Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Primary palatogenesis involves an intricate array of events. Cell migration, proliferation, differentiation, programmed death, and fusion occur. Prior to fusion, the morphology of the epithelium undergoes marked changes. Epithelial projections form and extend across the fusion site attaching by filopodia to the opposite prominence. By appearance, the epithelium plays a critical role in facial development. In order to monitor epithelial activities, a study was done to isolate and characterize epithelial cells derived from the primary palate. The primary palate was microdissected from day 13 Sprague-Dawley rat embryos, and the epithelium and mesenchyme were separated by enzymatic digestion with a 3% trypsin-pancreatin solution (3:1). All explants were cultured in Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 medium (1:1) supplemented with 10% fetal calf serum (FCS), 20 ng/ml epidermal growth factor (EGF), and antibiotics. Explant cells were gathered by trypsin harvesting and sub-cultured. These sub-cultured cells were further characterized. Transmission and scanning electron microscopy showed that the cells retained many morphological features observed in vivo. In passaged cells, type IV collagen, laminin, and cytokeratins were visualized by immunocytochemistry. Gel electrophoresis analysis of the water-insoluble extracts demonstrated major bands of proteins of 50 kD and 44 kD that were synthesized by the epithelial cells but not by the mesenchymal cells. These cytokeratin types are suggestive of a simple undifferentiated embryonic epithelium. The effect of all-trans retinoic acid (RA) on cell number and [3H]-proline incorporation was assessed. At [10(-4)M] and [10(-6)M] retinoic acid resulted in significant inhibition in cell proliferation and amount of proline incorporated, with the greater inhibition occurring in the mesenchymal cells. In the concentrations studied, retinoic acid has an inhibitory effect on the two differently derived cell types. This study established that sub-cultured epithelial cells maintain their phenotype and can be used to study fusion processes. Part 2 will demonstrate how the morphology of the epithelial cells can be modified to produce the changes that are observed during fusion of the primary palate.
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PMID:Epithelial bridging of the primary palate: I. Characterization of sub-cultured epithelial cells. 263 81

The organization of intermediate-sized filaments (IF) of the cytokeratin type was studied in cultures of PtK2 cells in which typical IF structures are maintained during mitosis, using a monoclonal antibody (KG 8.13). This antibody reacts, in immunoblotting experiments, with the larger of the two major cytokeratin polypeptides present in these cells but, using standard immunofluorescence microscopy procedures, does not react with the cytokeratin filaments abundant in interphase cells, in striking contrast to various antisera and other monoclonal cytokeratin antibodies. In the same cell cultures, however, the antibody does react with cytokeratin filaments of mitotic and early postmitotic cells. The specific reaction with cytokeratin filaments of mitotic cells only is due to the exposure of the specific immunologic determinant in mitosis and its masking in interphase cells. Treatment of interphase cells with both Triton X-100 as well as with methanol and acetone alters the cytokeratin filaments and allows them to react with this monoclonal antibody. A similar unmasking was noted after treatment with buffer containing 2 M urea or low concentrations of trypsin. We conclude that the organization of cytokeratin, albeit still arranged in typical IF, is altered during mitosis of PtK2 cells.
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PMID:Change of cytokeratin filament organization during the cell cycle: selective masking of an immunologic determinant in interphase PtK2 cells. 619 64

Intermediate-sized filaments have been noted in epithelioid sarcoma by previous investigators, two of whom have reported that the filaments represent vimentin. We utilized polyclonal antibodies directed against keratin and immunoperoxidase techniques (PAP) to stain 32 of the more than 300 cases accumulated at the AFIP . All of our material was formalin-fixed, paraffin-embedded. Seventy-five percent of our cases (24/32) showed positive immunoreactivity, a feature that may be of diagnostic help in distinguishing epithelioid sarcoma from modular fasciitis, benign and malignant fibrous histiocytoma, malignant melanoma, and necrotizing granuloma. In these cases, the reaction was enhanced using predigestion with trypsin. The immunoreactivity varied from tumor to tumor, perhaps due to formalin fixation. Since synovial sarcoma and mesothelioma may also be cytokeratin-positive, our findings indicate that keratin immunoreactivity is not confined to epithelial tumors and may also occur in neoplasms traditionally regarded as mesenchymal.
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PMID:Keratin in epithelioid sarcoma. An immunohistochemical study. 620 17

We investigated distant recurrence and S-phase fraction (SPF), estimated by flow cytometry with and without selection of the epithelial cell population, in 201 stage II breast carcinomas. The tumour tissue was disintegrated mechanically by scissors and one part of the cell suspension was treated with a detergent-trypsin method for single-parameter analysis, and the other part, for immunological selection of epithelial cells, was incubated with a monoclonal antibody (CAM 5.2) recognising cytokeratins 8 and 18 and a secondary fluorescein isothiocyanate-labelled antibody. DNA was stained with propidium iodide. In order to compare the methods, statistical analysis was performed on the 152 tumours with S-phase fraction estimated by both methods. Sixty-five tumours were diploid, 81 were aneuploid and six tumours had different ploidy determined by the two methods. Using univariate regression analysis, SPF of the epithelial cell population predicted recurrence more effectively than SPF from single-parameter analysis. In multivariate regression analysis, SPF of the cytokeratin-containing population added significant prognostic information to the SPF of the non-selected cells. We concluded that the use of flow cytometric selection of epithelial breast carcinoma cells enhances the predictability value of SPF.
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PMID:Flow cytometric analysis of S-phase fraction in breast carcinomas using gating on cells containing cytokeratin. South East Sweden Breast Cancer Group. 751 Jan 19

Like most epithelial cells that are isolated from tissues and placed in culture, the phenotype of human retinal pigment epithelial cells (RPE) in vitro is more variable than for the same cells in situ. The phenotypic heterogeneity of the cultures has been attributed to varying stages of differentiation of the cells induced by the culture environment. In this study we show that within the same cultures RPE cells exist as phenotypic variants which are distinct and stable. Two phenotypically distinct subpopulations were identified, one epithelioid and one fusiform, that were present from the first spreading event in primary culture through multiple serial passages and when maintained for extended periods at confluency. Cell aggregation studies indicated differences in cellular adhesions, known determinants of cell shape, between the subpopulations. Methods to separate the subpopulations were developed which are based on the differential trypsin sensitivity of adhesions. The separated subpopulations had the same RPE cytokeratins by immunoblotting, but cytokeratin filaments (and actin filaments) had different organizations. The study indicates that RPE cell cultures contain at least two subpopulations of phenotypically distinct cells under identical culture conditions that can be separated and propagated independently. The phenotypic variants offer a model system for investigating determinants of epithelial cell shape in RPE. Further, the separation methods might be applied to test for phenotypic variants in other types of cultured epithelia.
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PMID:Separation of phenotypically distinct subpopulations of cultured human retinal pigment epithelial cells. 751 70

We have compared the prediction of distant recurrence for S-phase fraction (SPF) and DNA-ploidy, as estimated by flow cytometry, on an epithelial cell population and an unselected cell population from 268 node-negative breast-cancer patients diagnosed between 1985 and 1988. The tumor tissue was mechanically disintegrated and divided for flow cytometric analysis using both gated cells containing cytokeratin 8 and 18 and ungated cells treated with a detergent-trypsin solution. The relationship to distant recurrence was investigated for flow cytometric data, tumor size and estrogen and progesterone receptor content in univariate and multivariate Cox's regression analysis. The regression analyses were performed on 209 cases with S-phase fractions estimated by both methods. In 11 cases, DNA-ploidy classification differed, reflecting increased sensitivity to minor aneuploid peaks but a decreased ability to separate peaks in the near-diploid region for the gated populations. When SPF were used in univariate analysis as a continuous parameter or the upper tertile was used as cut-off value, SPF from the cytokeratin-gated cell population were most closely associated with recurrence and contributed additional prognostic information to SPF from the unselected cell population in the multivariate analysis. Out of the following variables:tumor size, ER and PR status, SPF and DNA ploidy, only SPF from immunoselected cells contributed prognostic information in multivariate analysis. These results indicate that SPF from immunoselected cell populations improves the prediction of recurrence in node-negative breast cancer.
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PMID:S-phase fraction after gating on epithelial cells predicts recurrence in node-negative breast cancer. 752 14

Barrett's oesophagus is a preneoplastic condition in which the squamous mucosa of the oesophagus is replaced by columnar epithelium. Epithelial cells of Barrett's oesophagus were isolated from resected oesophagus specimens by two methods not previously applied to the culture of Barrett's oesophagus cells. These techniques included trypsinisation of small fragments of mucosa, followed by plating in tissue culture dishes, and a direct tissue explant technique. A modified MCDB-153 growth medium was used. Primary trypsin technique cultures were plated on uncoated plastic, or plastic coated with type I collagen, type IV collagen, or fibronectin. Growth on type IV collagen and fibronectin plates was slower but produced less contamination from fibroblasts. By 20-40 days most cultures formed confluent monolayers made up of cells with epithelial morphology. The cells were cytokeratin positive, vimentin negative, and contained alcian blue positive vacuoles, confirming their epithelial origin and suggesting their derivation from Barrett's oesophagus. Electron microscopy showed tonofilaments, microvilli, and desmosomes. Cells proliferated through up to eight subcultures before growth slowed and cells showed senescent changes. This study shows that epithelial cells from Barrett's oesophagus can be grown by comparatively simple tissue culture techniques. These methods can provide sufficient material for a variety of molecular biology and biochemical studies of epithelial cells from Barrett's oesophagus.
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PMID:Tissue culture of epithelium derived from Barrett's oesophagus. 806 13

A new human cell line, LC-2/ad was established from pleural effusion of pulmonary adenocarcinoma of a 51 year old Japanese female. The LC-2/ad cells exhibit an epithelial appearance and a tendency to form small domes as observed with phase-contrast microscopy. The modal chromosome number was 53-56. Plating efficiency and doubling time were 6.8% and 58 h, respectively (32th passage). Immunocytochemically, the cells were strongly positive for CEA and cytokeratins including cytokeratin no. 18 which is present in simple epithelia. Ultrastructurally, the cultured cells were characterized by well-formed junctional complexes and microvilli. Subcutaneous injection of 5 x 10(6) cells into a nude mouse resulted in tumor formation classified histologically as a moderately differentiated adenocarcinoma. This cell line produced at least two functionally active trypsin inhibitors together with several proteinases in vitro. The main inhibitor was purified partially from the serum-free conditioned medium and confirmed immunologically as human alpha 1-antitrypsin (AAT). Immunohistochemically, the xenografted tumor was also positive for AAT. The cell line LC-2/ad is useful for the study of tumor-derived serine proteinase inhibitors, in particular AAT.
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PMID:Establishment and characterization of a human lung adenocarcinoma cell line (LC-2/ad) producing alpha 1-antitrypsin in vitro. 829 44

An assay of epithelial barrier function was developed to monitor immune-mediated changes in lung permeability that may be occurring during pulmonary allograft rejection and inflammatory lung diseases. Lung tissue was obtained from minipigs, digested with collagenase (1 mg/ml) overnight, and propagated in RPMI 1640 tissue culture medium. Cells with an epithelioid morphology were purified by differential detachment using trypsin-ethylenediaminetetraacetic acid and were characterized as epithelial by positive staining with an anti-cytokeratin monoclonal antibody. Monolayers of these epithelial cells were cultured on porous tissue culture inserts, and transmonolayer resistance values were measured. Transmonolayer resistance values reached a mean of 5487 +/- 2882 omega (mean +/- 95% confidence interval; n = 9) after 5 days in culture. These values indicated the presence of functional intercellular tight junctions between the cells. Addition of cytotoxic immune effector cells to the cultured monolayers caused a rapid reduction in the transmonolayer resistance values, whereas unstimulated splenocytes failed to produce this effect. Comparison of these results with those obtained in parallel experiments performed with standard isotopic cytotoxicity assays indicated the sensitivity of the transmonolayer resistance technique. The assay described in this report will enable in vitro modeling of epithelial permeability damage mediated by both activated lymphoid cells and their soluble products.
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PMID:An in vitro system to model pulmonary epithelial barrier dysfunction mediated by immune effector cells. 832 23

Establishment of cell culture systems for the study of organogenesis during human embryonic development could provide the basis for the study of molecular mechanisms that regulate cellular proliferation and organ morphogenesis. We have developed a cell culture system for undifferentiated mesenchymal cells isolated from the human fetal kidney, which retain the potential for conversion to differentiated epithelia in vitro. Microdissected marginal zone nephroblasts were treated with trypsin and plated on gelatin prior to unlimited serial passage in suspension. An absolute requirement for the indefinite proliferation of these undifferentiated progenitors was nephroblast growth factor (NB-GF), a growth factor activity secreted by a Wilms tumor cell line. The mitogenic effects of NB-GF were not reproduced by previously described growth factors known to be mitogenic for renal cells or by leukemia inhibitory factor. In addition, cultured nephroblasts were shown to retain their ability to differentiate into epithelia when exposed to 10% serum-containing medium in the absence of NB-GF. Immunocytochemical cytoskeletal protein marker analysis showed mutually exclusive staining of vimentin in nephroblasts and cytokeratin in epithelia. These findings suggest that NB-GF may play an important role in the regulation of nephroblast proliferation during renal development and in Wilms tumor biology.
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PMID:A putative Wilms tumor-secreted growth factor activity required for primary culture of human nephroblasts. 839 86


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