scholarly journals Detection of CD1 mRNA in Paneth cells of the mouse intestine by in situ hybridization.

1992 ◽  
Vol 40 (10) ◽  
pp. 1527-1534 ◽  
Author(s):  
J Lacasse ◽  
L H Martin
Keyword(s):  
In Situ Hybridization ◽  
Cell Biology ◽  
Paneth Cell ◽  
Intestinal Flora ◽  
Intraepithelial Lymphocytes ◽  
Paneth Cells ◽  
Peripheral T Cells ◽  
Mouse Intestine ◽  
Cluster Of Differentiation

Cluster of differentiation 1 (CD1) antigens are a family of non-MHC but Class I-like molecules that have been identified in humans and rodents. Although their function(s) remains unknown, it has been proposed that CD1 may present antigens to specific subsets of peripheral T-cells. We now provide evidence in support of this hypothesis through the demonstration by in situ hybridization that Paneth cells of the mouse intestine express CD1 mRNA. These cells are thought to be involved in the immunological regulation of intestinal flora and could accomplish this task through interactions with intestinal intraepithelial lymphocytes. The expression and localization of CD1 mRNA was confirmed by both autoradiographic and non-isotopic techniques. The relevance of these results to CD1 function as well as to Paneth cell biology is discussed.

Abnormal Paneth cell granule dissolution and compromised resistance to bacterial colonization in the intestine of CF mice

2004 ◽  
Vol 286 (6) ◽  
pp. G1050-G1058 ◽  
Author(s):  
Lane L. Clarke ◽  
Lara R. Gawenis ◽  
Emily M. Bradford ◽  
Louise M. Judd ◽  
Kathryn T. Boyle ◽  
...  
Keyword(s):  
Northern Blot Analysis ◽  
Clinical Symptoms ◽  
Bacterial Colonization ◽  
Paneth Cell ◽  
Light And Electron Microscopy ◽  
Paneth Cells ◽  
Real Time Quantitative Pcr ◽  
Granule Morphology ◽  
Mouse Intestine ◽  
Osmotic Laxative

Paneth cells of intestinal crypts contribute to host defense by producing antimicrobial peptides that are packaged as granules for secretion into the crypt lumen. Here, we provide evidence using light and electron microscopy that postsecretory Paneth cell granules undergo limited dissolution and accumulate within the intestinal crypts of cystic fibrosis (CF) mice. On the basis of this finding, we evaluated bacterial colonization and expression of two major constituents of Paneth cells, i.e., α-defensins (cryptdins) and lysozyme, in CF murine intestine. Paneth cell granules accumulated in intestinal crypt lumens in both untreated CF mice with impending intestinal obstruction and in CF mice treated with an osmotic laxative that prevented overt clinical symptoms and mucus accretion. Ultrastructure studies indicated little change in granule morphology within mucus casts, whereas granules in laxative-treated mice appear to undergo limited dissolution. Protein extracts from CF intestine had increased levels of processed cryptdins compared with those from wild-type (WT) littermates. Nonetheless, colonization with aerobic bacteria species was not diminished in the CF intestine and oral challenge with a cryptdin-sensitive enteric pathogen, Salmonella typhimurium, resulted in greater colonization of CF compared with WT intestine. Modest downregulation of cryptdin and lysozyme mRNA in CF intestine was shown by microarray analysis, real-time quantitative PCR, and Northern blot analysis. Based on these findings, we conclude that antimicrobial peptide activity in CF mouse intestine is compromised by inadequate dissolution of Paneth cell granules within the crypt lumens.

scholarly journals Nonspecific binding of nucleic acid probes to Paneth cells in the gastrointestinal tract with in situ hybridization.

1992 ◽  
Vol 40 (10) ◽  
pp. 1613-1618 ◽  
Author(s):  
K L Garrett ◽  
M D Grounds ◽  
M W Beilharz
Keyword(s):  
In Situ Hybridization ◽  
Gastrointestinal Tract ◽  
Nucleic Acid ◽  
Paneth Cells ◽  
Proteinase K ◽  
Hybridization Technique ◽  
Nonspecific Binding ◽  
Nucleic Acid Probes ◽  
Hybridization Data

Nonspecific binding of a number of unrelated nucleic acid probes to cells in the crypts of Lieberkuhn was observed in the small intestine of mice with the in situ hybridization technique. Hybridization signal was localized to cells which, by virtue of their histological position, represented Paneth cells. This signal could not be removed by RNAse, DNAse, or proteinase K treatment, and was not removed after high-stringency washing conditions. This report indicates that caution must be exercised in the interpretation of in situ hybridization data when looking for nucleic acid sequences in the gastrointestinal tract.

DANCE in developing and injured lung

2002 ◽  
Vol 282 (1) ◽  
pp. L75-L82 ◽  
Author(s):  
Jyh-Chang Jean ◽  
Ifeanyi Eruchalu ◽  
Yu Xia Cao ◽  
Martin Joyce-Brady
Keyword(s):  
In Situ Hybridization ◽  
Cell Biology ◽  
Exposure Period ◽  
Subtractive Hybridization ◽  
Recovery Phase ◽  
Mrna Abundance ◽  
Northern Analysis ◽  
Adult Lung ◽  
Injured Lung

We identified rat developing arteries and neural crest derivatives with multiple epidermal growth factor-like domains ( DANCE) as a developmentally regulated gene using suppression-subtractive hybridization. Northern analysis confirmed a fivefold induction of this mRNA transcript between fetal day 18 and 20 that persisted through postnatal day 17. The level was declining at postnatal day 21 and was similar in adult lung to that at fetal day 18. In adults DANCE mRNA abundance was highest in lung, kidney, and spleen, lower in heart, skeletal muscle, and brain, but absent from liver and thymus. It was abundant in pulmonary artery endothelium and a lung epithelial type 2 cell line, barely detectable in vascular smooth muscle, and absent in fibroblasts. In situ hybridization revealed a regulated pattern of expression in endothelial cells of fetal, postnatal, and adult lung. Because DANCE mRNA was inducible in systemic arteries during recovery from injury, we searched for induction in lung injured by hyperoxia. Mouse DANCE mRNA abundance was unchanged during an acute 3-day exposure period, induced threefold 5 days into the recovery phase, and returned to baseline at days 8, 11, and 14. In situ hybridization at day 5 suggested a diffuse pattern of induction. DANCE may play a role in lung endothelial cell biology during development repair after injury.

scholarly journals Tumor necrosis factor mRNA localized to Paneth cells of normal murine intestinal epithelium by in situ hybridization.

1990 ◽  
Vol 171 (1) ◽  
pp. 327-332 ◽  
Author(s):  
S Keshav ◽  
L Lawson ◽  
L P Chung ◽  
M Stein ◽  
V H Perry ◽  
...  
Keyword(s):  
Small Intestine ◽  
In Situ Hybridization ◽  
Lamina Propria ◽  
Northern Blot Analysis ◽  
Surface Membrane ◽  
Paneth Cells ◽  
Intestinal Physiology ◽  
Tumor Necrosis Factor Mrna ◽  
Necrosis Factor

Paneth cells in normal murine small intestine contain TNF mRNA that is readily detectable by in situ hybridization, unlike resident macrophages in lamina propria, which are negative. Northern blot analysis of whole tissue shows the presence of mRNA that has the same electrophoretic mobility as TNF mRNA from activated macrophages. A low level of TNF bioactivity, but no immunoreactivity, was detected in normal small intestine, and TNF production in resting Paneth cells appears to be post-transcriptionally controlled. Typical leukocyte surface membrane markers were not found on Paneth cells, but were expressed by the surrounding lamina propria macrophages. Paneth cells are thus epithelial cells with leukocyte-like secretory potential that may be important in intestinal physiology and pathology.

scholarly journals Detection of Deoxyribonuclease I Along the Secretory Pathway in Paneth Cells of Human Small Intestine

1998 ◽  
Vol 46 (7) ◽  
pp. 833-840 ◽  
Author(s):  
Osamu Shimada ◽  
Harunori Ishikawa ◽  
Hisami Tosaka-Shimada ◽  
Toshihiro Yasuda ◽  
Koichiro Kishi ◽  
...  
Keyword(s):  
Small Intestine ◽  
In Situ Hybridization ◽  
Secretory Pathway ◽  
Dnase I ◽  
Paneth Cells ◽  
Tissue Homogenate ◽  
Immunogold Electron Microscopy ◽  
Human Small Intestine ◽  
Deoxyribonuclease I

The expression and distribution of deoxyribonuclease I (DNase I) in human duodenum, jejunum and ileum were examined by DNase I activity assay and the reverse transcriptase-polymerase chain reaction (RT-PCR), immunofluorescence, in situ hybridization, and immunocytochemical ultrastructural analyses. High levels of DNase I were detected in the cytoplasm of Paneth cells in human small intestine. A tissue homogenate fraction rich in Paneth cells showed strong DNase I-specific enzymatic activity. Immunofluorescence analysis using several specific anti-human DNase I antibodies showed very strong immunoreactivity in the cytoplasm of every Paneth cell. In situ hybridization demonstrated high levels of DNase I mRNA in Paneth cells. Immunogold electron microscopy revealed gold particles localized along the secretory pathway, with the exocrine secretory granules mostly labeled. Our findings strongly suggest that Paneth cells synthesize and secrete DNase I into the intestinal lumen.

Eukaryotic genome organization analyzed by electron microscope in situ hybridization

1991 ◽  
Vol 49 ◽  
pp. 40-41
Author(s):  
Barbara A. Hamkalo ◽  
Sandya Narayanswami ◽  
Nadja Dvorkin
Keyword(s):  
In Situ Hybridization ◽  
Electron Microscope ◽  
Genome Organization ◽  
Cell Biology ◽  
Eukaryotic Genome ◽  
Dna Denaturation ◽  
Metaphase Chromosomes ◽  
Rna Sequences ◽  
Gold Particles

In situ hybridization is a powerful tool for the localization of DNA/RNA sequences in nuclei and chromosomes. The introduction of nonisotopic labelling methodologies in conjunction with fluorescent or enzyme-linked detection have resulted in a dramatic increase in the application of this technique at the light microscope (LM) level and has placed it in a pivotal role in cell biology, development and genetics. Development of equivalent mapping protocols at the EM level offers increased spatial resolution. We have combined the use of nonisotopic probes with invmunogold labelling to investigate eukaryotic genome organization at high resolution.Metaphase chromosomes released from mitotically-arrested cells are deposited on gold EM grids by centrifugation through a sucrose cushion. After fixation (0.1% glutaraldehyde, 20 min) and DNA denaturation, chromosomes are hybridized to cloned probes enzymatically labelled with biotin-dUTP, digoxigenin-dUTP, dinitrophenyl-dUTP or covalently coupled to N-acetoxyacetoaminofluorene. Hybrid sites typically are detected by a two-step antibody incubation and 1-30 nm colloidal gold particles.

scholarly journals A novel triple-color detection procedure for brightfield microscopy, combining in situ hybridization with immunocytochemistry.

1994 ◽  
Vol 42 (10) ◽  
pp. 1299-1307 ◽  
Author(s):  
E J Speel ◽  
M P Jansen ◽  
F C Ramaekers ◽  
A H Hopman
Keyword(s):  
In Situ Hybridization ◽  
Horseradish Peroxidase ◽  
Cell Biology ◽  
Enzyme Reaction ◽  
Color Contrast ◽  
Reaction Products ◽  
Red Color ◽  
Fast Red ◽  
Fast Light

We describe a fast light microscopic procedure for the simultaneous enzyme cytochemical detection of three different DNA target sequences in contrasting colors in both interphase and metaphase cell preparations. Chromosome-specific DNA probes labeled with either biotin, digoxygenin, or fluorescein were hybridized as a mixture and detected clearly and accurately by precipitates of the horseradish peroxidase-diaminobenzidine (PO-DAB, brown color), alkaline phosphatase-Fast Red (APase-Fast Red, red color), or horseradish peroxidase-tetramethylbenzidine (PO-TMB, green color) reaction, respectively. The PO-TMB reaction product was stabilized effectively by the addition of sodium tungstate to the reaction mixture, thus making the PO-TMB reaction now generally applicable to in situ hybridization (ISH). To avoid mixing of the precipitates of the two PO reactions used in the triple-color ISH method, the first detected PO activity was always completely inactivated by a mild acid treatment before the second one was applied. Finally, the cell preparations were embedded in a thin protein layer cross-linked by formaldehyde to ensure permanent stabilization of the enzyme reaction products and optimal visualization of color contrast. The triple-color ISH detection procedure could be combined with beta-galactosidase-5-bromo-4-chloro-3-indolyl-beta- D-galactoside (beta-Gal-BCIG) immunocytochemistry (ICC), leading to the simultaneous localization of multiple DNA targets and a protein target in the same cell. The described procedure may therefore be a valuable tool in the areas of cytogenetics, cell biology, and molecular pathology.

scholarly journals Role of lipopolysaccharide in colonization of the mouse intestine by Salmonella typhimurium studied by in situ hybridization.

1996 ◽  
Vol 64 (9) ◽  
pp. 3811-3817 ◽  
Author(s):  
T R Licht ◽  
K A Krogfelt ◽  
P S Cohen ◽  
L K Poulsen ◽  
J Urbance ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Salmonella Typhimurium ◽  
Mouse Intestine

Cryptdin gene expression in developing mouse small intestine

1997 ◽  
Vol 272 (1) ◽  
pp. G197-G206 ◽  
Author(s):  
D. Darmoul ◽  
D. Brown ◽  
M. E. Selsted ◽  
A. J. Ouellette
Keyword(s):  
Paneth Cell ◽  
Paneth Cells ◽  
Intestinal Lumen ◽  
Intestinal Epithelial ◽  
Rt Pcr ◽  
Adult Mice ◽  
Mucosal Surfaces ◽  
Mouse Small Intestine ◽  
Pcr Products ◽  
Mouse Intestine

In rodents, the four intestinal epithelial cell lineages differentiate and become morphologically distinct during the first 2-3 postnatal wk. In studies reported here, reverse transcriptase-polymerase chain reaction (RT-PCR)-based assays detected Paneth cell defensin mRNAs in intestinal RNA from 1-day-old (P1) mice before crypt formation and maturation of the epithelium. Analysis of these defensin-coding RT-PCR products from P1 mice showed that 69% of clones sequenced coded for cryptdin-6, suggesting that it is the most abundant enteric defensin mRNA in the newborn. Paneth cell mRNAs, including cryptdins-4 and -5, lysozyme, matrilysin, and defensin-related sequences, also were detected in RNA from P1 mouse intestine. Unlike adult mice, where only Paneth cells are immunopositive for cryptdin, cryptdin-containing cells were distributed throughout the newborn intestinal epithelium and not in association with rudimentary crypts. Cryptdin immunoreactivity in the P1 mouse intestine was specific for intracellular granule contents, and immunofluorescent detection of cryptdins on mucosal surfaces suggested that the peptides are released into the intestinal lumen in P1 mice Defensin secretion may contribute to innate immunity of the neonatal intestine before the presence of distinguishable Paneth cells.

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