) signaling in murine osteoblasts. In vivo, Rs1 expression induces a dramatic anabolic skeletal response, with midfemur girth increasing 1,200% and femur mass increasing 380% in 9-week-old mice. Bone volume, cellularity, areal bone mineral density, osteoblast gene markers, and serum bone turnover markers were also elevated. No such phenotype developed when Rs1 was expressed after the first 4 weeks of postnatal life, indicating an exquisite temporal sensitivity of osteoblasts to Rs1 expression. This pathway may represent an important determinant of bone mass and may open future avenues for enhancing bone repair and treating metabolic bone diseases.

PMID: 18212126 [PubMed - indexed for MEDLINE]

NF-kappaB signaling pathway read more learn more

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 signaling pathway NF-kB pathway NF-kB signaling

2012年9月12日星期三

Neoceptors: reengineering GPCRs to recognize tailored ligands.

Neoceptors: reengineering GPCRs to recognize tailored ligands.

Trends Pharmacol Sci. 2007 Mar;28(3):111-6

Authors: Jacobson KA, Gao ZG, Liang BT

Abstract
Efforts to model and reengineer the putative binding sites of G-protein-coupled receptors (GPCRs) have led to an approach that combines small-molecule 'classical' medicinal chemistry and gene therapy. In this approach, complementary structural changes (e.g. based on novel ionic or H-bonds) are made in the receptor and ligand for the selective enhancement of affinity. Thus, a modified receptor (neoceptor) is designed for activation by tailor-made agonists that do not interact with the native receptor. The neoceptor is no longer activated by the native agonist, but rather functions as a scaffold for the docking of novel small molecules (neoligands). In theory, the approach could verify the accuracy of GPCR molecular modeling, the investigation of signaling, the design of small molecules to rescue disease-related mutations, and small-molecule-directed gene therapy. The neoceptor-neoligand pairing could offer spatial specificity by delivering the neoceptor to a target site, and temporal specificity by administering neoligand when needed.

PMID: 17280720 [PubMed - indexed for MEDLINE]

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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 pathway NF-kB signaling NF-kappaB signaling pathway

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]

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2012年9月11日星期二

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]

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Tbr2 deficiency in mitral and tufted cells disrupts excitatory-inhibitory balance of neural circuitry in the mouse olfactory bulb.

Tbr2 deficiency in mitral and tufted cells disrupts excitatory-inhibitory balance of neural circuitry in the mouse olfactory bulb.

J Neurosci. 2012 Jun 27;32(26):8831-44

Authors: Mizuguchi R, Naritsuka H, Mori K, Yoshihara Y

Abstract
The olfactory bulb (OB) is the first relay station in the brain where odor information from the olfactory epithelium is integrated, processed through its intrinsic neural circuitry, and conveyed to higher olfactory centers. Compared with profound mechanistic insights into olfactory axon wiring from the nose to the OB, little is known about the molecular mechanisms underlying the formation of functional neural circuitry among various types of neurons inside the OB. T-box transcription factor Tbr2 is expressed in various types of glutamatergic excitatory neurons in the brain including the OB projection neurons, mitral and tufted cells. Here we generated conditional knockout mice in which the Tbr2 gene is inactivated specifically in mitral and tufted cells from late embryonic stages. Tbr2 deficiency caused cell-autonomous changes in molecular expression including a compensatory increase of another T-box member, Tbr1, and a concomitant shift of vesicular glutamate transporter (VGluT) subtypes from VGluT1 to VGluT2. Tbr2-deficient mitral and tufted cells also exhibited anatomical abnormalities in their dendritic morphology and projection patterns. Additionally, several non-cell-autonomous phenotypes were observed in parvalbumin-, calbindin-, and 5T4-positive GABAergic interneurons. Furthermore, the number of dendrodendritic reciprocal synapses between mitral/tufted cells and GABAergic interneurons was significantly reduced. Upon stimulation with odorants, larger numbers of mitral and tufted cells were activated in Tbr2 conditional knockout mice. These results suggest that Tbr2 is required for not only the proper differentiation of mitral and tufted cells, but also for the establishment of functional neuronal circuitry in the OB and maintenance of excitatory-inhibitory balance crucial for odor information processing.

PMID: 22745484 [PubMed - indexed for MEDLINE]

G-protein Receptors gpcr pathway NF-κB

Heterotrimeric G-protein complex and G-protein-coupled receptor from a legume (Pisum sativum): role in salinity and heat stress and cross-talk with phospholipase C.

Heterotrimeric G-protein complex and G-protein-coupled receptor from a legume (Pisum sativum): role in salinity and heat stress and cross-talk with phospholipase C.

Plant J. 2007 Aug;51(4):656-69

Authors: Misra S, Wu Y, Venkataraman G, Sopory SK, Tuteja N

Abstract
Heterotrimeric G-proteins transduce signals from activated G-protein-coupled receptors (GPCR) to appropriate downstream effectors and thereby play an important role in signaling. A role of G-proteins in salinity and heat stress tolerance has not heretofore been described. We report isolation of cDNAs of two isoforms of Galpha (Galpha1, 1152 bp; Galpha2, 1152 bp), one Gbeta (1134 bp), two isoforms of Ggamma (Ggamma1, 345 bp; Ggamma2, 303 bp) and a GPCR (1008 bp) from Pisum sativum, and purification of all the encoded recombinant proteins (Galpha, 44 kDa; Gbeta, 41 kDa; Ggamma, 14 kDa; GPCR, 35 kDa). The transcript levels of Galpha and Gbeta were upregulated following NaCl, heat and H(2)O(2) treatments. Protein-protein interaction studies using an in vitro yeast two-hybrid system and in planta co-immunoprecipitation showed that the Galpha subunit interacted with the pea Gbeta subunit and pea phospholipase C (PLCdelta) at the calcium-binding domain (fn1). The GTPase activity of the Galpha subunit increased after interaction with PLCdelta. The GPCR protein interacted with all the subunits of G-proteins and with itself. Transgenic tobacco plants (T(0) and T(1)) constitutively over-expressing Galpha showed tolerance to salinity and heat, while Gbeta-over-expressing plants showed only heat tolerance, as tested by leaf disk senescence assay and germination/growth of T(1) seeds/seedlings. These findings provide direct evidence for a novel role of Galpha and Gbeta subunits in abiotic stress tolerance and possible cross-talk between PLC- and G-protein-mediated signaling pathways.

PMID: 17587233 [PubMed - indexed for MEDLINE]

find out more info GPCR Signaling G-protein Receptors

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 NF-kappaB signaling pathway NF-kB signaling pathway

Epigallocatechin-3-O-gallate inhibits the production of thymic stromal lymphopoietin by the blockade of caspase-1/NF-?B pathway in mast cells.

Epigallocatechin-3-O-gallate inhibits the production of thymic stromal lymphopoietin by the blockade of caspase-1/NF-?B pathway in mast cells.

Amino Acids. 2012 Jun;42(6):2513-9

Authors: Moon PD, Choi IH, Kim HM

Abstract
The cytokine thymic stromal lymphopoietin (TSLP) has been implicated in the development and progression of allergic diseases such as atopic dermatitis, asthma, and chronic obstructive pulmonary disease. However, it has not yet been clarified the effect of epigallocatechin-3-O-gallate (EGCG) on the production of TSLP. Thus, we investigated how EGCG inhibits the production of TSLP in the human mast cell line (HMC-1) cells. Enzyme-linked immunosorbent assay, reverse transcription-polymerase chain reaction, luciferase assay, and Western blot analysis were used to investigate the effects of EGCG. EGCG inhibited the production and mRNA expression of TSLP in HMC-1 cells. EGCG also inhibited the nuclear factor-?B luciferase activity induced by phorbol myristate acetate plus A23187. Furthermore, EGCG inhibited the activation of caspase-1 in HMC-1 cells. These results provide evidence that EGCG can help us to treat inflammatory and atopic diseases through the inhibition of TSLP.

PMID: 21833768 [PubMed - indexed for MEDLINE]

gpcr pathway NF-κB NF-kB signaling pathway

2012年9月10日星期一

Selection and characterization of DARPins specific for the neurotensin receptor 1.

Selection and characterization of DARPins specific for the neurotensin receptor 1.

Protein Eng Des Sel. 2009 Jun;22(6):357-66

Authors: Milovnik P, Ferrari D, Sarkar CA, Pl�ckthun A

Abstract
We describe here the selection and characterization of designed ankyrin repeat proteins (DARPins) that bind specifically to the rat neurotensin receptor 1 (NTR1), a G-protein coupled receptor (GPCR). The selection procedure using ribosome display and the initial clone analysis required <10 microg of detergent-solubilized, purified NTR1. Complex formation with solubilized GPCR was demonstrated by ELISA and size-exclusion chromatography; additionally, the GPCR could be detected in native membranes of mammalian cells using fluorescence microscopy. The main binding epitope in the GPCR lies within the 33 amino acids following the seventh transmembrane segment, which comprise the putative helix 8, and additional binding interactions are possibly contributed by the cytoplasmic loop 3, thus constituting a discontinuous epitope. Since the selected binders recognize the GPCR both in detergent-solubilized and in membrane-embedded forms, they will be potentially useful both in co-crystallization trials and for signal transduction experiments.

PMID: 19389717 [PubMed - indexed for MEDLINE]

Ursolic acid inhibits T-cell activation through modulating nuclear factor-? B signaling.

Ursolic acid inhibits T-cell activation through modulating nuclear factor-? B signaling.

Chin J Integr Med. 2012 Jan;18(1):34-9

Authors: Zeng G, Chen J, Liang QH, You WH, Wu HJ, Xiong XG

Abstract
OBJECTIVE: To investigate the effects of ursolic acid (UA) on T-cell proliferation and activation, as well as to examine its effect on nuclear factor-?B (NF-?B) signaling pathway in T cells.
METHODS: T-cells isolated from BALB/c mice were incubated with UA at concentrations ranging from 5-30 ?mol/L in the presence of phorbol 12-myristate 13-acetate (PMA) or PMA plus ionomycin. The proliferation of T cells was measured by the MTT assay. The expressions of CD69, CD25, and CD71 on T-cell surface were analyzed using flow cytometry. The level of interleukin-2 (IL-2) in the culture supernatant of activated T cells was quantified by enzyme-linked immunosorbent assay (ELISA). The level of phosphorylated I?B-? (p-I?B-?) in total protein and p65, a subunit of NF-?B, nuclear translocation were measured by Western blot analysis.
RESULTS: UA in a dose-dependent manner significantly decreased the proliferation and inhibited the surface expressions of CD69, CD25, and CD71 in murine T lymphocytes upon in vitro activation (P<0.01). Significant reduction of IL-2 production was found in activated T cells treated with UA (P<0.01). The PMA-induced increase in p-I?B-? protein was inhibited, and nuclear translocation of p65 from the cytoplasm was blocked by UA.
CONCLUSION: UA is a potent inhibitor for T cell activation and proliferation; these effects are associated with the inhibition of NF-?B signaling pathway.

PMID: 21994027 [PubMed - indexed for MEDLINE]

gpcr pathway NF-κB NF-kB signaling pathway

Plant signaling in stress: G-protein coupled receptors, heterotrimeric G-proteins and signal coupling via phospholipases.

Plant signaling in stress: G-protein coupled receptors, heterotrimeric G-proteins and signal coupling via phospholipases.

Plant Signal Behav. 2008 Feb;3(2):79-86

Authors: Tuteja N, Sopory SK

PMID: 19516978 [PubMed - in process]

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Fucosterol isolated from Undaria pinnatifida inhibits lipopolysaccharide-induced production of nitric oxide and pro-inflammatory cytokines via the inactivation of nuclear factor-?B and p38 mitogen-activated protein kinase in RAW264.7 macrophages.

Fucosterol isolated from Undaria pinnatifida inhibits lipopolysaccharide-induced production of nitric oxide and pro-inflammatory cytokines via the inactivation of nuclear factor-?B and p38 mitogen-activated protein kinase in RAW264.7 macrophages.

Food Chem. 2012 Dec 1;135(3):967-75

Authors: Yoo MS, Shin JS, Choi HE, Cho YW, Bang MH, Baek NI, Lee KT

Abstract
It has been reported that fucosterol has anti-diabetic, anti-oxidant, and anti-osteoporotic effects. We investigated the anti-inflammatory effects and the underlying molecular mechanism of fucosterol in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. Fucosterol suppressed the expressions of inducible nitric oxide synthase (iNOS), tumour necrosis factor-? (TNF-?), and interleukin-6 (IL-6) by downregulating their transcriptions, and subsequently inhibited the productions of nitric oxide, TNF-?, and IL-6. In addition, fucosterol attenuated LPS-induced DNA binding and the transcriptional activity of nuclear factor-?B (NF-?B). These reductions were accompanied by parallel reductions in the phosphorylation and nuclear translocation of NF-?B. Furthermore, fucosterol attenuated the phosphorylations of mitogen-activated protein kinase kinases 3/6 (MKK3/6) and mitogen-activated protein kinase-activated protein kinase 2 (MK2), which are both involved in the p38 MAPK pathway. These results suggest that the anti-inflammatory effects of fucosterol are associated with the suppression of the NF-?B and p38 MAPK pathways.

PMID: 22953812 [PubMed - in process]

NF-kB signaling NF-kappaB signaling pathway read more

2012年9月9日星期日

IL-1? and compressive forces lead to a significant induction of RANKL-expression in primary human cementoblasts.

IL-1? and compressive forces lead to a significant induction of RANKL-expression in primary human cementoblasts.

J Orofac Orthop. 2012 Sep 7;

Authors: Diercke K, Kohl A, Lux CJ, Erber R

Abstract
AIM: The aim of this study was to investigate the response of primary human cementoblasts to conditions as they occur on the pressure side during orthodontic tooth movement. METHODS: In our previous study, the cementoblasts were characterized using markers for osteoblastogenic differentiation and the cementoblast-specific marker CEMP-1. Initially, primary human cementoblasts were compressed for 1�h, 4�h, and 6�h (30�g/cm(2)). In the second experiment, the cementoblasts were stimulated with interleukin (IL)-1? for 24�h and for 96�h with 1�ng/ml and 10�ng/ml and subsequently compressed for 1�h and 6�h. Changes in mRNA expression for receptor activator of NF-?B (RANK), RANK ligand (RANKL), osteoprotegerin (OPG), and cyclooxygenase-2 (COX-2) were measured by quantitative real-time polymerase chain reaction (RT-PCR). RANK and RANKL were also examined by immunocytochemical staining at the protein level. RESULTS: Compression (30�g/cm(2)) led to a significant increase in RANKL expression after 6�h. OPG expression in compressed cementoblasts was significantly reduced after 1�h. RANK remained unchanged during the course of the experiment. Stimulation with IL-1? induced RANKL and OPG expression. However, IL-1?-dependent induction of RANKL was more prominent than the induction of OPG, leading to a (significant) increase in the RANKL/OPG ratios. The expression of RANK remained unchanged after 24�h of stimulation with IL-1? and decreased significantly after 96�h. Compression of the prestimulated cells resulted in a further increase in RANKL expression significant after 6�h. OPG and RANK expression remained unchanged compared to the unstimulated sample. COX-2 increased significantly after both compression and stimulation with IL-1?. Combined stimulation and compression resulted in a significant further increase after 6�h compared to IL-1? stimulation alone. CONCLUSION: Primary human cementoblasts in vitro express increased levels of RANKL, in particular during the combination of inflammation and compression. The increase in RANKL expression is not compensated by an increase in OPG expression. The induction of RANKL expression was associated with a significant increase in COX-2 expression. Since RANKL attracts osteoclasts, its increase might be associated with the progression of root resorption. The in vitro alterations in cementoblasts we observed may be indicators of cellular mechanisms that lead to the increased root resorption during orthodontic treatment.

PMID: 22955577 [PubMed - as supplied by publisher]

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

Kinetics of G-protein-coupled receptor endosomal trafficking pathways revealed by single quantum dots.

Kinetics of G-protein-coupled receptor endosomal trafficking pathways revealed by single quantum dots.

Proc Natl Acad Sci U S A. 2010 Oct 26;107(43):18658-63

Authors: Fichter KM, Flajolet M, Greengard P, Vu TQ

Abstract
G-protein-coupled receptors (GPCRs) are the largest protein superfamily in the human genome; they comprise 30% of current drug targets and regulate diverse cellular signaling responses. The role of endosomal trafficking in GPCR signaling regulation is gaining substantial consideration. However, this process remains difficult to study due to the inability to distinguish among many individual receptors, simultaneously trafficking within multiple endosomal pathways. Here we show accurate measurement of the internalization and endosomal trafficking of single groups of serotonin (5-hydroxytryptamine, 5-HT) receptors using single quantum dot (QD) probes and quantitative colocalization. We demonstrate that the presence of a QD tag does not interfere with 5-HT receptor internalization or endosomal recycling. Direct measurements show simultaneous trafficking of the 5-HT1A receptor in two distinct endosomal recycling pathways. Single-molecule imaging of endosomal trafficking will significantly impact the understanding of cellular signaling and provide powerful tools to elucidate the actions of GPCR-targeted therapeutics.

PMID: 20940319 [PubMed - indexed for MEDLINE]

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

Clara cell 10-kDa protein gene transfection inhibits NF-?B activity in airway epithelial cells.

Clara cell 10-kDa protein gene transfection inhibits NF-?B activity in airway epithelial cells.

PLoS One. 2012;7(4):e35960

Authors: Long XB, Hu S, Wang N, Zhen HT, Cui YH, Liu Z

Abstract
BACKGROUND: Clara cell 10-kDa protein (CC10) is a multifunctional protein with anti-inflammatory and immunomodulatory effects. Induction of CC10 expression by gene transfection may possess potential therapeutic effect. Nuclear factor ?B (NF-?B) plays a key role in the inflammatory processes of airway diseases.
METHOD/RESULTS: To investigate potential therapeutic effect of CC10 gene transfection in controlling airway inflammation and the underlying intracellular mechanisms, in this study, we constructed CC10 plasmid and transfected it into bronchial epithelial cell line BEAS-2B cells and CC10 knockout mice. In BEAS-2B cells, CC10's effect on interleukin (IL)-1? induced IL-8 expression was explored by means of RT-PCR and ELISA and its effect on NF-?B classical signaling pathway was studied by luciferase reporter, western blot, and immunoprecipitation assay. The effect of endogenous CC10 on IL-1? evoked IL-8 expression was studied by means of nasal explant culture. In mice, CC10's effect on IL-1? induced IL-8 and nuclear p65 expression was examined by immunohistochemistry. First, we found that the CC10 gene transfer could inhibit IL-1? induced IL-8 expression in BEAS-2B cells. Furthermore, we found that CC10 repressed IL-1? induced NF-?B activation by inhibiting the phosphorylation of I?B-? but not I?B kinase-?/? in BEAS-2B cells. Nevertheless, we did not observe a direct interaction between CC10 and p65 subunit in BEAS-2B cells. In nasal explant culture, we found that IL-1? induced IL-8 expression was inversely correlated with CC10 levels in human sinonasal mucosa. In vivo study revealed that CC10 gene transfer could attenuate the increase of IL-8 and nuclear p65 staining in nasal epithelial cells in CC10 knockout mice evoked by IL-1? administration.
CONCLUSION: These results indicate that CC10 gene transfer may inhibit airway inflammation through suppressing the activation of NF-?B, which may provide us a new consideration in the therapy of airway inflammation.

PMID: 22558282 [PubMed - indexed for MEDLINE]

TOXICITY PROFILE OF SMALL-MOLECULE IAP ANTAGONIST GDC-0152 IS LINKED TO TNF-ALPHA PHARMACOLOGY.

TOXICITY PROFILE OF SMALL-MOLECULE IAP ANTAGONIST GDC-0152 IS LINKED TO TNF-ALPHA PHARMACOLOGY.

Toxicol Sci. 2012 Sep 5;

Authors: Erickson RI, Tarrant J, Cain G, Lewin-Koh SC, Dybdal N, Wong H, Blackwood E, West K, Steigerwalt R, Mamounas M, Flygare JA, Amemiya K, Dambach D, Fairbrother WJ, Diaz D

Abstract
Inhibitor-of-apoptosis (IAP) proteins suppress apoptosis and are overexpressed in a variety of cancers. Small-molecule IAP antagonists are currently being tested in clinical trials as novel cancer therapeutics. GDC-0152 is a small-molecule drug that triggers tumor cell apoptosis by selectively antagonizing IAPs. GDC-0152 induces NF-?B transcriptional activity leading to expression of several chemokines and cytokines, of which tumor necrosis factor alpha (TNF-?) is the most important for single-agent tumor activity. TNF-? is a pleiotropic cytokine that drives a variety of cellular responses, comprising inflammation, proliferation, and cell survival or death depending on the cellular context. As malignant and normal cells produce TNF-? upon IAP antagonism, increased TNF-? could drive both efficacy and toxicity. The toxicity profile of GDC-0152 in dogs and rats was characterized after intravenous dose administration once every two weeks for four doses. Findings in both species consisted of a dose-related, acute, systemic inflammatory response and hepatic injury. Laboratory findings included elevated plasma cytokines, an inflammatory leukogram, and increased liver transaminases with histopathological findings of inflammatory infiltrates and apoptosis/necrosis in multiple tissues; a toxicology profile consistent with TNF-?-mediated toxicity. Dogs exhibited more severe findings than rats, and humans did not exhibit these findings, at comparable exposures across species. Furthermore, elevations in blood neutrophil count, serum MCP-1 and other markers of inflammation corresponded to GDC-0152 exposure and toxicity and thus may have utility as safety biomarkers.

PMID: 22956632 [PubMed - as supplied by publisher]

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

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