Characteristics of rodent intestinal mucin Muc3 and alterations in a mouse model of human cystic fibrosis

2001 ◽  
Vol 280 (6) ◽  
pp. G1321-G1330 ◽  
Author(s):  
Ismat A. Khatri ◽  
Catherine Ho ◽  
Robert D. Specian ◽  
Janet F. Forstner
Keyword(s):  
Cystic Fibrosis ◽  
Mouse Model ◽  
Oligonucleotide Probe ◽  
Goblet Cells ◽  
Soluble Form ◽  
Density Fractions ◽  
Intestinal Mucin ◽  
Apical Membranes ◽  
Columnar Cells

Human mucin MUC3 and rodent Muc3 are widely assumed to represent secretory mucins expressed in columnar and goblet cells of the intestine. Using a 3′-oligonucleotide probe and in situ hybridization, we observed expression of rat Muc3 mostly in columnar cells. Two antibodies specific for COOH-terminal epitopes of Muc3 localized to apical membranes and cytoplasm of columnar cells. An antibody to the tandem repeat (TR) sequence (TTTPDV)3, however, localized to both columnar and goblet cells. On CsCl gradients, Muc3 appeared in both light- and heavy-density fractions. The lighter species was immunoreactive with all three antibodies, whereas the heavier species reacted only with anti-TR antibody. Thus Muc3 is expressed in two forms, a full-length membrane-associated form found in columnar cells (light density) and a carboxyl-truncated soluble form present in goblet cells (heavy density). In a mouse model of human cystic fibrosis, both soluble Muc3 and goblet cell Muc2 were increased in amount and hypersecreted. Thus Muc2 and Muc3 contribute to the excess intestinal luminal mucus of cystic fibrosis mice.

scholarly journals Tissue-specific expression of a rat intestinal mucin-like peptide

1992 ◽  
Vol 286 (2) ◽  
pp. 335-338 ◽  
Author(s):  
G Xu ◽  
D Wang ◽  
L J Huan ◽  
E Cutz ◽  
G G Forstner ◽  
...  
Keyword(s):  
Splice Variants ◽  
Goblet Cells ◽  
Rat Tissues ◽  
Rat Intestine ◽  
Rich Region ◽  
Specific Expression ◽  
Intestinal Mucin ◽  
C Terminus ◽  
Hybridization In Situ

Expression of the gene for a rat intestinal mucin-like peptide (MLP) was studied by Northern-blot analyses of RNA prepared from a panel of rat tissues. Four probes (A-D) were constructed so as to span a 3.5 kb-long cDNA for rat MLP, and used for hybridization. Positive signals were obtained in intestine and colon, whereas lung, liver, stomach, submandibular gland and spleen were negative. The only transcript detected was approx. 9.5 kb in size. No mRNA splice variants were found. Hybridization in situ using probe B1, which corresponds to a cysteine-rich region near the C-terminus of MLP, confirmed that the gene for MLP is expressed by goblet cells of rat intestine and colon.

scholarly journals TMOD-24. GENERATION AND CHARACTERIZATION OF A NOVEL TRANSGENIC IDO REPORTER MOUSE FOR IDO POSTTRANSLATIONAL MODIFICATION ANALYSIS IN SITU

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii233-ii233
Author(s):  
April Bell ◽  
Lijie Zhai ◽  
Erik Ladomersky ◽  
Kristen Lauing ◽  
Lakshmi Bollu ◽  
...  
Keyword(s):  
Mouse Model ◽  
Tumor Cell ◽  
Central Nervous System Tumor ◽  
Post Translational Modifications ◽  
Reporter Mouse ◽  
C Terminus ◽  
Human Gbm

Abstract Glioblastoma (GBM) is the most common and aggressive primary central nervous system tumor in adults with a median survival of 14.6 months. GBM is a potently immunosuppressive cancer due in-part to the prolific expression of immunosuppressive indoleamine 2,3 dioxygenase 1 (IDO). Tumor cell IDO facilitates the intratumoral accumulation of regulatory T cells (Tregs; CD4+CD25+FoxP3+). Although immunosuppressive IDO activity is canonically characterized by the conversion of tryptophan into kynurenine, we have utilized transgenic and syngeneic mouse models and mutant glioma lines to demonstrate that tumor cell IDO increases Treg accumulation independent of tryptophan metabolism. Here, we address the gap in our understanding of IDO signaling activity in vivo. Subcutaneously-engrafted human GBM expressing human IDO-GFP cDNA was isolated from immunodeficient humanized NSG-SGM3 mice. The tumor was immunoprecipitated for the GFP tag using GFP-TRAP followed by mass spectrometry which revealed a novel methylation site on a lysine residue at amino acid 373 in the IDO C-terminus region. Western blot analysis of IDO protein also revealed the presence of tyrosine phosphorylation. Additionally, we recently created a new transgenic IDO reporter mouse model whereby endogenous IDO is fused to GFP via a T2A linker (IDO→GFP). This model allows for the isolation of IDO+ cells in real-time and without causing cell death, thereby creating the opportunity for downstream molecular analysis of in situ-isolated GFP+ cells. Collectively, our work suggests that IDO non-enzyme activity may involve the post-translational modifications we recently identified. As IDO activity may differ between in vitro and in vivo modeling systems, we will use the new IDO→GFP reporter mouse model for an improved mechanistic understanding of how immunosuppressive IDO facilitates Treg accumulation in vivo.

scholarly journals Abnormal mucus in cap polyposis

Gut ◽  
1998 ◽  
Vol 42 (1) ◽  
pp. 135-138 ◽  
Author(s):  
M P Buisine ◽  
J F Colombel ◽  
M Lecomte-Houcke ◽  
P Gower ◽  
J P Aubert ◽  
...  
Keyword(s):  
In Situ Hybridisation ◽  
Clinical Manifestations ◽  
Goblet Cells ◽  
Biopsy Specimens ◽  
Mucin Genes ◽  
Cap Polyposis ◽  
Abnormal Glycosylation ◽  
Histochemical Examination ◽  
Mrna In Situ Hybridisation

Background—Cap polyposis is a rare disease characterised by mucoid and bloody diarrhoea, with polyps covered by a cap of mucoid and fibrinopurulent exudate. The pathogenesis is not known.Aims—To pour some light on cap polyposis pathogenesis, by examining the mucus of patients and analysing the expression of five mucin genes, MUC2, MUC3,MUC4, MUC5AC, and MUC5B.Patient and methods—The study was performed on biopsy specimens taken from a patient with recurrent cap polyposis. Histochemical examination, electron microscopy, and mRNA in situ hybridisation were used.Results—The mucus of cap polyposis differed in three respects from that of normal adult colon: abnormal ultrastructure of the mucus in the goblet cells, predominance of non-sulphated mucins, abnormal expression of the MUC4, MUC3, andMUC5AC genes.Conclusions—Most of these abnormalities have been reported for other pathological situations, suggesting that the abnormalities observed in the mucus of this patient with cap polyposis are probably secondary phenomena rather than primary. However, the mucin abnormalities detected, which reflect deregulation of the expression of three apomucin genes, abnormal glycosylation, and abnormalities of the secretion process, are also probably involved in the clinical manifestations of cap polyposis.

scholarly journals In situ detection of vitamin D-induced calcium-binding protein (9-kDa CaBP) messenger RNA in rat duodenum.

1986 ◽  
Vol 34 (2) ◽  
pp. 277-280 ◽  
Author(s):  
M Warembourg ◽  
O Tranchant ◽  
C Perret ◽  
C Desplan ◽  
M Thomasset
Keyword(s):  
Vitamin D ◽  
In Situ Hybridization ◽  
Calcium Binding ◽  
Binding Protein ◽  
Cdna Probe ◽  
Calcium Binding Protein ◽  
Pbr322 Dna ◽  
Rat Duodenum ◽  
Columnar Cells

We have previously described the molecular cloning of a cDNA fragment synthesized from rat duodenal mRNA coding for a 9000-dalton vitamin D-induced calcium-binding protein (9-kDa CaBP) (3). We now report the use of this cloned cDNA to study the cytological distribution of 9-kDa CaBP mRNA in rat duodenum by in situ hybridization. Tissue sections, fixed in ethanol:acetic acid, were hybridized to the 3H-cDNA probe and processed for autoradiography. The specificity of the CaBP mRNA-DNA hybrid formation was checked using 3H-labeled plasmid pBR322 DNA as a control probe. 9k-Da CaBP mRNA, visualized by silver grains, was found only in the absorptive epithelial cells, and the concentration was greater in the cells at the villous tips than in those of the crypts. The 9k-Da CaBP mRNA was observed mainly in the cytoplasm of the columnar cells and less frequently in the nucleus. Labeling was not seen in the brush border and goblet cells. The submucosa, with Brunner's glands and muscularis, also showed no specific 9-kDa CaBP mRNA concentration. This demonstration of 9-kDa CaBP gene activity in the columnar cells of the rat duodenum illustrates the usefulness of in situ hybridization for characterization of specific cells involved in the expression of 1,25(OH)2 D3 activity.

ID: 63: RAPID 3D PRECLINICAL QUANTITATIVE LUNG IMAGING WITH ULTRASHORT-ECHO TIME (UTE) MRI IN A MOUSE MODEL OF CYSTIC FIBROSIS LUNG DISEASE

2016 ◽  
Vol 64 (4) ◽  
pp. 975.1-975
Author(s):  
C Anderson ◽  
C Flask
Keyword(s):  
Cystic Fibrosis ◽  
Magnetic Resonance ◽  
Mouse Model ◽  
Lung Disease ◽  
Lung Tissue ◽  
Lung Imaging ◽  
Early Disease ◽  
Lung Anatomy ◽  
Echo Time ◽  
Regional Disease

Currently, the life expectancy for cystic fibrosis (CF) lung disease is less than 40 years due to decreasing lung function despite significant advances in the care and treatment of these patients. As patients live longer, the preservation of healthy lung tissue becomes of paramount importance to improve patient quality of life and increase life span. To do this, an understanding of the early disease processes is needed as is an ability to monitor the efficacy of therapeutic interventions early in life. CF lung disease, similar to other lung diseases, is a regional disease causing local dysfunction in the lung tissue and changes in lung anatomy. It is important for any monitoring or diagnostic tool to be sensitive to early regional disease which current methods (spirometry) are not. This lack of sensitivity to regional disease limits the ability of physicians and researchers to track the earliest stages of disease and assess treatment efficacy in these initial disease stages, ideally in infants and young children. Three dimensional imaging presents a unique solution to this problem by providing a non-invasive, volumetric investigation of the lung tissue. Computed tomography has long been the first choice in clinical lung imaging offering excellent resolution and fast imaging times but results in repeated exposure to ionizing radiation. Because the patient populations of interest are infants and children, avoidance of unnecessary, repeated radiation exposure during longitudinal monitoring is desirable. This combination of clinical and research need has led us to the exploration of rapid MRI techniques for lung imaging. We are interested in developing a novel, robust quantitative Magnetic Resonance Imaging technique that allows for 3D investigation of the lung tissue and is sensitive to early disease changes. Our hypothesis is that quantitative imaging will be able to detect changes in regional lung anatomy as an indication of early disease before disease is detected by standard methods. To accomplish this goal, we are proposing the implementation of multiple advanced quantitative MRI techniques including T1-mapping using Saturation-Recovery Look-Locker mapping and simultaneous multiple parameter mapping (combinations of T1, T2, T2*) using the recently developed Magnetic Resonance Fingerprinting method. An ultra-short echo time acquisition will be used to ensure imaging of the rapidly decaying MRI signal in the lung is possible. Using a radial acquisition, we plan to include an undersampled acquisition to reduce imaging time and generate an imaging method that is rapid and insensitive to patient motion. Our goal is to initially apply these quantitative measures in a mouse model of cystic fibrosis to establish the ability of the imaging methods to be sensitive to regional disease in CF mice. We expect to see changes in the quantitative parameters in areas that correspond to diseased areas of the lung upon histological investigation. These quantitative measurements should give unambiguous indications of disease and allow identification of changes in lung anatomy early in the disease process. This work will lay the foundation for translation of clinical CF monitoring in a pediatric population. Translational studies such as these will hopefully provide a measurement of disease progression and provide a new opportunity to evaluate early disease therapeutics offering insight into the earliest manifestations of CF lung disease.

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