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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To establish a suitable control for pancreatic tumor cell lines, we have isolated and cultured primary human pancreatic duct cells from transplant donors. Duct cells were isolated by dissecting the main pancreatic duct and first-degree branches and enzymatic digestion. Aggregates of cells were cultured for 1 up to 5 weeks and monitored for changes in morphology and growth by phase contrast microscopy. Contaminating fibroblasts were mechanically removed from day 4 on and by cloning of epithelial cells. Cultured cells were characterized by phase contrast microscopy, electron microscopy, and immunofluorescence with antibodies against intermediate filaments (cytokeratins, vimentin, desmin), mucins (Du-Pan-2, CA 19-9), carbonic anhydrase II, acinar cell enzymes (amylase, lipase, trypsin), and islet cells. About 90% of the cultured cells could be identified as ductal epithelial cells by their expression of cytokeratins, mucins, and carbonic anhydrase II. These cells showed the ultrastructural features of duct cells. After 3-5 weeks of culture, most of the cultured cells showed co-expression of cytokeratins and vimentin in addition to duct cell markers. About 10% of cells were contaminating fibroblasts (vimentin positive, cytokeratin negative). The cultured normal human duct cells as the postulated cells of origin of the pancreatic adenocarcinoma may serve as a useful control for cultured pancreatic tumor cell lines.
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PMID:Isolation, culture, and characterization of human pancreatic duct cells. 846 98

A new method for the long-term culture of pure rat thymic epithelial cells was established. The cultures were characterized by immunocytochemistry, electron microscopy and proliferation assays. Non-epithelial thymic cells were eliminated with a reliable and reproducible pre-plating method, by differential trypsin treatment of the cultures and by addition of horse serum to the culture medium instead of fetal calf serum. The final cultures contained more than 95% pure epithelial cells as evidenced by immunostaining for cytokeratin. Ultrastructural studies indicated that these cells are physiologically active epithelial cells with tonofilaments, desmosomes and filopods. The subsets of the thymic epithelial cells in vitro were investigated by comparing their staining pattern with that obtained in situ using several subtype-selective antibodies. Thymic epithelial cells in vitro showed a preferential expression of subcapsular/perivascular and medullary markers. Only few cultivated cells were of cortical origin. In the first to the fourth subcultures, some cells were immunopositive for the thymus hormone/factor thymulin. The proliferation of thymic epithelial cells was stimulated by horse serum and to a lesser extend by fetal calf serum. The adenylate cyclase activators isoproterenol and forskolin, and the glucocorticoid cortisol inhibited the proliferation.
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PMID:Rat thymic epithelial cells in vitro and in situ: characterization by immunocytochemistry and morphology. 859 52

Cytokeratin expression in mammals is generally restricted to epithelial cells and has been utilized to differentiate epithelial from nonepithelial tissues in these species. Since cytokeratins have been shown to be highly conserved during vertebrate evolution, the objective of the present study has been to ascertain the expression pattern of cytokeratins in tissues of the common carp (Cyprinus carpio). A panel of 10 anti-human cytokeratin antibodies was evaluated using a streptavidin-biotin-peroxidase complex detection system. Tissues were fixed in 10% neutral-buffered formalin, 100% ethanol or methacarn. Only formalin-fixed tissues were pre-digested with trypsin prior to immunostaining. Formalin-fixed tissues generally resulted in a less intense, more diffuse staining pattern with considerable background compared with ethanol and methacarn and was therefore the least desirable fixative. The diverse staining pattern observed with the various antibodies used in this study was consistent with previous findings in other teleosts. The results confirm that cytokeratin expression in teleosts is fundamentally different from that in mammals and therefore should be used as a method to differentiate epithelial cell types in these species only with discretion.
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PMID:Cytokeratin-filament expression in epithelial and non-epithelial tissues of the common carp (Cyprinus carpio). 899 9

In the present study we describe a novel multiparameter flow cytometric (FCM) assay to estimate the fraction of cycling cells in epithelial tumors derived from fresh frozen as well as archival material. To this end, MCF-7 cells as well as a series of breast carcinomas (n = 10; fresh frozen as well as formalin fixed and paraffin embedded) were stained using a panel of different antibodies directed against the Ki67-Ag (DAKO/PC, MIB-1, Ki-S5, and poly-Ki67) for a 3-parameter cytokeratin/Ki67-Ag/DNA FCM analysis. Whereas all Ki67-Ag antibodies work equally well in the methanol fixed cell line, MIB-1 and Ki-S5 epitopes are retained in cell suspensions mechanically derived from fresh frozen tissue. Only antibody Ki-S5 shows specific nucleolar staining patterns in cell suspensions prepared by trypsin digestion of formalin fixed, paraffin embedded tissue sections. A good correlation was found between the fractions of Ki67-Ag-positive epithelial cells measured in cell suspensions derived from fresh frozen and the corresponding formalin fixed and paraffin embedded tumor samples. Furthermore, the fraction of Ki67-Ag-positive epithelial cells as determined by 3-parameter FCM correlated very well with the Ki67-Ag-labeling index in paraffin embedded tissue sections.
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PMID:Multi-parameter flow cytometric analysis with detection of the Ki67-Ag in paraffin embedded mammary carcinomas. 904 Nov 18

We established a retinal pigment epithelium-derived cell line from transgenic mouse harboring temperature-sensitive simian virus 40 T-antigen gene (tsSV40T) and examined its characteristics. We enucleated both eyes from a 2-month-old transgenic mouse and removed the retinal pigment epithelial (RPE) cells and neuroretinal cells. After cloning the RPE cells, we obtained a cell line (RPET). RPET cells grew well at 33 degrees C but not at 37 degrees C, expressing on the temperature-sensitive character of tsSV40T, and maintained characters of RPE cells such as T1-tyrosinase production, phagocytosis of rod outer segments, and presence of cytokeratin, microvilli on the cell surface and lysosome-like granules around the Golgi apparatus in the cytoplasm. Conditioned medium (CM) from a culture of neuroretinal cells harboring tsSV40T was essentially required for growth. The factor(s) in CM was heat-and acid labile, but was resistant to trypsin digestion. In the presence of 3% CM, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) and strong effects on RPET cells, whereas insulin, insulin-like growth factor I (IGF-I), and IGF-II had moderate growth effects. Interestingly, none of these growth factors stimulated the RPET cells in the absence of CM. EHS-Matrix had growth effect, whereas laminin, collagen types I and IV, and fibronectin had no marked growth effects on RPET cells. RPET cells were morphologically changed on a laminin-coated dish. They could not spread on the coated dish, and the majority of the cells floated. But when the floating cells were transferred to non-coated dishes, they immediately attached themselves. These results suggest that RPET cells are a good model for for finding novel growth factor(s) and for investigating the mechanism of cell-laminin attachment.
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PMID:A retinal pigment epithelium-derived cell line from transgenic mouse harboring temperature-sensitive simian virus 40 large T-antigen gene. 907 3

The "gold standard" inflow cytometric DNA analysis of breast cancer uses fresh tumor cells simultaneously labeled for cytokeratin (CK) and DNA. We developed a 2-parameter CK-DNA flow assay suitable for archival, paraffin-embedded tissue (PT). Six anti-CK monoclonal antibodies were tested by immunocytochemistry and our assay for staining of nuclei extracted from PT breast cancers by combination pepsin-trypsin digestion. Clone CAM 5.2 was inadequate for PT nuclear suspensions, but a cocktail of 2 anti-CK clones (AE1/AE3 and KL-1) distinguished epithelial from nonepithelial nuclei in 2-parameter flow dot plots. We studied 82 routine PT breast tumors by our assay and used a univariate flow DNA histogram based on fresh biopsy tissue for comparison. Three histogram data quality indicators were improved. A trend toward higher S-phase fractions was found for DNA diploid PT tumors, although when inflammation was evident histologically, the increment in S-phase fraction with gating was often marked. CK gating identified PT tumors containing concurrent CK-positive DNA diploid and nondiploid populations (27 of 56 DNA nondiploid histograms). By excluding nonepithelial nuclei, 2-color CK-DNA flow methods may increase the accuracy of ploidy and S-phase fraction measurements. Our method appears superior to previous techniques using clone CAM 5.2 for labeling of archival breast cancers.
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PMID:Flow cytometric DNA analysis with cytokeratin gating of formalin-fixed deparaffinized breast cancer nuclei. 970 23

The mechanism of malonyl-CoA-independent acute control of hepatic carnitine palmitoyltransferase I (CPT-I) activity was investigated. In a first series of experiments, the possible involvement of the cytoskeleton in the control of CPT-I activity was studied. The results of these investigations can be summarized as follows. (i) Very mild treatment of permeabilized hepatocytes with trypsin produced around 50% stimulation of CPT-I activity. This effect was absent in cells that had been pretreated with okadaic acid (OA) and seemed to be due to the action of trypsin on cell component(s) distinct from CPT-I. (ii) Incubation of intact hepatocytes with 3, 3'-iminodipropionitrile, a disruptor of intermediate filaments, increased CPT-I activity in a non-additive manner with respect to OA. Taxol, a stabilizer of the cytoskeleton, prevented the OA- and 3, 3'-iminodipropionitrile-induced stimulation of CPT-I. (iii) CPT-I activity in isolated mitochondria was depressed in a dose-dependent fashion by the addition of a total cytoskeleton fraction and a cytokeratin-enriched cytoskeletal fraction, the latter being 3 times more potent than the former. In a second series of experiments, the possible link between Ca2+/calmodulin-dependent protein kinase II (Ca2+/CM-PKII) and the cytoskeleton was studied in the context of CPT-I regulation. The data of these experiments indicate that (i) purified Ca2+/CM-PKII activated CPT-I in permeabilized hepatocytes but not in isolated mitochondria, (ii) purified Ca2+/CM-PKII abrogated the inhibition of CPT-I of isolated mitochondria induced by a cytokeratin-enriched fraction, and (iii) the Ca2+/CM-PKII inhibitor KN-62 prevented the OA-induced phosphorylation of cytokeratins in intact hepatocytes. Results thus support a novel mechanism of short-term control of hepatic CPT-I activity which may rely on the cascade Ca2+/CM-PKII activation --> cytokeratin phosphorylation --> CPT-I de-inhibition.
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PMID:Malonyl-CoA-independent acute control of hepatic carnitine palmitoyltransferase I activity. Role of Ca2+/calmodulin-dependent protein kinase II and cytoskeletal components. 970 78

The existence of progenitor (stem) cells in the human liver remains a matter of debate. In rodent models of hepatocarcinogenesis and injury, oval cells proliferate in the periportal regions of the portal tracts and are suggested to derive from a stem cell compartment, because they are capable of differentiating into hepatocytes or biliary epithelial cells. In this study, the rat oval cell marker, OV-6 has been used to investigate the hypothesis that there are stem cells present in fetal and pediatric human liver. The pattern of OV-6 expression was compared with the established adult biliary cell markers human epithelial antigen-125 (HEA-125) and cytokeratin-19 (CK-19). In normal pediatric liver (n = 7), bile ducts and ductules were immunostained with CK-19 and HEA-125, whereas OV-6 staining was consistently negative. In fetal tissue (n = 10), ductal plate cells, primitive bile ducts, and hepatoblasts were stained with CK-19 and HEA-125 although only some of the ductal plate cells and hepatoblasts were OV-6 positive. In biliary atresia (n = 6) and 1, anti-trypsin deficiency (1,AT) (n = 4), CK-19 and HEA-125 immunostained ductular proliferative cells that tended to form finely anastomosing ductules, whereas OV-6 staining was found more on discrete cells confined to portal tract margins. Additionally, in diseased liver, OV-6 was strongly positive in hepatocyte lobules with greatest intensity in the periseptal regions. This widespread hepatocyte OV-6 positivity suggests that the antibody may identify cells of a less differentiated phenotype (transitional hepatocytes) that have replaced the mature cells. Therefore, it is proposed that in human liver, OV-6 is recognizing cells with a progenitor stem cell-like phenotype with the capacity to differentiate into OV-6 positive ductular cells or lobular hepatocytes.
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PMID:Immunolocalization of OV-6, a putative progenitor cell marker in human fetal and diseased pediatric liver. 975 34

A method for primary cell culture of fetal rat gastric fundic epithelial cells was developed. The tissue was incubated with 0.125% trypsin at 4 degrees C for 8-10 hours. The epithelial cells isolated were then cultured in DMEM/F12 medium supplemented with 20% fetal calf serum. Within 24 hours the cells attached to the culture plate and became confluent in 3 days. On phase contrast microscopy, over 90% of cell possessed epithelial characteristics. Immunocytochemical studies showed: (a) 90% of cells were positive in anti-cytokeratin antibody staining; (b) 90% of the epithelial cells contained PAS positive granules; (c) 20% of epithelial cells gave a strong reaction for succinic dehydrogenase activity. Electron microscopy (EM) showed microvilli on the surface of cells, junctional complexes (tight juntion and desmosome), glycogen and mitochondria. Autoradiographic studies showed that these cells possessed the capability to synthesize DNA and this ability was maximum on day 2. This in vitro system may provide a valuable model for studies of cellular functions of gastric mucosa.
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PMID:[Primary culture of fetal rat gastric mucosal epithelium]. 1007 56

To analyze the regulation of water channels in the peritoneum, we tried to establish a primary mesothelial cell culture system. Male Sprague-Dawley rats weighing about 250 g were anesthetized, and 10 mL of phosphate-buffered saline (PBS) containing 0.25% trypsin and 1 mmol/L ethylenediamine tetraacetic acid (EDTA) was infused into the peritoneal cavity for 15 minutes. Sediments from the recovered fluid were cultured in medium M199 supplemented with 10% fetal bovine serum (FBS). The culture was succeeded 4-6 times before experiments commenced. After exposure to the test medium, RNA was extracted and subjected to reverse transcriptase polymerase chain reaction (RT-PCR) for 10-19 cycles, then was measured by Southern blot analysis with a digoxin-labeled probe. Cultured cells were positively stained with mouse monoclonal anti-cytokeratin antibody, confirming their characteristics as mesothelial cells. Aquaporin-1 (AQP-1) message in the cultured cells increased with increases in glucose and mannitol concentrations when beta-actin message was used as an internal control. Tranexamic acid effected no change in AQP-1 message in the cultured mesothelial cells. This system offers potential as a simple approach to test the effects of osmolytes, cytokines, and vasoactive hormones on aquaporin expression and water transport in the peritoneum.
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PMID:Water channel AQP-1 in the primary cell culture of rat peritoneum. 1068 62


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