Cell line adaptation Changes in the proteome level during a step-wise adaptation of an adherent Madin Darby canine kidney (MDCK) cell line
ABSTRACT: In this work, changes in the proteome level during a step-wise adaptation of an adherent Madin Darby canine kidney (MDCK) cell line to suspension growth and chemically defined medium were analyzed.
Project description:The current study aimed to detect and identify significant differentially expressed proteins between a virulent and an attenuated Histomonas meleagridis strain which was in vitro co-cultivated with Escherichia coli DH5α. Two-dimensional gel electrophoresis (2-DE) was used for proteome visualization , gel image software for computational detection of significantly up-regulated protein spots and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) for protein identification. The statistical analysis fulfilling two criteria (> or = 3-fold up-regulation and P<0.05) detected 119 differentially expressed protein spots out of which 62 spots were located in gels of the virulent strain and 57 spots in gels of the attenuated strain. The mass spectrometric analysis of 32 spots, up-regulated in gels of the virulent strain, showed that they are of H. meleagridis origin. As opposed to this, the mass spectrometric analysis of 49 protein spots , up-regulated in the gels of the attenuated strain , identified 32 spots as specific to the protozoan. Additionally, the analysis identified a number of E. coli DH5α proteins which were detected as differentially expressed by the computational gel image and statistical analysis.
Project description:A custom multi-species microarray was used to study gene expression in wild hornyhead turbot (Pleuronichthys verticalis), collected from polluted and clean coastal waters in Southern California and in laboratory male zebrafish (Danio rerio) following exposure to estradiol and 4-nonylphenol. A multi-gene cross species microarray was fabricated as a diagnostic tool to screen the effects of environmental chemicals in fish, for which there is minimal genomic information. The microarray measurement of gene expression in zebrafish, which are phylogenetically distant from turbot, indicates that this multi-species microarray will be useful for measuring endocrine responses in Pleuronectiformes and other fish for which there is minimal genomic sequence information.
Project description:Nuclear proteins of developing wheat grains collected during the cellularization, effective grain-filling and maturation phases of development were analysed.. Nuclear proteins were extracted and separated by two-dimensional gel electrophoresis. Image analysis revealed 371 and 299 reproducible spots in gels with first dimension separation along pH 4-7 and pH 6-11 isoelectric gradients, respectively. The relative abundance of 464 (67%) protein spots changed during grain development. Abundance profiles of these proteins clustered in six groups associated with the major phases and phase transitions of grain development. Using nano liquid chromatography-tandem mass spectrometry to analyse 387 variant and non-variant protein spots, 114 different proteins were identified that were classified into 16 functional classes. We noted that some proteins involved in the regulation of transcription, like HMG1/2-like protein and histone deacetylase HDAC2, were most abundant before the phase transition from cellularization to grain-filling, suggesting that major transcriptional changes occur during this key developmental phase. The maturation period was characterized by high relative abundance of proteins involved in ribosome biogenesis.
Project description:Global transcriptome patterns were determined in XVE-14 and wild-type seedlings induced for 45 min b-estradiol in order to identify the genes early regulated by EBE transcription factor. We used microarrays to identify genes differentially expressed in EST-inducible EBE over-expression line #14 compared to wild-type plants, 45 min after 2µM EST induction. Three independent biological replications were performed. In order to identify potential direct/early target genes of EBE transcription factor, estradiol inducible overexpression system was used. Three week old Arabidopsis EBE-XVE (line 14) and WT seedlings were treated with ß-estradiol (2µM) for 45 min. RNA was isolated from shoot and subjected to hybridization on Affymetrix microarrays. Experiment was performed in 3 biological replications and genes differentially expressed between estradiol treated EBE-XVE and WT plants were identified as potential early targets of EBE.
Project description:Ovarian cancer is the 5th main cause of death from cancer in females, worldwide. An early and accurate diagnosis of ovarian carcinoma is likely to improve clinical outcome of patients in terms of reduced morbidity and mortality. The objective of our project is the proteome profiling of ovarian malignant and non-malignant tissue samples to find potential biomarkers of disease diagnosis and prognosis. In this project, we have used a systematic protein profiling approach involving 2-dimensional gel electrophoresis coupled with MALDI-TOF MS analysis for identification of biomarkers of ovarian cancer. The associated research paper is in preparation.
Project description:The goal of this study is to identify ERalpha-target genes affected by overexpression of the histone arginine methyltransferase CARM1 in breast cancer cells. The roles of CARM1 in ERalpha+ breast cancer was not well characterized. Therefore, we created a Dox inducible CARM1 overexpressing MCF7 cell line where CARM1 is overexpressed by 2 fold to determine the created a Dox-inducible CARM1 overexpressing MCF7 cells for evaluation of the global effects of CARM1 on Eralpha-target gene expression. MCF7-tet-on-CARM1 clone 13 were treated under 4 conditions: DMSO; Dox; E2 (10nM); Dox+E2. In Dox+E2 condition, cells were pre-treated with Dox for 5 days before treating with E2 for 4 hours. 3 biological replicates were included and total of 12 samples were analyzed.
Project description:Global transcriptome patterns were performed using ORE1-IOE-2h (2h after Estradiol and Mock treatment) as well as transiently (6h) overexpressed Arabidopsis mesophyll cell protoplasts To identify genes more rapidly responding to elevated ORE1 expression we here repeated the previous experiment (Balazadeh et al., 2010), but shortened the EST induction time to 2 h (ORE1-IOE-2h dataset). Furthermore, to exclude potential misinterpretation due to EST treatment we also included an experiment where we transiently expressed a 35S::ORE1 construct in Arabidopsis mesophyll cell protoplasts and extracted RNA 6 h after the transfection (35S::ORE1-6h dataset);
Project description:Signal intensities of BC-GP array for 105 speimens The Verigene Gram-Positive Blood Culture (BC-GP) nucleic acid assay is an automated microarray-based test, which can detect 12 Gram-positive bacterial genes and 3 resistance determinants using blood culture broths. We investigated signal intensities of microarray spots, and reclassified undetermined results where the automated system failed and various errors were called in blood culture specimens and spiked samples. Signal intensity analysis of BC-GP assay. SAMPLE_077 [Blood culture, Escherichia coli, Gram-negative, BacTALERT, Supernatant, Peripheral blood] had no signal data in the array raw data. Thus, SAMPLE_077 is not represented in this Series.
Project description:Glucose-stimulated insulin secretion (GSIS) is suppressed through α-adrenergic receptor stimulation by catecholamines, epinephrine and norepinephrine, in pancreatic β-cells. Previous work has elucidated a bevy of adrenergic regulatory mechanisms beyond traditional Gi-coupled signaling including regulation of ion channels and interactions with exocytotic machinery. Glucose oxidation may also be an important site for adrenergic regulation of GSIS, but the link between epinephrine and glucose oxidation in β-cells is undefined. Here, we evaluate whether adrenergic stimulation decreases oxidative metabolism in β cells. Oxygen consumption rates were determined for Min6 and isolated rat islets in 20mM glucose complete media, then epinephrine was added at either 0 nM (vehicle control) or 100nM, followed by 10uM yohimbine (a selective Adrα2A antagonist). To identify glucose oxidation as the primary metabolic pathway affected by epinephrine, oxidation of 14C(U)-labeled glucose was determined in Min6 cells with epinephrine or vehicle. Oxygen consumption and glucose oxidation experiments were conducted in the presence of cAMP and insulin secretion blockers, respectively. Proteomics was performed on Min6 cells exposed to epinephrine for 4 hours and compared to controls. Epinephrine, but not vehicle, reduced (P<0.01) oxygen consumption rates in rat islets and Min6 cells to 64 ± 6% and 65 ± 1% of baseline, respectively, and yohimbine restored oxygen consumption to rates not different from baseline. In Min6 cells incubated with epinephrine rates of 14C glucose oxidation were reduced (P<0.01) 66 ± 4% compared to vehicle controls. These results demonstrate that acute epinephrine exposure suppresses glucose oxidation in β cells via the specific adrenergic receptor, Adrα2A, and indicate a new role for adrenergic regulation in GSIS.