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)

Microwave (MW) energy permits rapid tissue fixation for light and electron microscopy but its effects on antigen preservation have not been fully evaluated. We, therefore, fixed three samples of human skin, uterus, and cervix, and two samples of human colon and breast by MW irradiation (5 to 8 seconds) during simultaneous immersion in a dilute aldehyde mixture (2% formaldehyde and 0.05% glutaraldehyde). For comparison, similar portions of each specimen were fixed in formalin. Specimens were processed routinely and embedded in paraffin for light microscopy. Sections from each specimen were stained with hematoxylin and eosin and, by immunoperoxidase techniques, for epithelial membrane antigen, leukocyte common antigen, S-100 keratin, carcinoembryonic antigen, and factor VIII-related antigen, the latter three with and without preliminary trypsinization. Colon sections were also stained for chromogranin. In all cases, light microscopic morphology was comparable for tissues fixed by the MW method and formalin-fixed specimens, as was immunostaining for epithelial membrane antigen, leukocyte common antigen, S-100 protein, and chromogranin. Formalin-fixed tissues required trypsinization for optimal detection of keratin, carcinoembryonic antigen, and factor VIII-related antigen. In contrast, trypsin-pretreatment was not necessary to demonstrate these antigens in MW-fixed specimens and, in fact, resulted in tissue digestion. We conclude that this MW fixation method provides a means for rapidly fixing tissues for immunoperoxidase staining while preserving excellent light microscopic morphology.
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PMID:Rapid microwave fixation of human tissues for light microscopic immunoperoxidase identification of diagnostically useful antigens. 331 39

The present study examined the effect of aging on epithelium and on its ability to respond to an inductive stimulus provided by murine dental papillae. In fetal CD-1 mice, 15- to 17-day molar mesenchyme was combined with 15- to 19-day epithelium from the secondary palates. Enamel organs were separated from the dental papillae, and palatal epithelium was peeled away from its underlying mesenchyme after treatment with 1% trypsin. Recombinants of epithelium and papillae were initially cultured on a solidified complex medium for 24 hr followed by an additional 10-14 days of intraocular explanation. Control specimens consisted of isolated molar papillae. Nineteen of 88 isochronal, heterotypic recombinations formed teeth. None of the 46 heterochronal, heterotypic grafts of 18- and 19-day palatal epithelium combined with 15- to 17-day molar papillae-produced teeth. Instead, keratin-filled epithelial cysts and bone spicules were formed. Isolated control molar papillae often formed bone in the intraocular sites but did not form teeth or contain epithelium. These results show that palatal epithelium is first restricted to its developmental pathway at 18 days of gestation. Younger epithelium can convert to functional ameloblasts that secrete enamel protein. In addition to the change in gene expression, normal tooth morphology is attained. The loss of competence of the palatal epithelium at 18 days gestation coincided with the acquisition of stratum corneum and the attainment of the fully differentiated state. The oral surface of palatal epithelium appears to be determined histogenically and morphogenically at 18 days of gestation in mice.
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PMID:Tooth induction and temporal patterning in palatal epithelium of fetal mice. 371 51

From our long experience with primary cultures of human differentiated cells, we have been able to come to the following conclusions. The culture media routinely employed have been developed using established cell lines but not primary cells as the growth marker. Therefore the culture media for primary cultures should be modified from those routinely employed. In addition the concentrated supplementation of serum to basal media dose not contribute to the growth of primary differentiated cells, and in fact on the contrary, is advantageous for the growth of non-target fibroblasts which would hamper the growth of the target cells. A hypoxic culture environment is more favorable for primary cultures especially when smaller cell numbers are used as the inoculum. The primary culture cells are more fragile in comparison with established cell lines. Therefore, the low-temperature cell diopersion procedure is recommended with the use of diluted crystalline trypsin saline. On the basis of the above findings, we have successfully carried out primary and transfer cultures of human esophageal and gall bladder epithelial cells, skin keratinocytes and endothelial cells. Their epithelial mature was proved by the presence of keratin in their cytoplasm, and the phenotype of endothelial cells was evident from the presence of Factor VIII-related antigen in their cytoplasm. Culture of endothelial cells requires the supplementation of a specific growth factor which we have utilized and isolated from conditioned medium of human diploid fibroblasts. Tiny amounts of the factor were shown to, induce the vascularization of rabbit eye cornea.
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PMID:[Primary cultures of various differentiated human cells and their transfer]. 380 Apr 6

Solid and papillary epithelial neoplasms of the pancreas from six female patients were studied using immunohistochemistry and electron microscopy to define better their histogenesis. The tumors ranged in diameter from 5 to 15 cm (average: 9 cm), and, on cross section, most had areas of hemorrhage and necrosis, sometimes extensive. Microscopically, there was a solid and pseudopapillary pattern, with tumor cells typically having ovoid nuclei with delicate folding and indistinct nucleoli. Of note were the following: a relatively low mitotic rate (range: 0-6/20 hpf), the presence of hyaline globules (four of six cases), and collections of foam cells (three of six cases). Staining for cytoplasmic argyrophil granules was negative in each case. Ultrastructurally, the solid and papillary epithelial neoplasms of the pancreas showed evidence of acinar or ductular differentiation. Two contained zymogen granules, one had intermediate filaments (probably keratin), and three had abundant rough endoplasmic reticulum and mitochondria. Immunostaining was positive for chymotrypsin (six of six cases), trypsin (four of six), and amylase (three of six). None was positive for alpha-1-antitrypsin, neuron-specific enolase, pancreatic polypeptide, gastrin, glucagon, somatostatin, or insulin. The findings support an origin from exocrine pancreas, and follow-up indicates a low rate of malignancy, with local recurrence in two of the six patients.
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PMID:Solid and papillary epithelial neoplasm of the pancreas. An ultrastructural and immunocytochemical study of six cases. 381 76

Permeability barriers must exist in transitional epithelium to prevent the free flow of water from underlying blood capillaries through the epithelium into the hypertonic urine, and such a barrier has now been demonstrated in isolated bladders. This barrier is passive in function and can be destroyed by damaging the luminal surface of the transitional epithelium with sodium hydroxide and 8 M urea solutions, by digesting it with trypsin, lecithinase C, and lecithinase D, or by treating it with lipid solvents such as Triton x 100 and saponin. From this it is concluded that the barrier depends on the integrity of lipoprotein cell membranes. The barrier function is also destroyed by sodium thioglycollate solutions, and electron microscope investigations show that sodium thioglycollate damages the thick asymmetric membrane which limits the luminal face of the superficial squamous cell. Cytochemical staining shows the epithelium to contain disulfide and thiol groups and to have a concentration of these groups at the luminal margin of the superficial cells. It thus appears that the permeability barrier also depends on the presence of disulfide bridges in the epithelium, and it is presumed that these links are located in keratin. Because of the effect of thioglycollates, both on the barrier function and on the morphology of the membrane, it is suggested that keratin may be incorporated in the thick barrier membrane. It is proposed that the cells lining the urinary bladder and ureters should be regarded as a keratinizing epitheluim.
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PMID:The permeability of rat transitional epithelium. Kertinization and the barrier to water. 590 98

Insoluble epidermal proteins (possibly keratin), previously considered inert to enzyme action, were solubilized by either trypsin or chymotrypsin. Cleavage of the disulfide bonds prior to enzymatic action is not necessary. In addition, the enzymatic action on intact epidermis is not influenced by the presence or absence of endogenous lipids, soluble proteins, peptides, or amino acids. Solubilization of epidermal protein by chymotrypsin is inhibited by the supernatant solution of the homogenized epidermis.
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PMID:Enzymatic solubilization of insoluble proteins at neutral pH. 602 48

Glycoproteins and proteoglycans synthesized by human keratinocytes in medium containing D-[1-14C]glucosamine were extracted and analysed by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Extraction of the labelled keratinocytes with 0.5% Triton X-100 removed most of the glycoconjugates and left the cytoskeleton and nuclear residue adherent to the substratum. In addition to the cytoskeletal proteins, there was a relatively simple profile of glycoproteins and glycosaminoglycans associated with this adherent cytoskeleton. These consisted of eight glycoproteins in the mol.wt. range 99000-232000, five proteins in the keratin region (mol.wt. 42000-61000), hyaluronic acid and a sulphated glycosaminoglycan. Surface labelling of the keratinocytes with galactose oxidase (with or without neuraminidase)/KB3H4 revealed that many of the glycoproteins were exposed on the cell surface. The importance of the glycoproteins and proteoglycans in attaching the keratinocytes to the substratum was examined by studying their expression after incubation in medium containing tunicamycin and their degradation after digestion with trypsin and hyaluronidase. These studies, together with an examination of the glycoconjugates released by sequential extraction with 0.5% Triton X-100 followed by 0.2% sodium dodecyl sulphate, revealed that the glycoprotein of mol.wt. 232000 has an important role in mediating the attachment of keratinocytes to the substratum.
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PMID:Glycoproteins and glycosaminoglycans synthesized by human keratinocytes in culture. Their role in cell-substratum adhesion. 619 5

We show that intermediate-sized filaments reconstituted from human epidermal keratins appear unraveled in the presence of phosphate ions. In such unraveling filaments, up to four "4.5-nm protofibrils" can be distinguished, which are helically twisted around each other in a right-handed sense. Lowering the pH of phosphate-containing preparations causes the unraveling filaments to further dissociate into "2-nm protofilaments." In addition, we find that reconstitution of keratin extracts in the presence of small amounts of trypsin yields paracrystalline arrays of 4.5-nm protofibrils with a prominent 5.4-nm axial repeat. Limited proteolysis of intact filaments immobilized on an electron microscope grid also unveils the presence of 4.5-nm protofibrils within the filament with the same 5.4-nm axial repeat. These results, together with other published data, are consistent with a 10-nm filament model based on three distinct levels of helical organization: (a) the 2-nm protofilament, consisting of multi-chain extended alpha-helical segments coiled around each other; (b) the 4.5-nm protofibril, being a multi-stranded helix of protofilaments; and (c) the 10-nm filament, being a four-stranded helix of protofibrils.
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PMID:The fibrillar substructure of keratin filaments unraveled. 619 61

The membranous fraction isolated from stratum corneum by 8M urea-beta ME containing alkaline buffer (pH 9.0) is quite crude when observed by electron microscopy. However, this procedure may be useful for clinical samples, as one can isolate and compare both the soluble (interfilamentous) fraction and the keratin filament from the same sample in addition to the residues (membranous fraction). A further purified membranous fraction was isolated by a new method. Human stratum corneum was chopped and treated with 8M urea-50 mM Tris-HCl (pH 9.0), digested by the use of trypsin, and the product fractionated by a sucrose density gradient to obtain separate single cells without the cytoplasm. One sample was then treated with trypsin for 1 hour and another with urea buffer for 24 hours. Observations revealed a thickened inner membrane (marginal band) of approximately 150A. Each of the membranous samples contained a level of half-cystine markedly higher in amount (around 100/1,000) and involved mostly in the epsilon-(gamma-glutamyl) lysine cross-linkages (around 30%). In order to compare the membranous fraction of horny and living cells (marginal bands and plasma membranes), the fraction was then isolated from living cells. The relative amino acid composition of the membranous fraction of the plasma membrane resembled that of human erythrocytes, but was quite different from that of the marginal band. These comparative studies of biochemical and morphological features suggested the importance of S-S cross-linking enzymes and transglutaminase in the transformation mechanism of the marginal band.
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PMID:Comparative studies of the marginal band and plasma membrane of the epidermis. 619 46

Placental villi of early and term human placentae were dissociated with trypsin and mixed cell cultures were established. The different cell types were identified and estimated over a 14-day culture period using antibodies to keratin and vimentin filaments and their capacity to phagocytose yeast. The three main cell types were found to be epithelial-, macrophage- and fibroblast-like cells. The epithelial-like cells can be further divided into the multinucleated and the small- and medium-sized round cells, and these are most likely to be derived from the trophoblast. The cellular composition of cultures were different for early and term placentae and also varied characteristically over the 14-day cultures period.
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PMID:Identification and estimation of cell types in mixed primary cell cultures of early and term human placenta. 620 10


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