Image-based high-throughput screening for inhibitors of angiogenesis.

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Image-based high-throughput screening for inhibitors of angiogenesis.

Methods Mol Biol. 2013;931:139-51

Authors: Evensen L, Link W, Lorens JB

Abstract
Automated multicolor fluorescence microscopy facilitates high-throughput quantitation of cellular parameters of complex, organotypic systems. In vitro co-cultured vascular cells form capillary-like networks that model facets of angiogenesis, making it an attractive alternative for anti-angiogenic drug discovery. We have adapted this angiogenesis assay system to a high-throughput format to enable automated image-based high-throughput screening of live primary human vascular cell co-cultures with chemical libraries for anti-angiogenic drug discovery. Protocols are described for setup of a fluorescence-based co-culture assay, live cell image acquisition, image analysis of morphological parameters, and screening data handling.

PMID: 23027002 [PubMed - indexed for MEDLINE]

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A burst of ABC genes in the genome of the polyphagous spider mite Tetranychus urticae.

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A burst of ABC genes in the genome of the polyphagous spider mite Tetranychus urticae.

BMC Genomics. 2013 May 10;14(1):317

Authors: Dermauw W, Osborne EJ, Clark RM, Grbi M, Tirry L, Van Leeuwen T

Abstract
BACKGROUND: The ABC (ATP-binding cassette) gene superfamily is widespread across all living species. The majority of ABC genes encode ABC transporters, which are membrane-spanning proteins capable of transferring substrates across biological membranes by hydrolyzing ATP. Although ABC transporters have often been associated with resistance to drugs and toxic compounds, within the Arthropoda ABC gene families have only been characterized in detail in several insects and a crustacean. In this study, we report a genome-wide survey and expression analysis of the ABC gene superfamily in the spider mite, Tetranychus urticae, a chelicerate ~ 450 million years diverged from other Arthropod lineages. T. urticae is a major agricultural pest, and is among of the most polyphagous arthropod herbivores known. The species resists a staggering array of toxic plant secondary metabolites, and has developed resistance to all major classes of pesticides in use for its control. RESULTS: We identified 103 ABC genes in the T. urticae genome, the highest number discovered in a metazoan species to date. Within the T. urticae ABC gene set, all members of the eight currently described subfamilies (A to H) were detected. A phylogenetic analysis revealed that the high number of ABC genes in T. urticae is due primarily to lineage-specific expansions of ABC genes within the ABCC, ABCG and ABCH subfamilies. In particular, the ABCC subfamily harbors the highest number of T. urticae ABC genes (39). In a comparative genomic analysis, we found clear orthologous relationships between a subset of T. urticae ABC proteins and ABC proteins in both vertebrates and invertebrates known to be involved in fundamental cellular processes. These included members of the ABCB-half transporters, and the ABCD, ABCE and ABCF families. Furthermore, one-to-one orthologues could be distinguished between T. urticae proteins and human ABCC10, ABCG5 and ABCG8, the Drosophila melanogaster sulfonylurea receptor and ecdysone-regulated transporter E23. Finally, expression profiling revealed that ABC genes in the ABCC, ABCG ABCH subfamilies were differentially expressed in multi-pesticide resistant mite strains and in mites transferred to challenging (toxic) host plants. CONCLUSIONS: In this study we present the first comprehensive analysis of ABC genes in a polyphagous arthropod herbivore. We demonstrate that the broad plant host range and high levels of pesticide resistance in T. urticae are associated with lineage-specific expansions of ABC genes, many of which respond transcriptionally to xenobiotic exposure. This ABC catalogue will serve as a basis for future biochemical and toxicological studies. Obtaining functional evidence that these ABC subfamilies contribute to xenobiotic tolerance should be the priority of future research.

PMID: 23663308 [PubMed - as supplied by publisher]

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ATF4 and IRE1? inhibit DNA repair protein DNA-dependent protein kinase 1 induced by heat shock.

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ATF4 and IRE1? inhibit DNA repair protein DNA-dependent protein kinase 1 induced by heat shock.

Mol Cell Biochem. 2012 Dec;371(1-2):225-32

Authors: Zhu H, Guo FJ, Zhao W, Zhou J, Liu Y, Song F, Wang Y

Abstract
With the increase of environment temperature, more and more attentions are payed to the effects of heat stress. Cells under heat shock either are adapted to the condition or are damaged and dead. In this paper, we found that heat shock induced endoplasmic reticulum (ER) stress. ATF4, PERK, and IRE1? were induced by heat shock of 45 �C in the transcriptional level. Under the stress of 45 �C, PERK was phosphorylated and XBP1s was detected. The result indicated that heat shock could induce the ER stress. We found that heat shock of 45 �C induced the dysregulation of HSP70 and DNA-PKcs, and downregulated the expression of PARP1 and XRCC1. Further results showed that after the knockdown of ATF4 or IRE1?, the expression of DNA-PKcs and XRCC1 were increased. It was indicated that ATF4 and IRE1? could inhibit the expression of DNA-PKcs and XRCC1 under the heat stress. Our results suggested that heat shock could activate ER stress. IRE1? and ATF4, as the important ER stress molecules, could inhibit the expression of DNA repair proteins DNA-PKcs, XRCC1, and HSP70 under heat shock. Downregulation of DNA repair proteins could aggravate the cell damage that may cause cell apoptosis. This may explain that heat shock could increase the lethality of chemotherapeutic drugs on tumor cells.

PMID: 23001845 [PubMed - indexed for MEDLINE]

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ATF4 and IRE1? inhibit DNA repair protein DNA-dependent protein kinase 1 induced by heat shock.

Related Articles

ATF4 and IRE1? inhibit DNA repair protein DNA-dependent protein kinase 1 induced by heat shock.

Mol Cell Biochem. 2012 Dec;371(1-2):225-32

Authors: Zhu H, Guo FJ, Zhao W, Zhou J, Liu Y, Song F, Wang Y

Abstract
With the increase of environment temperature, more and more attentions are payed to the effects of heat stress. Cells under heat shock either are adapted to the condition or are damaged and dead. In this paper, we found that heat shock induced endoplasmic reticulum (ER) stress. ATF4, PERK, and IRE1? were induced by heat shock of 45 �C in the transcriptional level. Under the stress of 45 �C, PERK was phosphorylated and XBP1s was detected. The result indicated that heat shock could induce the ER stress. We found that heat shock of 45 �C induced the dysregulation of HSP70 and DNA-PKcs, and downregulated the expression of PARP1 and XRCC1. Further results showed that after the knockdown of ATF4 or IRE1?, the expression of DNA-PKcs and XRCC1 were increased. It was indicated that ATF4 and IRE1? could inhibit the expression of DNA-PKcs and XRCC1 under the heat stress. Our results suggested that heat shock could activate ER stress. IRE1? and ATF4, as the important ER stress molecules, could inhibit the expression of DNA repair proteins DNA-PKcs, XRCC1, and HSP70 under heat shock. Downregulation of DNA repair proteins could aggravate the cell damage that may cause cell apoptosis. This may explain that heat shock could increase the lethality of chemotherapeutic drugs on tumor cells.

PMID: 23001845 [PubMed - indexed for MEDLINE]

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ATF4 and IRE1? inhibit DNA repair protein DNA-dependent protein kinase 1 induced by heat shock.

Related Articles

ATF4 and IRE1? inhibit DNA repair protein DNA-dependent protein kinase 1 induced by heat shock.

Mol Cell Biochem. 2012 Dec;371(1-2):225-32

Authors: Zhu H, Guo FJ, Zhao W, Zhou J, Liu Y, Song F, Wang Y

Abstract
With the increase of environment temperature, more and more attentions are payed to the effects of heat stress. Cells under heat shock either are adapted to the condition or are damaged and dead. In this paper, we found that heat shock induced endoplasmic reticulum (ER) stress. ATF4, PERK, and IRE1? were induced by heat shock of 45 �C in the transcriptional level. Under the stress of 45 �C, PERK was phosphorylated and XBP1s was detected. The result indicated that heat shock could induce the ER stress. We found that heat shock of 45 �C induced the dysregulation of HSP70 and DNA-PKcs, and downregulated the expression of PARP1 and XRCC1. Further results showed that after the knockdown of ATF4 or IRE1?, the expression of DNA-PKcs and XRCC1 were increased. It was indicated that ATF4 and IRE1? could inhibit the expression of DNA-PKcs and XRCC1 under the heat stress. Our results suggested that heat shock could activate ER stress. IRE1? and ATF4, as the important ER stress molecules, could inhibit the expression of DNA repair proteins DNA-PKcs, XRCC1, and HSP70 under heat shock. Downregulation of DNA repair proteins could aggravate the cell damage that may cause cell apoptosis. This may explain that heat shock could increase the lethality of chemotherapeutic drugs on tumor cells.

PMID: 23001845 [PubMed - indexed for MEDLINE]

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Diseases associated with defective responses to DNA damage.

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Diseases associated with defective responses to DNA damage.

Cold Spring Harb Perspect Biol. 2012 Dec;4(12)

Authors: O'Driscoll M

Abstract
Within the last decade, multiple novel congenital human disorders have been described with genetic defects in known and/or novel components of several well-known DNA repair and damage response pathways. Examples include disorders of impaired nucleotide excision repair, DNA double-strand and single-strand break repair, as well as compromised DNA damage-induced signal transduction including phosphorylation and ubiquitination. These conditions further reinforce the importance of multiple genome stability pathways for health and development in humans. Furthermore, these conditions inform our knowledge of the biology of the mechanics of genome stability and in some cases provide potential routes to help exploit these pathways therapeutically. Here, I will review a selection of these exciting findings from the perspective of the disorders themselves, describing how they were identified, how genotype informs phenotype, and how these defects contribute to our growing understanding of genome stability pathways.

PMID: 23209155 [PubMed - indexed for MEDLINE]

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A burst of ABC genes in the genome of the polyphagous spider mite Tetranychus urticae.

Related Articles

A burst of ABC genes in the genome of the polyphagous spider mite Tetranychus urticae.

BMC Genomics. 2013 May 10;14(1):317

Authors: Dermauw W, Osborne EJ, Clark RM, Grbi M, Tirry L, Van Leeuwen T

Abstract
BACKGROUND: The ABC (ATP-binding cassette) gene superfamily is widespread across all living species. The majority of ABC genes encode ABC transporters, which are membrane-spanning proteins capable of transferring substrates across biological membranes by hydrolyzing ATP. Although ABC transporters have often been associated with resistance to drugs and toxic compounds, within the Arthropoda ABC gene families have only been characterized in detail in several insects and a crustacean. In this study, we report a genome-wide survey and expression analysis of the ABC gene superfamily in the spider mite, Tetranychus urticae, a chelicerate ~ 450 million years diverged from other Arthropod lineages. T. urticae is a major agricultural pest, and is among of the most polyphagous arthropod herbivores known. The species resists a staggering array of toxic plant secondary metabolites, and has developed resistance to all major classes of pesticides in use for its control. RESULTS: We identified 103 ABC genes in the T. urticae genome, the highest number discovered in a metazoan species to date. Within the T. urticae ABC gene set, all members of the eight currently described subfamilies (A to H) were detected. A phylogenetic analysis revealed that the high number of ABC genes in T. urticae is due primarily to lineage-specific expansions of ABC genes within the ABCC, ABCG and ABCH subfamilies. In particular, the ABCC subfamily harbors the highest number of T. urticae ABC genes (39). In a comparative genomic analysis, we found clear orthologous relationships between a subset of T. urticae ABC proteins and ABC proteins in both vertebrates and invertebrates known to be involved in fundamental cellular processes. These included members of the ABCB-half transporters, and the ABCD, ABCE and ABCF families. Furthermore, one-to-one orthologues could be distinguished between T. urticae proteins and human ABCC10, ABCG5 and ABCG8, the Drosophila melanogaster sulfonylurea receptor and ecdysone-regulated transporter E23. Finally, expression profiling revealed that ABC genes in the ABCC, ABCG ABCH subfamilies were differentially expressed in multi-pesticide resistant mite strains and in mites transferred to challenging (toxic) host plants. CONCLUSIONS: In this study we present the first comprehensive analysis of ABC genes in a polyphagous arthropod herbivore. We demonstrate that the broad plant host range and high levels of pesticide resistance in T. urticae are associated with lineage-specific expansions of ABC genes, many of which respond transcriptionally to xenobiotic exposure. This ABC catalogue will serve as a basis for future biochemical and toxicological studies. Obtaining functional evidence that these ABC subfamilies contribute to xenobiotic tolerance should be the priority of future research.

PMID: 23663308 [PubMed - as supplied by publisher]

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