Biomedical subjects

Ann Harris

Publications and source records attributed to Ann Harris.

At least 19 recordsLinked to original sources

PubMed2025

Cellular imbalance in proximal and distal lung of CFTR-/- sheep in utero and at birth.

BACKGROUND: The Lung is the major focus of therapeutic approaches for the inherited disorder cystic fibrosis (CF) as without treatment lung disease is life-limiting. However, the initiating events that predispose the CF lung to cycles of infection, inflammation and resultant tissue damage are still unclear. Inflammation may occur in the CF lung prior to birth in human and several large animal models suggesting an in utero origin for the disease and encouraging further studies prior to birth. METHODS: Here we used the sheep model of CF (CFTR-/-) and age-matched wild-type (WT) sheep of the same breed to investigate the single cell transcriptomes of proximal and distal lung tissue at 80 days and 120 days of gestation and at term (147 days). Single cell RNA-seq was performed on tissues from 4 to 7 animals of each genotype (WT and CFTR-/-) at each time point. RESULTS: At term, FOXJ1-expressing ciliated cells are overrepresented in both lung regions from CFTR-/- lambs, while secretory epithelial and basal cells are underrepresented in proximal lung, as are T cells and monocytes in distal lung. The imbalance in ciliated and basal cells was confirmed by immunohistochemistry. At 120 days of gestation, lymphoid cells are slightly more abundant in proximal and distal lung from CFTR-/- animals compared to WT, consistent with the transient CF-associated inflammatory response in utero. At 80 days of gestation, T and B cells are underrepresented in both lung regions. CONCLUSIONS: The differences in epithelial cell abundance observed in the CFTR-/- lambs at term may reflect sequelae from the loss of CFTR on lung development and differentiation in utero. These findings provide novel insights into the cellular mechanisms of pathology and may be relevant to the design of new therapeutic approaches for CF lung disease.

Animals↗

PubMed2006

Extended lung cancer incidence follow-up in the Mayo Lung Project and overdiagnosis.

BACKGROUND: A troubling aspect of cancer screening is the potential for overdiagnosis, i.e., detection of disease that, in the absence of screening, would never have been diagnosed. Overdiagnosis is of particular concern in lung cancer screening because newer screening modalities can identify small nodules of unknown clinical significance. Previously published analyses of data from the Mayo Lung Project, a large randomized controlled trial conducted among 9211 male cigarette smokers in the 1970s and early 1980s indicated that overdiagnosis might exist in lung cancer screening. At the end of follow-up (July 1, 1983), no difference in lung cancer mortality was observed, but an excess of 46 cases in the intervention arm suggested overdiagnosis. Because that excess could instead have resulted from short follow-up time, we investigated this possibility by conducting long-term lung cancer incidence follow-up. METHODS: We investigated the lung cancer status through 1999 of the 7118 participants in the Mayo Lung Project who were alive and without diagnosed lung cancer in 1983 by use of medical records, surveys mailed to participants or next-of-kin, and state death certificates. RESULTS: Information was available for 6101 participants, including 811 with inconclusive lung cancer status. From November 1971 through December 31, 1999, 585 participants in the intervention arm and 500 in the usual-care arm were diagnosed with lung cancer. CONCLUSIONS: The persistence of excess cases in the intervention arm after 16 additional years of follow-up provides continued support for overdiagnosis in lung cancer screening.

Death Certificates↗

PubMed2006

N-Glycosylation of the MUC1 mucin in epithelial cells and secretions.

The MUC1 mucin is an important tumor-associated antigen that shows extensive glycosylation in vivo. The O-glycosylation of this molecule, which has been well characterized in many cell types and tissues, is important in conferring the unusual biochemical and biophysical properties on a mucin. N-Glycosylation is crucial to the folding, sorting, membrane trafficking, and secretion of many proteins. Here, we evaluated the N-glycosylation of MUC1 derived from two sources: endogenous MUC1 isolated from human milk and a recombinant epitope-tagged MUC1F overexpressed in Caco2 colon carcinoma cells. N-Glycans on purified MUC1F/MUC1 were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), gas chromatography-mass spectrometry (GC-MS), and CAD-ESI-MS/MS. The spectra indicate that MUC1F N-glycans have compositions consistent with high-mannose structures (Hex(5-9)HexNAc(2)) and complex/hybrid-type glycans (NeuAc(0-3)Fuc(0-3)Hex(3-8)HexNAc(3-7)). Many of the N-glycan structures are identical on MUC1F and native MUC1; however, a marked difference is seen between the N-glycans on membrane-bound and secreted forms of the native molecule.

Caco-2 Cells↗

PubMed2005

MZF-1 and DbpA interact with DNase I hypersensitive sites that correlate with expression of the human MUC1 mucin gene.

The MUC1 mucin is a large membrane-tethered glycoprotein that shows differential expression in many adenocarcinomas, where it contributes to their invasive and metastatic properties. We previously identified DNase I hypersensitive sites at -750 and -250 bp in the human MUC1 gene promoter and showed concordance between the -250 site and MUC1 mRNA levels in vivo. Transient expression assays using promoter constructs, in which the core DHS was deleted, to drive reporter gene expression revealed in vivo evidence for their activity. DNase I footprinting using nuclear extracts from HPAF human pancreatic carcinoma cells and MCF7 breast carcinoma cells identified three protein-binding elements in these regions (-250FP1, FP2 and -750FP). Electrophoretic mobility shift assays detected several complexes between HPAF nuclear proteins and labeled FP DNA probes. Southwestern blots and UV cross-linking experiments identified myeloid zinc finger-1 (MZF-1) as a candidate transcription factor among proteins binding to the -250FP1 and FP2 sequences. Another candidate that was identified by screening an HPAF cDNA expression library with the -250FP1 probe is DNA binding protein A (DbpA). Exogenous DbpA expression in COS-7 cells was accompanied by upregulation of MUC1 promoter activity via the -250 DHS, suggesting that DbpA binding to the -250 DHS can influence human MUC1 gene expression.

Animals↗

PubMed2005

Mucin glycosylation and sulphation in airway epithelial cells is not influenced by cystic fibrosis transmembrane conductance regulator expression.

Abnormalities in mucus properties and clearance make a major contribution to the pathology of cystic fibrosis (CF). Our aim was to test the hypothesis that the defects in CF mucus are a direct result of mutations in the CF transmembrane conductance regulator (CFTR) protein. We evaluated a single mucin molecule MUC1F/5ACTR that carries tandem repeat sequence from MUC5AC, a major secreted airway mucin, in a MUC1 mucin vector. To establish whether the presence of mutant or normal CFTR directly influences the O-glycosylation and sulphation of mucins in airway epithelial cells, we used the CFT1-LC3 (DeltaF508 CFTR mutant) and CFT1-LCFSN (wild-type CFTR corrected) human airway epithelial cell lines. MUC1F/5ACTR mucin was immunoprecipitated, centricon purified, and O-glycosylation was evaluated by Matrix-assisted laser desorption ionization and electrospray tandem mass spectrometry to determine the composition of different carbohydrate structures. Mass spectrometry data showed the same O-glycans in both CFTR mutant and wild-type CFTR corrected cells. Metabolic labeling assays were performed to evaluate gross glycosylation and sulphation of the mucins and showed no significant difference in mucin synthesized in six independent clones of these cell lines. Our results show that the absence of functional CFTR protein causes neither an abnormality in mucin O-glycosylation nor an increase in mucin sulphation.

Carbohydrate Conformation↗

PubMed2005

A T3 allele in the CFTR gene exacerbates exon 9 skipping in vas deferens and epididymal cell lines and is associated with Congenital Bilateral Absence of Vas Deferens (CBAVD).

The different alleles at the (TG)m(T)n polymorphic loci at the 3' end of the human CFTR intron 8 determine the efficiency by which exon 9 is spliced. We identified a novel TG12T3 allele in a congenital bilateral absence of vas deferens (CBAVD) patient who carries a [TG11T7; p.Phe508Cys; p.Met470Val] haplotype on the other chromosome. To better understand the complex regulation of exon 9 splicing, we analyzed the levels of correctly spliced CFTR transcripts in six CFTR-expressing epithelial cell lines derived from lung, colon, testis, vas deferens, and epididymis transiently transfected with four CFTR minigenes (pTG11T7, pTG12T7, pTG12T5, and pTG12T3). In this work, we show that a decrease in the Ts at the polymorphic locus in a TG12 background determines a cell-type dependent reduction in exon 9+ transcripts that is not related to the basal splicing efficiency in the cell line. These data emphasize the role of the T5 allele in CBAVD and identify the T3 allele as a severe cystic fibrosis (CF) disease-causing mutation. Finally, UV cross-linking experiments demonstrated that tissue-specific trans-acting splicing factors do not contribute to the different patterns of exon 9 splicing found between the cell lines. However, we observed that lower numbers of Ts can alter the binding of TDP-43 (TDP43 or TARDBP) to its specific target ug12 in a tissue-specific manner. Our results support the idea that the ratio of general splicing factors plays a role in the tissue variability of exon 9 alternative splicing.

Adult↗

PubMed2005

The CFTR gene and regulation of its expression.

The cystic fibrosis transmembrane conductance regulator gene (CFTR) shows clear temporal and developmental regulation of its expression. However, there are few well-defined regulatory elements that control this pattern of expression, and their mechanism of action is poorly understood. We review the structure and organization of the CFTR gene and what is known about its regulation. The CFTR gene promoter is clearly important for maintaining levels of CFTR gene expression, but apparently it does not contain any tissue-specific elements. Thus tissue-specificity is probably controlled by sequences lying elsewhere in this large gene. We discuss data from our group and others implicating additional regions of CFTR in regulatory functions, and evaluate candidate transcription factors that may be involved. Further, we summarize aspects of the regulation of the developmental expression of CFTR. Definition of CFTR gene regulatory elements could be of considerable therapeutic significance, since only a small increase in CFTR expression in the correct cell type could alleviate the disease phenotype.

Animals↗

PubMed2005

The role of the SEA (sea urchin sperm protein, enterokinase and agrin) module in cleavage of membrane-tethered mucins.

The membrane-tethered mucins are cell surface-associated dimeric or multimeric molecules with extracellular, transmembrane and cytoplasmic portions, that arise from cleavage of the primary polypeptide chain. Following the first cleavage, which may be cotranslational, the subunits remain closely associated through undefined noncovalent interactions. These mucins all share a common structural motif, the SEA module that is found in many other membrane-associated proteins that are released from the cell surface and has been implicated in both the cleavage events and association of the subunits. Here we examine the SEA modules of three membrane-tethered mucins, MUC1, MUC3 and MUC12, which have significant sequence homology within the SEA domain. We previously identified the primary cleavage site within the MUC1 SEA domain as FRPG/SVVV a sequence that is highly conserved in MUC3 and MUC12. We now show by site-directed mutagenesis that the F, G and S residues are important for the efficiency of the cleavage reaction but not indispensable and that amino acids outside this motif are probably important. These data are consistent with a new model of the MUC1 SEA domain that is based on the solution structure of the MUC16 SEA module, derived by NMR spectroscopy. Further, we demonstrate that cleavage of human MUC3 and MUC12 occurs within the SEA domain. However, the SEA domains of MUC1, MUC3 and MUC12 are not interchangeable, suggesting that either these modules alone are insufficient to mediate efficient cleavage or that the 3D structure of the hybrid molecules does not adequately re-create an accessible cleavage site.

Agrin↗

PubMed2004

Evaluation of MUC6 mucin tandem repeats.

The MUC6 mucin was originally isolated from stomach mucus and is one of the major secreted mucins of the digestive tract. A full-length cDNA has not been isolated for this large molecule (greater than 15 kb) and it remains poorly studied. To circumvent the lack of reagents for investigating MUC6, we isolated a cDNA clone from a human fetal pancreatic duct cDNA library that encodes 282 amino acids of the MUC6 tandem repeat. A blast search with the sequence of this cDNA clone showed 90% homology with the original MUC6 (L07517) derived from a human stomach cDNA library and 95% homology both with AK096772, a MUC6-related protein isolated from a human prostate cDNA library and the human genome project clone AC083984. The MUC6 partial cDNA clone isolated from fetal pancreas was inserted into an epitope-tagged MUC1 mucin molecule in place of the native tandem repeat. This chimeric mucin was expressed in human pancreatic (Panc1) and colon (Caco2) carcinoma cell lines and purified for analysis of O-glycosylation by fast atom bombardment mass spectrometry (FAB-MS). The FAB-MS spectra showed O-glycans that had been detected previously on chimeric mucins carrying different tandem repeats, though the spectra for MUC1F/6TR mucins expressed in the Panc1 and Caco2 cells were very different. There was a paucity of O-glycosylation in Panc1 cells in comparison to Caco2 cells where many more structures were evident, and the most abundant glycans in Panc1 cells were sialylated.

Amino Acid Sequence↗

PubMed2004

HNF1alpha is involved in tissue-specific regulation of CFTR gene expression.

The CFTR (cystic fibrosis transmembrane conductance regulator) gene shows a complex pattern of expression with tissue-specific and temporal regulation. However, the genetic elements and transcription factors that control CFTR expression are largely unidentified. The CFTR promoter does not confer tissue specificity on gene expression, suggesting that there are regulatory elements outside the upstream region. Analysis of potential regulatory elements defined as DNase 1-hypersensitive sites within introns of the gene revealed multiple predicted binding sites for the HNF1alpha (hepatocyte nuclear factor 1alpha) transcription factor. HNF1alpha, which is expressed in many of the same epithelial cell types as CFTR and shows similar differentiation-dependent changes in gene expression, bound to these sites in vitro. Overexpression of heterologous HNF1alpha augmented CFTR transcription in vivo. In contrast, antisense inhibition of HNF1 alpha transcription decreased the CFTR mRNA levels. Hnf1 alpha knockout mice showed lower levels of CFTR mRNA in their small intestine in comparison with wild-type mice. This is the first report of a transcription factor, which confers tissue specificity on the expression of this important disease-associated gene.

Animals↗

PubMed2004

Analysis of genomic CFTR DNA.

There are numerous methodologies available for the analysis of genomic CFTR DNA. We present here the basic tools to allow a thorough investigation of the CFTR gene, beginning with the identification of potential regulatory regions using DNase I hypersensitive sites, and continuing with methods for the detection of mutations: denaturing High Performance Liquid Chromatography (dHPLC), Single Strand Conformation Polymorphism (SSCP), and allele-specific oligonucleotide (ASO) hybridisation. Also provided is a comprehensive set of PCR primers for the amplification of most regions of the CFTR gene. Full details of the methodologies given are to be found at the European Working Group on CFTR Expression website http://central.igc.gulbenkian.pt/cftr/vr/transcripts.html.

Cystic Fibrosis Transmembrane Conductance Regulato↗

PubMed2004

Methods for RNA extraction, cDNA preparation and analysis of CFTR transcripts.

The scope of this article is to outline some of the basic methods for good quality RNA preparation from mammalian tissues and cells (including epithelial cells). Additionally, we give an outline of common techniques of measuring CFTR gene expression such as quantitative and semi-quantitative reverse transcription (RT) PCR and ribonuclease protection assay (RPA). These methods are designed to detect low abundance transcripts, which apply to CFTR mRNA in most cell types and tissues.

Cystic Fibrosis Transmembrane Conductance Regulato↗

PubMed2004

Non-PCR methods for the analysis of CFTR transcripts.

Cystic fibrosis transmembrane conductance regulator gene (CFTR) shows a complex mechanism of tissue-specific and temporal regulation. CFTR mRNA detection and measurement are extremely difficult because of the low to very low levels of its endogenous expression. In this paper, we describe four different non-PCR methods optimized to analyze CFTR transcripts in epithelial cell lines, primary cell lines and native tissues that express significant amounts of CFTR transcript.

Blotting, Northern↗

PubMed2004

Quantitative methods for the analysis of CFTR transcripts/splicing variants.

In cystic fibrosis (CF), transcript analysis and quantification are important for diagnosis, prognosis and also as surrogate markers for some therapies including gene therapy. Classical RNA-based methods require significant expression levels in target samples for appropriate analysis, thus PCR-based methods are evolving towards reliable quantification. Various protocols for the quantitative analysis of CFTR transcripts (including those resulting from splicing variants) are described and discussed here.

Cystic Fibrosis↗

PubMed2003

Alternative 5' exons of the CFTR gene show developmental regulation.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene shows a complex mechanism of tissue-specific and temporal regulation. Expression of the sheep and human CFTR genes shows a gradual decline during lung development, from the early mid-trimester through to term. Alternative upstream exons of CFTR have been identified in several species but their functional role remains obscure. We identified a novel 5' exon of the sheep CFTR gene (ov1a) that occurs in two splice forms (ov1aL and ov1aS), which are both mutually exclusive with exon 1. CFTR transcripts including ov1aL and ov1aS are present at low levels in many sheep tissues, however ov1aS shows temporal and spatial regulation during fetal lung development, being most abundant when CFTR expression levels start to decline. Alternative 5' exons -1a and 1a in the human CFTR gene also show changes in expression levels through lung development. Evaluation of ov1aL and ov1aS by Mfold reveals the potential to form extremely stable secondary structures which would cause ribosomal subunit detachment. Further, the loss of exon 1 from the CFTR transcript removes motifs that are crucial for normal trafficking of the CFTR protein. Recruitment of these alternative upstream exons may represent a novel mechanism of developmental regulation of CFTR expression.

Alternative Splicing↗

PubMed2003

Evaluation of gene targeting by homologous recombination in ovine somatic cells.

Mouse models for some human genetic diseases are limited in their applications since they do not accurately reproduce the phenotype of the human disease. It has been suggested that larger animals, for example sheep, might produce more useful models, as some aspects of sheep physiology and anatomy are more similar to those of humans. The development of methods to clone animals from somatic cells provides a potential novel route to generate such large animal models following gene targeting. Here, we assess targeting of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in ovine somatic cells using homologous recombination (HR) of targeting constructs with extensive (>11 kb) homology. Electroporation of these constructs into ovine fetal and post-natal fibroblasts generated G418-resistant clones, but none analyzed had undergone HR, suggesting that at least for this locus, it is an extremely inefficient process. Karyotyping of targeted ovine fetal fibroblasts showed them to be less chromosomally stable than post-natal fibroblasts, and, moreover, extended culture periods caused them to senesce, adversely affecting their viability for use as nuclear transfer donor cells. These data stress the importance of donor cell choice in somatic cell cloning and suggest that culture time be kept to a minimum prior to nuclear transfer in order to maximize cell viability.

Animals↗

PubMed2003

Alternative splicing of the ovine CFTR gene.

Alternative splicing of the human CFTR gene was studied previously and shown not to generate functional CFTR-like chloride ion channels. However, it is possible that some of the alternatively spliced forms may encode CFTR proteins with different functions. The ovine CFTR gene is very similar to the human gene and has regulatory mechanisms in common. To evaluate whether the alternatively spliced forms of human CFTR are conserved in the sheep, the splice forms of the ovine CFTR gene were examined. A transcript lacking exon 9 was observed in the sheep, but unlike the human exon 9-transcript, it did not result from a polymorphic intron 8 splice acceptor site. Sheep CFTR transcripts lacking exon 17b were seen and have also been described in the human. Transcripts lacking 98 bp of the 5' end of exon 13, the whole of exon 13, and both exons 14b and 15 respectively were seen in sheep but have not been reported in human. Splice site donor and acceptor sequences were isolated, and alternative transcripts were shown to result from a combination of aberrant sites and competition of 5' splice donor sequences.

Alternative Splicing↗

PubMed2003

A comparative genomic analysis of the cow, pig, and human CFTR genes identifies potential intronic regulatory elements.

The identification of sequences within noncoding regions of genes that are conserved between several species may indicate potential regulatory elements. This is important for genes with complex control mechanisms such as the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR demonstrates similar patterns of temporal and spatial expression in human and sheep, but these differ significantly in mouse cftr. The complete sheep CFTR sequence is unavailable so we annotated BAC clones encompassing the CFTR gene from two other artiodactyl species (cow and pig) for comparative sequence analysis. Regions of introns 2, 3, 10, 17a, 18, and 21 and 3' flanking sequence corresponding to human CFTR DNase I hypersensitive sites (DHS) showed high homology in the cow and pig. Cross-species sequence conservation also enabled finer mapping of other human DHS, including those in introns 1, 16, and 20. Additional potential regulatory elements not associated with human DHS were also identified.

Animals↗