Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The polydispersity of most human secretory mucin messages has made them difficult to detect specifically and quantitatively, impeding the evaluation of the relative expression of the various mucin genes and their role in normal and pathological conditions. For this reason, we developed competitive reverse transcriptase-polymerase chain reaction (PCR) methods to measure the airway mucins MUC2 and MUC5AC. Oligonucleotide pairs were designed that specifically detect MUC2 and MUC5AC, as demonstrated by the size and sequence of the PCR product and the expected tissue distribution. The mucin oligonucleotide primers were used to synthesize internal competitive standards, called MIMIC. Using this assay, the relative expression of these messages was analyzed in retinoid-replete or -deprived cultures of normal human tracheobronchial epithelial (NHTBE) cells. Retinoid deficiency induces squamous metaplasia in vivo and in vitro. Consistent with these observations and in contrast to a previous report, retinoid-deprived cultures produced at least an order of magnitude less MUC2 and MUC5AC message than retinoid-replete cultures. In summary, this paper describes methodology that can be applied to the specific and quantitative measurement of mucin messages and demonstrates that, in NHTBE cells, the level of MUC2 and MUC5AC mRNA is increased by retinoids.
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PMID:Quantitation of mucin RNA by PCR reveals induction of both MUC2 and MUC5AC mRNA levels by retinoids. 899 74

Human submandibular/sublingual gland secretions contain a multimeric high molecular weight mucin (MG1) and a monomeric low molecular weight mucin (MG2). MG2 is the product of the MUC7 gene, whereas the gene for MG1 has not been identified. Previously, we isolated a clone (pSM2-1) from a human sublingual gland cDNA expression library using an antibody against deglycosylated MG1 (Troxler et al., Biochem. Biophys. Res Commun., 217, 1112-1119, 1995). In order to identify the mucin gene from which pPM2-1 was derived, Northern blots of human submandibular and sublingual gland RNA were hybridized with a series of probes for tandem repeats found in the high molecular weight secreted mucins MUC2, MUC3, MUC4, MUC5AC, MUC5B, and MUC6. The only known mucin expressed at high levels in sublingual gland was MUC5B, and no known mucin was expressed at high levels in submandibular gland. A series of overlapping clones was obtained by rescreening the sublingual gland cDNA library and by reverse transcriptase-polymerase chain reaction. The resulting clones connected pSM2-1 to a series of MUC5B tandem repeats at the 3' end of the repeat domain and provided the complete nucleotide and deduced amino sequence of the carboxyl terminal region of MG1. This region is enriched with respect to cysteine (approximately 10 mol %) and contained a D domain and a carboxyl terminal domain that could be aligned with the corresponding domains in human intestinal MUC2, human tracheobronchial MUC5AC, and human von Willebrand factor. The limited expression of known mucin genes, together with the considerable mucin synthesizing capacity of submandibular gland, suggests that a novel (previously not described) mucin gene is expressed in this gland and constitutes a portion of MG1 in salivary secretions.
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PMID:Molecular characterization of a major high molecular weight mucin from human sublingual gland. 936 39

A primary cell culture of human gastric mucous cells was developed using enzymatic treatment of surgically obtained gastric mucosal specimens. Preferential attachment of gastric mucous cells during a preincubation step resulted in the enrichment of mucous cells [over 90% stained with periodic acid-Schiff (PAS) and mucin-type lectins] in the primary cell culture. Gastric mucous cells could be maintained in culture for 10 days. DNA synthesis peaked during the first 2 days in culture (8+/-1% bromodeoxyuridine-positive cells). During the entire culture period gastric mucous cells released high-molecular-weight glycoproteins into the medium, as determined by gel chromatography on a Sepharose CL-4B column and by metabolic labelling with [14C]-N-acetylglucosamine. Gastric mucin was verified by gas chromatographic analysis of the carbohydrate composition and fractionation of the void-volume fraction by density gradient centrifugation. Determination of the terminal glycosylation of the secreted glycoproteins by a lectin-ELISA revealed that there was a high quantity of alpha-l-fucose. Prostaglandin E2 significantly stimulated glycoprotein secretion during the entire cultivation period by 29-60%. Analysis of mucin-encoding MUC mRNA expression by reverse transcriptase polymerase chain reaction revealed that gastric mucous cells predominantly express MUC1 and MUC5AC, and to a lesser extent MUC6, which reflects the expression pattern obtained following analysis of biopsied samples of gastric mucosa. This primary cell culture model enables the regulation of mucin secretion and mucin gene expression in man to be investigated.
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PMID:Morphological and molecular characterization of human gastric mucous cells in long-term primary culture. 979 1

The levels of mRNA corresponding to the MUC1, MUC2, MUC5AC, MUC5B, and MUC6 genes were determined in 19 human colon adenocarcinoma cell lines by the reverse transcriptase-polymerase chain reaction method using specific primers in an attempt to correlate to the levels of cell surface carbohydrate epitopes. All 19 cell lines expressed MUC1 and MUC5B mRNA, whereas MUC2, MUC5AC, or MUC6 mRNA were only detected in 8, 3, or 2 of 19 cell lines, respectively. Sialyl Lewis a carbohydrates, identified by the monoclonal antibody (mAb) CA19-9, and sialyl Lewis X carbohydrates. identified by mAb KM93, were observed, with most of the cell lines expressing multiple mucin core polypeptide genes but with few cell lines expressing only MUC1 and MUC5B. Sialyl Tn epitopes identified by mAb B195.3R11 and by mAb TKH-2 were strongly expressed on both of two MUC6-positive cells, whereas only a small portion of MUC6-negative cells expressed these epitopes. Strict correlation between mucin gene expression and any carbohydrate epitopes examined was not observed.
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PMID:Expression of mucin genes and carbohydrate epitopes in 19 human colon carcinoma cell lines. 1010 Jul 57

Mucus hypersecretion is characteristic of chronic airway diseases. However, regulatory mechanisms are poorly understood. Human airway epithelial cells grown on permeable supports at the air-liquid interface (ALI) develop a mucociliary morphology resembling that found in vivo. Such cultures provide a model for studying secretory cell lineage, differentiation, and function, and may provide insight regarding events leading to mucus hypersecretion. The mucin gene expression profile of well-differentiated human airway epithelial cells in culture has not yet been established. We compared expression of all the currently described mucin genes in poorly differentiated (conventional cultures on plastic) and well-differentiated (ALI) human nasal and bronchial epithelial cells. Differentiation-dependent upregulation of MUC3, MUC5AC, MUC5B, and MUC6 messenger RNA (mRNA) was demonstrated using reverse transcriptase-polymerase chain reaction (RT-PCR). Northern blot analysis showed a similar increase for MUC4 and demonstrated that induction of MUC4 and MUC5B expression depended on retinoic acid. MUC1, MUC2, MUC7, and MUC8 mRNAs were also detected by RT-PCR, but these genes did not appear to be strongly regulated as a function of differentiation. Mucin gene expression was similar in bronchial and nasal cells. Thus, mucociliary differentiation of human airway epithelia in vitro entails upregulation of several mucin genes.
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PMID:Mucin gene expression during differentiation of human airway epithelia in vitro. Muc4 and muc5b are strongly induced. 1010 Sep 90

Our goal was to determine the effect of transdermal nicotine on cytokine and mucin gene transcription in ulcerative colitis (UC). Sixty-four nonsmoking patients with active UC were randomly assigned to transdermal nicotine (maximum dose 22 mg/day) or placebo for 4 weeks. Clinical assessment and colonic mucosal biopsies were obtained at entry and after 4 weeks. Inflammatory and immunoregulatory cytokines were assessed by qualitative reverse transcriptase-polymerase chain reaction (RT-PCR). Based on this initial screen. IL-8 mRNA levels were measured by RT-competitive PCR. MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, and MUC6 mRNA concentrations were measured by quantitative dot blot analysis. Cytokine mRNA expression, except for IL-8, was similar in all patients. IL-8 mRNA levels were significantly decreased in the colonic mucosa of nicotine-treated patients who improved (p = 0.04). IL-8 mRNA values were similar before and after treatment in nonresponding nicotine-treated patients and in all placebo-treated patients. Mucin gene expression was similar in all patient groups. Beneficial effects of transdermal nicotine in active UC may result from decrease of IL-8 expression at the transcriptional level. Transdermal nicotine has no effect on mucin gene transcription.
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PMID:Transdermal nicotine decreases mucosal IL-8 expression but has no effect on mucin gene expression in ulcerative colitis. 1045 73

For the advanced study of the cell and molecular biology of middle ear mucosa, an in vitro cell culture system is required. Although middle ear epithelial cells have been cultured from various species of laboratory animal, there have been no reports concerning a serial subculture system of human middle ear epithelial cells. In this paper, we describe the establishment of a primary culture system of human middle ear epithelial cells using a serum-free conditioned medium and the characterization of these cells by the expression of phenotypic characteristics of epithelial cells and mucin genes. Cultured cells were anchorage-dependent in terms of growth and showed a polygonal cobblestone-like appearance: desmosomes in the cell junction were observed by electron microscopy. In the immunocytochemical study, cytokeratin (epithelial cell marker) was expressed in all cultured cells. but von Willebrand factor (endothelial cell marker) was not. Unexpectedly, vimentin (fibroblast marker) was locally expressed, and a double stain showed the co-expression of both cytokeratin and vimentin in the same cell. The products of reverse transcriptase polymerase chain reaction from cultured cells yielded distinct bands compatible with the expected sizes of the MUC1, MUC2, MUC5AC and MUC5B genes. This culture system will allow us to prepare the cell line and to perform advanced studies of human middle ear mucosal biology.
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PMID:Mucin gene expression in cultured human middle ear epithelial cells. 1120 May 87

Hypersecretion of mucin is a common feature of chronic and mucoid otitis media which may play an important role in hearing loss. The mechanisms controlling mucin secretion in the middle ear are not completely understood. Our reverse transcriptase-polymerase chain reaction results demonstrate that mRNAs of MUC1, MUC2, MUC3, MUC4 and MUC5AC are expressed in normal rat middle ear mucosa. Moreover, the expression of mRNA of the secretory mucins MUC2, MUC3 and MUC5AC was threefold lower in normal middle ear mucosa than that in the intestine or trachea. In contrast, expression of the membrane-bound mucins MUC1 and MUC4 was approximately the same in both middle ear mucosa and the intestine or trachea. MUC5AC proteins were also identified immunohistochemically in normal rat middle car epithelium. The methodology used in this study provides useful baseline information for investigation of the mechanisms of regulation of mucin gene expression during otitis media.
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PMID:Detection of mucin gene expression in normal rat middle ear mucosa by reverse transcriptase-polymerase chain reaction. 1127 Apr 93

In gastric cancer, altered expression of MUC1, MUC2, MUC5AC, and MUC6 mucin genes has already been described. We show in this report by the means of in situ hybridization, reverse transcriptase-polymerase chain reaction, and transfection assays that MUC5B is also abnormally expressed in gastric carcinomatous tissues and cell lines. We thus undertook to elucidate the molecular mechanisms that regulate the transcription of MUC5B in gastric cancer cells. To this end, high expressing (KATO-III) and low expressing (AGS) gastric cancer cell lines were chosen to study human mucin gene MUC5B expression and promoter activity. Sequencing of the promoter region revealed a distal TATA box located 1 kilobase upstream of the proximal TATA box. Functional activity of the promoter was addressed by using deletion mutants covering 2044 nucleotides upstream of the MUC5B transcription start site. We identified a distal promoter 10 times more active than the proximal promoter in KATO-III cells. In AGS cells, both promoters, much less active, showed the same range of activity. Binding assays allowed us to show that the transcription factor ATF-1 binds to a cis-element present in the distal promoter. Sp1, which binds to both promoters specifically transactivates the proximal promoter. Treatment of transfected cells with PMA, cholera toxin A subunit, and calcium ionophore showed that only PMA led to a substantial activation of the distal promoter. MUC5B 5'-flanking region having a high GC content, influence of methylation on the MUC5B expression was assessed. Our results indicate that repression of MUC5B expression visualized in AGS cells is due in part to the presence of numerous methylated cytosine residues throughout the 5'-flanking region. Altogether these results demonstrate that MUC5B expression in gastric cancer cells is governed by a highly active distal promoter that is up-regulated by protein kinase C and that repression is under the influence of methylation.
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PMID:Aberrant expression of human mucin gene MUC5B in gastric carcinoma and cancer cells. Identification and regulation of a distal promoter. 1127 96

We investigated the expression levels of MUC5AC in endotoxin-induced otitis media with effusion (OME) in the rat using competitive polymerase chain reaction (PCR) and the morphology of middle ear mucosa using transmission electron microscopy (TEM). Experimental OME in the rat was induced after middle ear instillation of Escherichia coli lipopolysaccharides (LPS). Middle ear mucosa were obtained at 0 h, 12 h, Day 1, Day 3, Day 7 and Day 14 and reverse transcriptase (RT)-PCRs were then performed for the identification of MUC1, MUC2, MUC5AC and submandibular mucin 1 expression, followed by competitive PCRs for MUC5AC and beta2-microglobulin expression. Normal middle ear mucosa revealed no expression of mucin genes, whereas endotoxin upregulated the expression of MUC5AC mRNA between 12 h and Day 7, with maximal expression at Days 1 and 3. Middle ears treated three times with LPS upregulated more MUC5AC mRNA expression, by a factor of approximately 3.5, than those 1 day after one instillation. On TEM, dark granulated cells were observed at Day 3 after endotoxin instillation, but mixed granulated cells were seen on the ears treated three times with LPS. These results suggest that MUC5AC could be one of the major mucin genes in the middle ear mucosa related to otitis media.
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PMID:Up-regulation of MUC5AC mRNA expression in endotoxin-induced otitis media. 1142 2


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