Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Effects of overexpression of EhMyb-dr on transcription in Entamoeba histolytica

ABSTRACT: A myb domain containing protein of the SHAQKY family, EhMyb-dr, was overexpressed in E. histolytica trophozoites, and effects on transcription were measured. Two arrays of trophozoites expressing Myc tagged EhMyb-dr were compared to two arrays performed on wild type trophozoites. The effects of transfection and drug were controlled for by including data from trophozoites expressing irrelevant plasmids, and grown in the same level of G418. All parasites were of strain HM-1:IMSS

ORGANISM(S): Entamoeba histolytica

SUBMITTER: Gretchen Ehrenkaufer

PROVIDER: E-GEOD-13023 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

A developmentally regulated Myb domain protein regulates expression of a subset of stage-specific genes in Entamoeba histolytica.

Ehrenkaufer Gretchen M GM Hackney Jason A JA Singh Upinder U

Cellular microbiology 20090222 6

Conversion between a cyst and trophozoite stage is essential to disease transmission and pathogenesis in the parasitic protist Entamoeba histolytica. A transcriptomic analysis of E. histolytica cysts and trophozoites has recently been accomplished, but the molecular basis of the regulation of encystation is not known. We have now identified a developmentally regulated Myb protein (belonging to the SHAQKY family of Myb proteins), which controls expression of a subset of amoebic stage-specific gen ...[more]

PMID: 19239479

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Project description:Objective: L-type calcium channels (LTCC) homeostatically regulate calcium on a beat by beat basis, but also provide Ca that over long time scales may contribute to transcriptional regulation. We previously showed that sustained LTCC blockade (CCB) elicits LTCC remodeling in ventricular cardiac myocytes (CM). Here we hypothesize that sustained CCB has broad effects on the expression of genes involved in calcium handling. Methods and Results: Therefore, we subjected adult mice to sustained CCB for 24 hours and performed gene expression profiling. In comparison to vehicle-only control animals, 231 genes were up-regulated, and 111 genes were down-regulated by sustained LTCC blockade (p <0.01). Gene ontology analysis suggested that the CaMKIIdelta signaling pathway was up-regulated in these cells. Unexpectedly, phosphorylation of phospholamban (PLN) at threonine17 (Thr17), an index of CaMKIIdelta activity, was not changed by sustained CCB; however, the degree of phosphorylation of the neighboring PLN-Ser16 substrate site for PKA was significantly reduced by sustained CCB compared to control. Gene expression profiling suggested no change in PKA, but it showed that protein phosphatase 2A (PP2A) mRNA increased, and immunoblots demonstrated that PP2Ac-alpha protein was significantly increased by sustained CCB. Consistent with elevated PP2Ac-alpha protein expression LTCC exhibited decreased phosphorylation of the C-terminal Ser1928 PKA substrate site. Conclusions: We conclude that sustained CCB elicits a spectrum of transcriptional events, including compensatory up-regulation of LTCC and PP2Ac-alpha. Although this study is restricted to mouse, these results suggest the new hypothesis that clinically-relevant sustained LTCC blockade in humans results in changes in gene regulation in the heart. Keywords: L-type calcium channel, calcium channel blockade, verapamil Female and male ICR mice (12-14 weeks age) weighing between 25 and 30 grams were anesthetized with a ketamine/xylazine mixture ( i.p.) allowing the subcutaneous implantation of miniosmotic pumps (Alzet, model 2001). The pumps were filled with either verapamil or vehicle (0.02% ascorbic acid). Control animals carried mini-pumps with vehicle and control animals were investigated in parallel with each set of experimental animals. Mini pumps delivered verapamil at 3.6 mg/kg/day for 24 (RNA)-48 (protein) hours. After treatment animals were anesthetized and weighed. Hearts were excised, rinsed, blotted dry, weighed, and then frozen on dry ice and the stored at -80oC until studied. Animals were anesthetized and euthanized according to animal protocols approved by the University of Kentucky Institutional Animal Care and Use Committee. This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication NO. 85-23, revised 1996). Left ventricular free wall from female mice was rapidly excised and either snap frozen at -80oC or used immediately for RNA isolation. Three VER treated mice and 3 vehicle treated mice were used to generate RNA for microarray. Total RNA was isolated using the RNAqueous -4PCR kit (Ambion) and quantitated spectrophotometrically at 260nm. Contaminating genomic DNA was eliminated by DNase treatment (Ambion). RNA quality was assessed using the Agilent 2100 Bioanalyzer. Microarray data was obtained using the Affymetrix 430 V2 GeneChip (representing 45,101 probe sets), in accordance with the manufacturer’s specifications.

Dataset Information

Effects of overexpression of EhMyb-dr on transcription in Entamoeba histolytica

Publications

A developmentally regulated Myb domain protein regulates expression of a subset of stage-specific genes in Entamoeba histolytica.

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