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Biomedical subjects

Barbara J Ebersole

Publications and source records attributed to Barbara J Ebersole.

11 recordsLinked to original sources
PubMed2006

Influenza virus evades innate and adaptive immunity via the NS1 protein.

Both antibodies and T cells contribute to immunity against influenza virus infection. However, the generation of strong Th1 immunity is crucial for viral clearance. Interestingly, we found that human dendritic cells (DCs) infected with influenza A virus have lower allospecific Th1-cell stimulatory abilities than DCs activated by other stimuli, such as lipopolysaccharide and Newcastle disease virus infection. This weak stimulatory activity correlates with a suboptimal maturation of the DCs following infection with influenza A virus. We next investigated whether the influenza A virus NS1 protein could be responsible for the low levels of DC maturation after influenza virus infection. The NS1 protein is an important virulence factor associated with the suppression of innate immunity via the inhibition of type I interferon (IFN) production in infected cells. Using recombinant influenza and Newcastle disease viruses, with or without the NS1 gene from influenza virus, we found that the induction of a genetic program underlying DC maturation, migration, and T-cell stimulatory activity is specifically suppressed by the expression of the NS1 protein. Among the genes affected by NS1 are those coding for macrophage inflammatory protein 1beta, interleukin-12 p35 (IL-12 p35), IL-23 p19, RANTES, IL-8, IFN-alpha/beta, and CCR7. These results indicate that the influenza A virus NS1 protein is a bifunctional viral immunosuppressor which inhibits innate immunity by preventing type I IFN release and inhibits adaptive immunity by attenuating human DC maturation and the capacity of DCs to induce T-cell responses. Our observations also support the potential use of NS1 mutant influenza viruses as live attenuated influenza virus vaccines.

Animals↗
PubMed2005

Transcript profiling of immediate early genes reveals a unique role for activating transcription factor 3 in mediating activation of the glycoprotein hormone alpha-subunit promoter by gonadotropin-releasing hormone.

Recent studies profiling immediate early gene responses to GnRH in the LbetaT2 gonadotrope cell model revealed increased expression of numerous genes including activating transcription factor (ATF) 3. The present studies demonstrate similar results with GnRH administration in vivo in ovariectomized mice. In this model, ATF3 mRNA was markedly up-regulated at 20, 40, and 60 min after in vivo administration of a GnRH analog. In alphaT3-1 gonadotrope cells, ATF3 mRNA and protein were induced by GnRH in a manner consistent with in vivo observations. Pharmacological studies implicated a combined role for the activities of protein kinase C isozymes, ERK and c-Jun N-terminal kinase, in modulating ATF3 expression. The role of ATF3 was further investigated in the activation of the human glycoprotein hormone alpha-subunit gene promoter. GnRH induced the alpha-subunit promoter-luciferase reporter approximately 16-fold, and this induction was completely abolished with mutations in the dual cAMP response elements (CREs) or the combined inhibition of GnRH-induced ERK and c-Jun N-terminal kinase. GnRH induced recruitment of ATF3, c-Jun, and c-Fos to the dual CREs. Overexpression and specific knockdown of ATF3 by small inhibitory RNA implicate a functional role for ATF3 in mediating activation of the alpha-subunit gene promoter. These studies provide clear evidence that ATF3 is a key immediate early gene induced by GnRH administration in vivo and in the alphaT3-1 gonadotrope cell model. These studies support the conclusion that the dual CREs of the human alpha-subunit promoter are the target of GnRH-induced MAPK regulation through ATF3.

Activating Transcription Factor 3↗
PubMed2004

Mutational analysis of the serotonin receptor 5HT2c in severe early-onset human obesity.

Deletion of the serotonin receptor 5HT2c in mice results in increased food intake and obesity. We screened 95 individuals with severe early-onset obesity for mutations in the coding sequence of this gene. We found a novel missense variant c.1255A > G (Thr419Ala) in a single Caucasian subject that was not found in 192 Caucasian control subjects. In transiently-transfected COS cells, the Thr419Ala variant was indistinguishable from the wild-type receptor in its ability to generate inositol phosphate, although differences in coupling to other pathways were not excluded. Three previously unreported silent variants: IVS3 + 30G > A, IVS3 + 80C > G and IVS4 - 31A > G were found with prevalences of 11.5%, 0.5% and 17.9%, respectively. In conclusion, mutations in 5HT2c are unlikely to be a common cause of severe early-onset human obesity. The identification of several novel polymorphisms at this locus may aid future genetic epidemiological studies.

Animals↗
PubMed2003

Transcriptome fingerprints distinguish hallucinogenic and nonhallucinogenic 5-hydroxytryptamine 2A receptor agonist effects in mouse somatosensory cortex.

Most neuropharmacological agents and many drugs of abuse modulate the activity of heptahelical G-protein-coupled receptors. Although the effects of these ligands result from changes in cellular signaling, their neurobehavioral activity may not correlate with results of in vitro signal transduction assays. 5-Hydroxytryptamine 2A receptor (5-HT2AR) partial agonists that have similar pharmacological profiles differ in the behavioral responses they elicit. In vitro studies suggest that different agonists acting at the same receptor may establish distinct patterns of signal transduction. Testing this hypothesis in the brain requires a global signal transduction assay that is applicable in vivo. To distinguish the cellular effects of the different 5-HT2AR agonists, we developed an assay for global signal transduction on the basis of high throughput quantification of rapidly modulated transcripts. Study of the responses to agonists in human embryonic kidney 293 cells stably expressing 5-HT2ARs demonstrated that each agonist elicits a distinct transcriptome fingerprint. We therefore studied behavioral and cortical signal transduction responses in wild-type and 5-HT2AR null-mutant mice. The hallucinogenic chemicals (+/-)-2,5-dimethoxy-4-iodoamphetamine (DOI) and lysergic acid diethylamide (LSD) stimulated a head-twitch behavioral response that was not observed with the nonhallucinogenic lisuride hydrogen maleate (LHM) and was absent in receptor null-mutant mice. We also found that DOI, LSD, and LHM each induced distinct transcriptome fingerprints in somatosensory cortex that were absent in 5-HT2AR null-mutants. Moreover, DOI and LSD showed similarities in the transcriptome fingerprints obtained that were not observed with the behaviorally inactive drug LHM. Our results demonstrate that chemicals acting at the 5-HT2AR induce specific cellular response patterns in vivo that are reflected in unique changes in the somatosensory cortex transcriptome.

Animals↗
PubMed2003

Molecular basis of partial agonism: orientation of indoleamine ligands in the binding pocket of the human serotonin 5-HT2A receptor determines relative efficacy.

Based on experiment and computational simulation, we present a structural explanation for the differing efficacies of indole agonists at the human serotonin 5-HT2A receptor (5HT2AR). We find that serotonin [5-hydroxytryptamine (5-HT)] forms hydrogen-bonds with Ser3.36 in helix 3 and Ser5.46 in helix 5. Disruption of these hydrogen bonds by methyl-substitution of the cationic primary amine or of the backbone N1-amine, respectively, leads to a reduction in agonist efficacy. Computational simulation predicts that mutation of Ser3.36 to Ala should allow a similar interaction with helix 3 both for agonists that have unmodified cationic amine side chains and for those with substituted amines. Experimentally, this mutation was found to largely eliminate the differences in efficacy caused by cationic amine substitution for a series of indole congeners. Similarly, substitution of the N1-amine, which interacts with Ser5.46, reduced efficacy more markedly at the wild-type (WT) than at the Ser5.46Ala mutant receptor. Computational modeling of binding pocket interactions of ligands with WT and mutant receptor constructs demonstrate how the Ser3.36 and Ser5.46 interactions serve to modify the agonist's favored position in the binding pocket. A striking correlation was found between differences in the position assumed by the indole ring and differences in agonist activity. These data support the hypothesis that the position of the agonist interacting with the receptor is influenced by specific interactions in helices 3 and 5 and determines the degree of receptor activation by agonist through a mechanism that is likely to be shared by other G-protein coupled receptors in this class.

Alanine↗
PubMed2002

Conserved helix 7 tyrosine acts as a multistate conformational switch in the 5HT2C receptor. Identification of a novel "locked-on" phenotype and double revertant mutations.

Studies in many rhodopsin-like G-protein-coupled receptors are providing a general scheme of the structural processes underlying receptor activation. Microdomains in several receptors have been identified that appear to function as activation switches. However, evidence is emerging that these receptor proteins exist in multiple conformational states. To study the molecular control of this switching process, we investigated the function of a microdomain involving the conserved helix 7 tyrosine in the serotonin 5HT2C receptor. This tyrosine of the NPXXY motif was substituted for all naturally occurring amino acids. Three distinct constitutively active receptor phenotypes were found: moderate, high, and "locked-on" constitutive activity. In contrast to the activity of the other receptor mutants, the high basal signaling of the locked-on Y7.53N mutant was neither increased by agonists nor decreased by inverse agonists. The Y7.53F mutant was uncoupled. Computational modeling based on the rhodopsin crystal structure suggested that Y7.53 interacts with the conserved aromatic ring at position 7.60 in the recently identified helix 8 domain. This provided a basis for seeking revertant mutations to correct the defective function of the Y7.53F receptor. When the Y7.53F receptor was mutated at position 7.60, the wild-type phenotype was restored. These results suggest that Y7.53 and Y7.60 contribute to a common functional microdomain connecting helices 7 and 8 that influences the switching of the 5HT2C receptor among multiple active and inactive conformations.

Amino Acid Motifs↗
PubMed2002

Accuracy and calibration of commercial oligonucleotide and custom cDNA microarrays.

We compared the accuracy of microarray measurements obtained with oligonucleotide arrays (GeneChip, Affymetrix) with a laboratory-developed cDNA array by assaying test RNA samples from an experiment using a paradigm known to regulate many genes measured on both arrays. We selected 47 genes represented on both arrays, including both known regulated and unregulated transcripts, and established reference relative expression measurements for these genes in the test RNA samples using quantitative reverse transcriptase real-time PCR (QRTPCR) assays. The validity of the reproducible (average coefficient of variation = 11.8%) QRTPCR measurements were established through application of a new mathematical model. The performance of both array platforms in identifying regulated and non-regulated genes was identical. With either platform, 16 of 17 definitely regulated genes were correctly identified, and no definitely unregulated transcript was falsely identified as regulated. Accuracy of the fold-change measurements obtained with each platform was assessed by determining measurement bias. Both platforms consistently underestimate the relative changes in mRNA expression between experimental and control samples. The bias observed with cDNA arrays was predictable for fold-changes <250-fold by QRTPCR and could be corrected by the calibration function F(c) = F(a(cDNA))(q), where F(a(cDNA)) is the microarray-determined fold-change comparing experimental with control samples, q is the correction factor and F(c) is the calibrated value. The bias observed with the commercial oligonucleotide arrays was less predictable and calibration was unfeasible. Following calibration, fold-change measurements generated by custom cDNA arrays were more accurate than those obtained by commercial oligonucleotide arrays. Our study demonstrates systematic bias of microarray measurements and identifies a calibration function that improves the accuracy of cDNA array data.

Animals↗
PubMed2002

Validated genomic approach to study differentially expressed genes in complex tissues.

Microarray-based genomic techniques allow the simultaneous determination of relative levels of expression of a large number of genes. Studies of the transcriptome in complex neurobiological systems are uniquely demanding due to the heterogeneous nature of these cells. Most brain regions contain a large variety of cell populations that are closely intermingled. The expression of any specific gene may be restricted to a subpopulation of cells, and changes in gene expression may occur in only a small fraction of the cells expressing that transcript. Due to this dilution effect, many genes of interest are expected to have relatively low levels of expression in tissue homogenates. Furthermore, biologically significant differences in expression may result in only small fold-changes. Therefore genomic approaches using brain dissections must be optimized to identify potentially regulated transcripts and differential expression should be confirmed using quantitative assays. We evaluated the effects of increasing tissue complexity on detection of regulated transcripts in focused microarray studies using a mouse cell line, mouse hypothalamus and mouse cortex. Regulated transcripts were confirmed by quantitative real-time PCR. As tissue complexity increased, distinguishing significantly regulated genes from background variation became increasingly more difficult. However, we found that cDNA microarray studies using regional brain dissections and appropriate numbers of replicates could identify genes showing less than 2-fold regulation and that most regulated genes identified fell within this range.

Animals↗
PubMed2002

Coupling of GnRH concentration and the GnRH receptor-activated gene program.

The initial waves of gene induction caused by GnRH in the LbetaT2 gonadotrope cell line have recently been identified using microarrays. We now investigate the relationship of the concentration of GnRH to the level of biosynthesis induced. Using an optimized custom cDNA microarray, we show that a large number of genes are induced in a concentration-dependent fashion. Detailed time course studies of the induction of six induced transcripts using quantitative real-time PCR suggest that the amplitude, but not the temporal pattern, depends on the concentration of GnRH. The early genes appear to show a delay in gene induction, followed by a linear phase of increase. The relationship of rate of synthesis and GnRH concentration was studied by mathematical modeling of the induction of two genes, gly96 and tis11. In both cases, only the rates of increase, but not the lag times, are influenced by the concentration of GnRH exposure. Western blot analyses for c-Jun and Egr1 show that the levels of nuclear protein for these transcription factors also depend on the concentration of GnRH. These studies indicate that, despite the complex signaling network connecting the receptor to the activated genes, the biosynthetic rate of RNA polymerase at induced genes is correlated with the concentration of GnRH at the GnRH receptor.

Blotting, Western↗