In the growth plate, the reserve and perichondral zones have been hypothesized to have similar functions, but their exact functions are poorly understood. Our hypothesis was that significant differential gene expression exists between perichondral and reserve chondrocytes that may differentiate the respective functions of these two zones. Normal Sprague-Dawley rat growth plate chondrocytes from the perichondral zone (PC), reserve zone (RZ), proliferative zone (PZ), and hypertrophic zone (HZ) were isolated by laser microdissection and then subjected to microarray analysis. In order to most comprehensively capture the unique features of the two zones, we analyzed both the most highly expressed genes and those that were most significantly different from the proliferative zone (PZ) as a single comparator.
Microarray analysis of perichondral and reserve growth plate zones identifies differential gene expressions and signal pathways.
No sample metadata fields
View SamplesPseudomonas aeruginosa is an opportunistic pathogen that can adapt to changing environments and can secrete an exopolysaccharide known as alginate as a protection response resulting in a colony morphology and phenotype referred to as mucoid. However how P. aeruginosa senses its environment and activates alginate overproduction is not fully understood. Previously, we showed that Pseudomonas isolation agar (PIA) supplemented with ammonium metavanadate (PIAAMV) induces P. aeruginosa to overproduce alginate. Vanadate is a phosphate mimic and causes protein misfolding by disruption of disulfide bonds. Here we used PIAAMV to characterize the pathways involved in inducible alginate production and tested the global effects of P. aeruginosa growth on PIAAMV by a mutant library screen, transcriptomics, and in a murine acute virulence model. The PA14 non-redundant mutant library was screened on PIAAMV to identify new genes that are required for the inducible alginate stress response. A functionally diverse set of genes encoding products involved in cell envelope biogenesis, peptidoglycan, uptake of phosphate and iron, phenazines biosynthesis, and other processes were identified as positive regulators of the mucoid phenotype on PIAAMV. Transcriptome analysis of P. aeruginosa growing in the presence of vanadate caused differential expression of genes involved in virulence, envelope biogenesis, and cell stress pathways. In this study, it was observed that growth on PIAAMV attenuates P. aeruginosa in a mouse pneumonia model. Induction of alginate overproduction occurs as a stress response to protect P. aeruginosa but it may be possible to modulate and inhibit these pathways based on the new genes identified in this study.
Genes required for and effects of alginate overproduction induced by growth of Pseudomonas aeruginosa on Pseudomonas isolation agar supplemented with ammonium metavanadate.
No sample metadata fields
View SamplesAlginate overproduction by P. aeruginosa, also known as mucoidy is associated with chronic endobronchial infections in cystic fibrosis (CF). Alginate biosynthesis in this bacterium is initiated by the extracytoplasmic function sigma factor (22, AlgU/T). In the wild type (wt) nonmucoid strains, such as PAO1, AlgU is sequestered by the anti-sigma factor MucA that inhibits alginate production. However, the degradation of MucA by activated intramembrane proteases AlgW and/or MucP can lead to the conversion from nonmucoid strains to mucoid. Previously we reported that the absence of the sensor kinase KinB in PAO1 causes the initiation of AlgW-dependent proteolysis of MucA resulting in alginate overproduction. In the kinB mutant this activation requires alternate sigma factor RpoN (54). To determine the RpoN-dependent KinB regulon, microarray and proteomic analyses were performed on a mucoid kinB mutant and an isogenic nonmucoid kinB rpoN double mutant. In the kinB mutant, RpoN controlled the expression of approximately 20% of the genome. Besides alginate biosynthesis and regulator genes such as AlgW, KinB, in concert with RpoN, also control a large number of genes including: those involved in carbohydrate metabolism, quorum sensing, iron regulation, rhamnolipid production, and motility. In an acute pneumonia murine infection model, mice exhibited better survival when challenged with the kinB mutant than wt PAO1. Together, these data strongly suggest that KinB controls virulence factors important for acute pneumonia and conversion to mucoidy.
Analysis of the Pseudomonas aeruginosa regulon controlled by the sensor kinase KinB and sigma factor RpoN.
Treatment
View SamplesThe Pseudomonas aeruginosa response regulator AlgR is critical for the organism's virulence and controls up to 155 different genes. In order to determine which genes are controlled by phosphorylated and unphosphorylated AlgR, phosphomimetic and phosphoablative alleles were recombined onto the chromosome of PAO1. The algR gene was mutated at aspartate 54 to asparagine (D54N) for the phosphoablative allele and mutated at aspartete 54 to glutamate (D54E) for the phosphomimetic allele. These alleles were recombined into the PAO1 chromosome.
<i>Pseudomonas aeruginosa</i> AlgR Phosphorylation Status Differentially Regulates Pyocyanin and Pyoverdine Production.
No sample metadata fields
View SamplesThis SuperSeries is composed of the SubSeries listed below.
Human decidual NK cells from gravid uteri and NK cells from cycling endometrium are distinct NK cell subsets.
Specimen part
View SamplesFragmented RNA cocktails from FACS sorted Human decidual NK cell, and peripheral blood CD56Bright and CD56Dim NK cells, previously hybridization to HGU95AV2 chips (Koopman et al J Exp Med. 2003 Oct 20;198(8):1201-1), were stored long term at -80C, thawed and hybridized to HG-U133A arrays.
Human decidual NK cells from gravid uteri and NK cells from cycling endometrium are distinct NK cell subsets.
Specimen part
View SamplesFragmented RNA cocktails from FACS sorted Human decidual NK cell, and peripheral blood CD56Bright and CD56Dim NK cells, previously hybridization to HGU95AV2 chips (Koopman et al J Exp Med. 2003 Oct 20;198(8):1201-1), were stored long term at -80C, thawed and hybridized to HG-U133B arrays.
Human decidual NK cells from gravid uteri and NK cells from cycling endometrium are distinct NK cell subsets.
Specimen part
View SamplesHuman NK cells from the decidua basalis of gravid uteri (dNK) and from cycling endometrium (eNK) of women undergoing hysterectomy were isolated and compared by gene expression profiling using Affymetrix microarrays with probes representing ~47,400 transcripts. Substantial differences indicate that these two types of NK cells represent distinct subsets.
Human decidual NK cells from gravid uteri and NK cells from cycling endometrium are distinct NK cell subsets.
Specimen part
View SamplesSpinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disorder, which is caused by an unstable CAG-repeat expansion in the SCA2 gene, that encodes a polyglutamine tract (polyQ-tract) expansion in ataxin-2 protein (ATXN2). The RNA-binding protein ATXN2 interacts with the poly(A)-binding protein PABPC1, localizing to ribosomes at the rough endoplasmic reticulum or to polysomes. Under cell stress ATXN2 and PABPC1 show redistribution to stress granules where mRNAs are kept away from translation and from degradation. It is unknown whether ATXN2 associates preferentially with specific mRNAs or how it modulates their processing. Here, we investigated Atxn2 knock-out (Atxn2-/-) mouse liver, cerebellum and midbrain regarding their RNA profile, employing oligonucleotide microarrays for screening and RNA deep sequencing for validation. Modest ~1.4-fold upregulations were observed for the level of many mRNAs encoding ribosomal proteins and other translation pathway factors. Quantitative reverse transcriptase PCR and immunoblots in liver tissue confirmed these effects and demonstrated an inverse correlation also with PABPC1 mRNA and protein. ATXN2 deficiency also enhanced phosphorylation of the ribosomal protein S6, while impairing the global protein synthesis rate, suggesting a block between the enhanced translation drive and the impaired execution. Furthermore, ATXN2 overexpression and deficiency retarded cell cycle progression. ATXN2 mRNA levels showed a delayed phasic twofold increase under amino acid and serum starvation, similar to ATXN3, but different from motor neuron disease genes MAPT and SQSTM1. ATXN2 mRNA levels depended particularly on mTOR signalling. Altogether the data implicate ATXN2 in the adaptation of mRNA translation and cell growth to nutrient availability and stress.
Genetic ablation of ataxin-2 increases several global translation factors in their transcript abundance but decreases translation rate.
Age, Specimen part
View SamplesPleomorphic adenoma gene 1 (PLAG1) encodes a transcription factor involved in cancer and growth. We study the role of PLAG1 in preimplantation embryos using STRT RNA-seq of single embryos from wild type and knockout mothers (both mated with wild type studs). The lack of maternal Plag1 led to delayed mouse 2-cell stage embryo development, compensatory expression of Plag1 from the paternal allele, and dysregulation of 1,089 genes. Half of these genes displayed a pattern of delayed activation and play roles in ribosome biogenesis and protein synthesis. These mouse genes further showed a significant overlap with human EGA genes with similar ontology, and an enrichment of the PLAG1 de novo motif. We conclude that Plag1 affects EGA through retrotransposons influencing ribosomes and protein synthesis, a mechanism that might also explain its roles in cancer and growth Overall design: Single wild type and maternal Plag1 knockout embryos at MII, 2-cell and 8-cell stage development in 14-16 biologicla replicas per developmental stage and genotype.
Pleomorphic Adenoma Gene 1 Is Needed For Timely Zygotic Genome Activation and Early Embryo Development.
Subject
View Samples