Maria-Cristina Keightley

Human Mineralocorticoid Receptor Genomic Structure and Identification of Expressed Isoforms

Most of the known effects of aldosterone are mediated by the mineralocorticoid receptor, an intracellular receptor belonging to the steroid/thyroid hormone/retinoic acid receptor superfamily. We determined the genomic structure of the human MR (hMR) and identified 10 exons in the gene, including two exons (1α and 1β) that encode different 5′-untranslated sequence. Expression of the two different hMR variants is under the control of two different promoters that contain no obvious TATA element, but multiple GC boxes. Our results indicate that hMR expression is regulated by alternative promoters perhaps in a tissue- or developmental-specific manner.

Diagnosis of Human Metapneumovirus Infection in Immunosuppressed Lung Transplant Recipients and Children Evaluated for Pertussis

ABSTRACT Human metapneumovirus (hMPV) is a recently discovered paramyxovirus that is known to cause respiratory tract infections in children and immunocompromised individuals. Given the difficulties of identifying hMPV by conventional culture, molecular techniques could improve the detection of this virus in clinical specimens. In this study, we developed a real-time reverse transcription-PCR (RT-PCR) assay designed to detect the four genetic lineages of hMPV. This assay and a commercial real-time nucleic acid sequence-based amplification (NASBA) assay (bioMérieux, Durham, NC) were used to determine the prevalence of hMPV in 114 immunosuppressed asymptomatic and symptomatic lung transplant recipients and 232 pediatric patients who were being evaluated for pertussis. hMPV was detected in 4.3% of the immunosuppressed lung transplant recipients and in 9.9% of children evaluated for pertussis. Both RT-PCR and NASBA assays were efficient in detection of hMPV infection in respiratory specimens. Even though hMPV was detected in a small number of the lung transplant recipients, it was still the most prevalent etiologic agent detected in patients with respiratory symptoms. In both of these diverse patient populations, hMPV infection was the most frequent viral respiratory tract infection identified. Given our findings, infection with hMPV infection should be determined as part of the differential diagnosis of respiratory illnesses.

Relationship of cytomegalovirus load assessed by real-time PCR to pp65 antigenemia in organ transplant recipients

BACKGROUND Cytomegalovirus (CMV) infection in immunocompromised patients can lead to viremia associated with morbidity and mortality. Monitoring of viral loads in blood is critical for initiating and monitoring antiviral treatment. OBJECTIVES Validate quantitative real-time PCR assay targeting the US17 and UL54 regions of the CMV genome for automated DNA and extraction and amplification. STUDY DESIGN 3422 blood specimens from organ transplant recipients, including longitudinal specimens from 12 organ transplant recipients, were tested by CMV PCR and pp65 antigenemia. RESULTS CMV PCR for both US17 and UL54, was more sensitive and detected CMV DNA earlier and for longer than the CMV pp65 antigenemia test. Using antigenemia results as a reference standard, an optimal cutoff of 500 normalized copies was calculated for both US17 and UL54 PCR targets based on high sensitivity, specificity, and positive and negative predictive values. CMV DNA levels tracked well with clinical symptoms, response to treatment, and antigenemia. CONCLUSIONS Detection of persistent increases in CMV DNA levels above 500 normalized copies by this real-time PCR assay is indicative of symptomatic CMV disease in organ transplant recipients. Quantitative real-time PCR for CMV DNA can be used in lieu of antigenemia for monitoring CMV infection and determining when to initiate preemptive treatment.

Cortisol resistance and the guinea pig glucocorticoid receptor.

The guinea pig has been employed as a model to study the structure/function relationships of the glucocorticoid receptor (GR), and to determine the regions of the receptor important for binding hormone and antihormone. Guinea pigs have high levels of circulating cortisol and GR with a approximately 20-fold lower affinity for dexamethasone than mouse GR. Cloning and sequencing of guinea pig GR has identified 24 amino acid changes in the ligand-binding domain (LBD) compared to the human GR. By substituting the guinea pig GR LBD for the human LBD in a human GR expression vector we have shown in cotransfection studies that guinea pig GR LBD confers glucocorticoid resistance as observed in vivo. In initial studies guinea pig GR LBD appeared constitutively active; in subsequent studies to determine which of the 24 amino acid changes present in the guinea pig GR LBD conferred resistance, it became apparent that the guinea pig LBD (LBD delta), amplified by PCR for subcloning into the human GR expression vector, contained a single adenine deletion in the hinge region within ten bases of the PCR primer. This single base deletion resulted in a frameshift bringing a stop codon into frame one codon after the deletion. While this now clearly accounts for the observed constitutive activity, since it is known that C-terminally truncated steroid receptors exhibit constitutive activation such a truncation is more difficult to reconcile with the repeatedly demonstrable hormone dose-response curves obtained with this guinea pig GR LBD delta.(ABSTRACT TRUNCATED AT 250 WORDS)

Real-time NASBA detection of SARS-associated coronavirus and comparison with real-time reverse transcription-PCR

Severe acute respiratory syndrome (SARS) exhibits a high mortality rate and the potential for rapid epidemic spread. Additionally, it has a poorly defined clinical presentation, and no known treatment or prevention methods. Collectively, these factors underscore the need for early diagnosis. Molecular tests have been developed to detect SARS coronavirus (SARS‐CoV) RNA using real time reverse transcription polymerase chain reaction (RT‐PCR) with varying levels of sensitivity. However, RNA amplification methods have been demonstrated to be more sensitive for the detection of some RNA viruses. We therefore developed a real‐time nucleic acid sequence‐based amplification (NASBA) test for SARS‐CoV. A number of primer/beacon sets were designed to target different regions of the SARS‐CoV genome, and were tested for sensitivity and specificity. The performance of the assays was compared with RT‐PCR assays. A multi‐target real‐time NASBA application was developed for detection of SARS‐CoV polymerase (Pol) and nucleocapsid (N) genes. The N targets were found to be consistently more sensitive than the Pol targets, and the real‐time NASBA assay demonstrates equivalent sensitivity when compared to testing by real‐time RT‐PCR. A multi‐target real‐time NASBA assay has been successfully developed for the sensitive detection of SARS‐CoV. J. Med. Virol. 77:602–608, 2005.

Molecular cloning and sequencing of a guinea-pig pro-opiomelanocortin cDNA

The guinea-pig has high levels of circulating cortisol. Though adrenocorticotropin (ACTH) levels are similar to those in other mammals, guinea-pig ACTH has been reported to have a single amino-acid substitution which results in increased bioactivity of the peptide. Pro-opiomelanocortin (POMC) is the precursor for ACTH, gamma-melanocyte-stimulating hormone (gamma-MSH) and the endogenous opioid peptide beta-endorphin. Both to confirm this substitution in guinea-pig ACTH and to establish whether other non-conservative substitutions occur elsewhere in the precursor we cloned guinea-pig POMC. The guinea-pig alanine for proline substitution at position 24 of ACTH was confirmed. Potentially significant mutations were also identified in gamma-MSH and beta-endorphin. A similar pattern of POMC mRNA expression was obtained for guinea-pig and rat as determined by Northern analysis and in situ hybridization. Southern blot analysis indicated that guinea-pig POMC is a single-copy gene. Cloning and sequencing of guinea-pig POMC thus further demonstrate the divergence of the New World hystricomorph peptides from those in New World primates, and underscore the differences observed in other endocrine axes in the guinea-pig.

In vivo mutation of pre-mRNA processing factor 8 (Prpf8) affects transcript splicing, cell survival and myeloid differentiation

Mutated spliceosome components are recurrently being associated with perturbed tissue development and disease pathogenesis. Cephalophŏnus (cph), is a zebrafish mutant carrying an early premature STOP codon in the spliceosome component Prpf8 (pre‐mRNA processing factor 8). Cph initially develops normally, but then develops widespread cell death, especially in neurons, and is embryonic lethal. Cph mutants accumulate aberrantly spliced transcripts retaining both U2‐ and U12‐type introns. Within early haematopoiesis, myeloid differentiation is impaired, suggesting Prpf8 is required for haematopoietic development. Cph provides an animal model for zygotic PRPF8 dysfunction diseases and for evaluating therapeutic interventions.

Minor class splicing shapes the zebrafish transcriptome during development

Significance The accurate removal of introns by pre-mRNA splicing is a critical step in proper gene expression. Most eukaryotic genomes, from plant to human, contain a tiny subset of “minor class” introns with unique sequence elements that require their own splicing machinery. The significance of this second splicing pathway has intrigued RNA biologists for two decades, but its biological relevance was recently underscored when defects in the process were firmly linked to human disease. Here, we use a novel zebrafish mutant with defective minor class splicing to investigate how this pathway shapes the transcriptome during vertebrate development. We link its pleiotropic phenotype to widespread changes in gene expression that disrupt essential cellular pathways, including mRNA processing. Minor class or U12-type splicing is a highly conserved process required to remove a minute fraction of introns from human pre-mRNAs. Defects in this splicing pathway have recently been linked to human disease, including a severe developmental disorder encompassing brain and skeletal abnormalities known as Taybi-Linder syndrome or microcephalic osteodysplastic primordial dwarfism 1, and a hereditary intestinal polyposis condition, Peutz-Jeghers syndrome. Although a key mechanism for regulating gene expression, the impact of impaired U12-type splicing on the transcriptome is unknown. Here, we describe a unique zebrafish mutant, caliban (clbn), with arrested development of the digestive organs caused by an ethylnitrosourea-induced recessive lethal point mutation in the rnpc3 [RNA-binding region (RNP1, RRM) containing 3] gene. rnpc3 encodes the zebrafish ortholog of human RNPC3, also known as the U11/U12 di-snRNP 65-kDa protein, a unique component of the U12-type spliceosome. The biochemical impact of the mutation in clbn is the formation of aberrant U11- and U12-containing small nuclear ribonucleoproteins that impair the efficiency of U12-type splicing. Using RNA sequencing and microarrays, we show that multiple genes involved in various steps of mRNA processing, including transcription, splicing, and nuclear export are disrupted in clbn, either through intron retention or differential gene expression. Thus, clbn provides a useful and specific model of aberrant U12-type splicing in vivo. Analysis of its transcriptome reveals efficient mRNA processing as a critical process for the growth and proliferation of cells during vertebrate development.

Delineating the roles of neutrophils and macrophages in zebrafish regeneration models.

The outcome following injury can be healing, scarring or regeneration, all of which initiate within a resolving inflammatory response. Regeneration, comprising the complete anatomical and functional restoration of lost tissue with minimal residual consequence of injury, is the outcome that most holistically restores prior function. Leukocytes are recognized as playing an important role in determining the balance between fully regenerative or only partially reparative outcomes. Although macrophages have attracted considerable attention for their capacity to direct pro-regenerative outcomes, neutrophils are also key players in initiating inflammation and in influencing its ensuing outcome. In the context of prior studies investigating the role of neutrophils and macrophages in wound healing and in tissue/organ regeneration (mostly wound repair/healing models in mice), we comprehensively review the experimental possibilities that zebrafish models offer for delineating the individual and interactive contributions of neutrophils and macrophages to the regenerative process in embryos and adults. Zebrafish are a highly regenerative vertebrate and have a myeloid system very analogous to that of less-regenerative mammalian models. There are well-characterized reporter lines for imaging and distinguishing neutrophil and macrophage behaviors in vivo, and tools enabling selective, independent manipulation of these two leukocyte lineages for functional studies. Zebrafish are an attractive model for delineating neutrophil and macrophage contributions not only to regeneration, but also to many other pathological processes. This article is part of a directed issue entitled: Regenerative Medicine: the challenge of translation.

Mediator subunit 12 is required for neutrophil development in zebrafish.

Hematopoiesis requires the spatiotemporal organization of regulatory factors to successfully orchestrate diverse lineage specificity from stem and progenitor cells. Med12 is a regulatory component of the large Mediator complex that enables contact between the general RNA polymerase II transcriptional machinery and enhancer bound regulatory factors. We have identified a new zebrafish med12 allele, syr, with a single missense mutation causing a valine to aspartic acid change at position 1046. Syr shows defects in hematopoiesis, which predominantly affect the myeloid lineage. Syr has identified a hematopoietic cell-specific requirement for Med12, suggesting a new role for this transcriptional regulator.