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.

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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]

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

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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]

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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.

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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]

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

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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]

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

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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]

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

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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.

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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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