The goal of this project is to identify genes preferentially expressed in inflammatory macrophages as compared with control macrophages.
Cutting Edge: IL-36 Receptor Promotes Resolution of Intestinal Damage.
Specimen part, Treatment
View SamplesTo determine spinal cord injury (SCI) induced changes in gene expression, laser capture microscopy (LCM) and Affymetrix microarrays were used to profiles three distinct populations of motor neurons (MNs) in five sets of littermates. In each set, one littermate received a lamenectomy (sham operated), and the other received a complete spinal cord transection. Each MN population was caudal to the transection and not axotomized. Thus, MNs in transected animals were responding to two major insults: deafferentation and changing microenvironments due to spreading immune and inflammatory responses. A total of 30 expression profiles from sham operated control animals have been uploaded to GEO database with accession number GSE2595. The current series contains 32 motoneuron expression profiles from spinalized mice. All chips (30 sham + 32 transection) were generated together using RMA in 2004.
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Sex, Specimen part
View SamplesThe overall cellular gene expression patterns engaged as a result of B virus (E2490) and HSV-1 (MacIntyre) infection at 1h, 3h, and 5h post infection were analyzed. Differentially regulated immune responsive genes during the early infection stage were identified.
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View SamplesThree cell types, intermediolateral column motoneurons, medial motoneurons, and lateral motoneurons were isolated from a single adult spinal cord using laser capture microscopy. Four hundred captures were collected for each cell type. For a given cell type, RNA was extracted from the 400 captures using an Arcturus picopure kit. RNA was split in half and two targets were produced using a double amplification protocol. Each target was hybridized to Affymetrix chips and signals were normalized with R-pack. Inverse logs are provided. Five animals were used in these experiments, and all three cell types were collected from each animal. Thus, for each cell type, there are five biological replicates, and for each biological replicate there are two technical replicates. In all thirty chips were analyzed. Techinical replicates are indicated as Set 1 and Set 2. Animal numbers are indicated by Pair1 through Pair 5.
Divergence between motoneurons: gene expression profiling provides a molecular characterization of functionally discrete somatic and autonomic motoneurons.
Specimen part
View SamplesAmong multiple interconnected pathways for L-Lysine (L-Lys) catabolism in pseudomonads, Pseudomonas aeruginosa PAO1 employed the decarboxylase and the transaminase pathways. However, up till now several genes involved in the operation and regulation of these pathways were still missing. Transcriptome analyses coupled with promoter activity measurements and mutant growth phenotype analysis lead us to identify several new members of the L-Lys and D-Lys catabolic pathways and their regulatory elements, including argR to trigger lysine decarboxylation into cadaverine, PauR for the -glutamylation pathway of polyamine catabolism into 5-aminovalerate, gcdR-gcdHG for glutarate utilization, dpkA, amaR-amaAB and PA2035 for D-Lys catabolism, lysR-lysXE for L-Lys efflux, and lysP for L-Lys uptake.
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View SamplesIn Caenorhabditis elegans, the six proteins that make up the REF-1 family are HES homologs that act in both Notch dependent and Notch-independent pathways to regulate embryonic events. To further our understanding of how the REF-1 family works to coordinate post-embryonic cellular events, we performed transcriptome analysis of HLH-25 and HLH-29 mutant strains.
Genome-wide microarrray analysis reveals roles for the REF-1 family member HLH-29 in ferritin synthesis and peroxide stress response.
Sex, Specimen part
View SamplesD-Glutamate (D-Glu), an essential component of peptidoglycans, can be utilized as carbon and nitrogen source by Pseudomonas aeruginosa. DNA microarrays were employed to identify genes involved in D-Glu catabolism. Through gene knockout and growth phenotype analysis, the divergent dguR-dguABC (D-glutamate utilization) gene cluster was shown to participate in D-Glu catabolism and regulation. The dguA gene encodes a FAD-dependent D-amino acid dehydrogenase with D-Glu as its preferred substrate, and its promoter was specifically induced by exogenous D-Glu and D-Gln. The function of DguR as transcriptional activator of the dguABC operon was demonstrated by promoter activity measurements in vivo and by mobility shift assays with purified His-tagged DguR in vitro. Although the DNA-binding activity of DguR did not require D-Glu, the presence of D-Glu in the binding reaction was found to stabilize a preferred nucleoprotein complex. The dguB gene encodes a putative enamine/imine deaminase of the RidA family, but its role in D-Glu catabolism remained to be determined. While a lesion in dguC encoding a periplasmic solute binding protein did not affect growth on D-Glu, the AatJMQP transporter for acidic amino acid uptake was found essential for D-Glu and L-Glu utilization. Expression of this uptake system was subjected to induction by exogenous DL-Glu, most likely via the AauSR two-component system. In summary, DguA was identified in this study as a new member of the FAD-dependent amino acid dehydrogenase family for D-amino acid catabolism. DguR serves as a D-Glu sensor and transcriptional activator of the dguA promoter.
Functional characterization of the dguRABC locus for D-Glu and d-Gln utilization in Pseudomonas aeruginosa PAO1.
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View SamplesPolyamines (putrescine, spermidine, and spermine) are major organic polycations essential for a wide spectrum of cellular processes. The cells require mechanisms to maintain homeostasis of intracellular polyamines to prevent otherwise severe adverse effects. We performed a detailed transcriptome profile analysis of P. aeruginosa in response to agmatine and putrescine with an emphasis in polyamine catabolism. Agmatine serves as precursor compound for putrescine (and hence spermidine and spermine), which was proposed to convert into GABA and succinate before entering the TCA cycle in support of cell growth as the sole source of carbon and nitrogen. Two acetylpolyamine amidohydrolases, AphA and AphB, were identified to be involved in the conversion of agmatine into putrescine. Enzymatic products of AphA were confirmed by mass spectrometry analysis. Interestingly, the alanine-pyruvate cycle was shown indispensable for polyamine utilization. The newly identified dadRAX locus, encoding the regulator, alanine transaminase and racemase respectively, coupled with SpuC, the major putrescine-pyruvate transaminase, were key components to maintain alanine homeostasis. Corresponding mutant strains were severely hampered in polyamine utilization. On the other hand, the alternative gamma-glutamylation pathway for the conversion of putrescine into GABA was also discussed. Subsequently, GabD, GabT and PA5313 were identified for GABA utilization. Growth defect of PA5313 gabT double mutant in GABA suggested the importance of these two transaminases. The succinic-semialdehyde dehydrogenase activity of GabD and its induction by GABA was also demonstrated in vitro. Polyamine utilization in general was proven independent of the PhoPQ two-component system even the expression of which was induced by polyamines. Multiple potent catabolic pathways as depicted in this study could serve pivotal roles in control of intracellular polyamine levels.
Transcriptome analysis of agmatine and putrescine catabolism in Pseudomonas aeruginosa PAO1.
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View SamplesPauA2 plays an essential role in spermine catabolism and that exogenous spermine exerts a bactericidal effect on the pauA2 mutant of P. aeruginosa. Not only subjected to growth inhibition by spermine, the pauA2 mutant without a functional -glutamylpolyamine synthetase PauA2 became more sensitive to -lactam antibiotics in human serum. To explore PauA2 as a potential target of drug development, suppressors of the pauA2 mutant were isolated from selection plates containing spermine. These suppressors share common changes in various phenotypes. Genome resequencing of a representative suppressor revealed a unique mutation at the phoU gene, and a constitutive expression of the Pho regulon as evidenced by measurements of transcriptome analysis.
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View SamplesHydroxy-L-proline (L-Hyp) mainly exists in animal collagens through post-translational modification on peptidyl proline. Utilization of L-Hyp as carbon and nitrogen sources in bacteria including P. aeruginosa requires conversion of L-Hyp to D-Hyp followed by the D-Hyp dehydrogenase pathway. However, the molecular mechanism in control of L-Hyp catabolism and transport at the genetic level was not clear. We performed a detailed transcriptome profile analysis of P. aeruginosa in response to L-Hyp and D-Hyp with an emphasis in catabolism and regulation, which revealed the presence of twelve genes in two adjacent loci that were induced by exogenous L-Hyp and D-Hyp. The first locus includes lhpABFE encoding a Hyp epimerase (LhpA) and D-Hyp dehydrogenase (LhpBEF), while the second locus covers genes for a putative ABC transporter (LhpPMNO), a protein of unknown function (LhpH), Hyp/Pro racemase (LhpK), and two enzymes in L-Hyp catabolism (LhpC and LhpG). In addition, proximal to these two loci, lhpR encodes a transcriptional regulator of the AraC family.
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