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)

A simple assay measuring degradation of human epidermal keratin and bovine tendon collagen is presented. Insoluble protein substrate (30 mg) was incubated with 1 ml buffer and enzyme sample for 1 h at 37 degrees C, following addition of 1 ml distilled water and removal of the remaining substrate by filtration/centrifugation. The protein content was determined in the filtrate/supernatant by the Lowry method. Keratin was prepared as follows: Freeze-drying, homogenization in a mortar or beetling mill, extraction in 0.9% (w/v) NaCl followed by water and 100% ethanol, drying at 37 degrees C. The assay was tested with pig pepsin, bovine trypsin, and crude extract of fish stomach, demonstrating that these preparations are effective in degrading human epidermal keratin.
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PMID:A simple assay for determining keratin and collagen degradation in vitro. 245 79

Dermal keratin bodies, consisting mainly of keratin intermediate filament aggregates (KIFA) coated with IgM anti-KIF autoantibodies, are present in normal human skin and occur in increased quantities in certain skin diseases. Keratin bodies are normally rapidly removed, but in primary localized cutaneous amyloidosis (PLCA) they are converted by an unknown mechanism to amyloid. Amyloid P component (AP), a glycoprotein identical to, and derived from, the normal plasma protein serum amyloid P component (SAP), is present in all forms of amyloid including PLCA. We investigated the interaction between SAP, keratin bodies, and KIFA. Immunofluorescence staining of normal skin using fluoresceinated anti-SAP and rhodamine-conjugated anti-IgM, or AE-1/AE-3 anti-keratin antibodies followed by Texas Red-conjugated anti-mouse immunoglobulin, showed that 52% +/- 4 (mean +/- sem, n = 6) of keratin bodies bound anti-SAP. Similar findings were present in a biopsy from a patient with lichen planus. Isolated KIFA, prepared by 8M urea extraction of normal human epidermis or cultured keratinocytes, were preincubated with normal human serum as a source of SAP and then stained with fluoresceinated anti-SAP. Bright fluorescence seen when the incubation medium contained Ca++ was absent in the presence of ethylenediamine tetraacetic acid. Specific Ca++-dependent binding of SAP to KIFA was confirmed using immunoblotting. Binding of SAP to KIFA did not prevent their degradation following exposure to trypsin or alpha-chymotrypsin. Similarly, partial enzymatic digestion of KIFA did not abrogate their ability to bind SAP. Our findings, that SAP is associated with keratin bodies in skin and exhibits Ca++-dependent binding to KIFA in vitro, identify keratin filaments as a newly recognized ligand for SAP.
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PMID:Amyloid P component binds to keratin bodies in human skin and to isolated keratin filament aggregates in vitro. 245 1

Stomach extract of Atlantic herring Clupea harengus, Atlantic salmon Salmo salar, cod Gadus morhua, redfish Sebastes marinus, and plaice Pleuronectes platessa, degraded human epidermal keratin effectively in vitro. The keratin-degrading activity of all extracts showed a pH optimum around 3.3-3.4, and sheets of plantar callus were degraded with about the same efficacy as keratin. Pepstatin sensitivity, heat lability, and the acidic pH optimum demonstrated that the keratin-degrading activity was pepsin. The keratin-degrading activity of cod stomach extract had a temperature optimum of around 42 degrees C at optimal pH, and showed a similar pH dependency with collagen as with keratin as substrate. The keratin-degrading activity of pepsin I and pepsin II purified from cod showed a pH optimum of 3.7 and 3.1, respectively, similar to that obtained with hemoglobin as substrate. Pig pepsin showed a pH optimum of about 2 with keratin, hemoglobin, and collagen as substrates. The present investigation demonstrates that fish pepsin is effective in degrading human epidermal keratin in vitro, and in a contemporary study the same was shown with fish trypsin. This may suggest a possible mechanism for the development of irritative hand eczema caused by exposure to fish and acidified fish material.
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PMID:Degradation of human epidermal keratin by fish pepsin. 245 43

Immunoperoxidase stains were performed on normal and neoplastic tissue from prostate, colon, thyroid, lung, nerve, uterus, and placenta embedded in both plastic (glycolmethacrylate [GMA]) and paraffin. Positive results in plastic section were obtained for carcinoembryonic antigen (CEA), keratin, epithelial membrane antigen (EMA), thyroglobulins, S-100, prostate-specific antigen, human chorionic gonadotrophin (HCG), and beta-HCG. More delicate staining with more precise localization of antigens is noted. Superior (paraformaldehyde) fixation and cold processing followed by GMA polymerization (4 degrees C) allow for optimum antigen survival. After fixation, tissue processing involves a series of 0.1 mol/L phosphate buffer rinses with sucrose and ammonium chloride in a conventional dip-and-dunk processor placed in a 4 degrees C cold room. Acetone dehydrations are used before GMA infiltration, cold polymerization, and sectioning. Before immunoperoxidase staining, the plastic section is digested in .25% bovine trypsin for ten minutes. The immunoperoxidase methods described can be useful when small biopsies are routinely embedded in plastic to obtain improved histologic (hematoxylin-eosin) sections. There may also be research applications in quantifying antigen expression in benign, dysplastic, and neoplastic tissues by examining the stains under high power.
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PMID:Antigen localization in immunoperoxidase-stained plastic-embedded soft tissues. 245 78

The internal epithelium of mouse forestomach represents a fully keratinized tissue that has many morphological aspects in common with the integumental epidermis. In the present study we have, therefore, analyzed keratin expression in the total epithelium, in subfractions of basal cells and in living and dead suprabasal cells that were obtained by Percoll density gradient centrifugation of trypsin-dissociated forestomach keratinocytes. The keratin analysis revealed that basal forestomach keratinocytes synthesize the same keratin types as basal epidermal cells (60000, 52,000 and 47,000 daltons), whereas differentiating cells contain both the epidermal suprabasal keratin pair (67,000 and 59,000 daltons) and the suprabasal keratin pair characteristic for other internal squamous epithelia (57,000 and 47,000 daltons). Indirect immunofluorescence using an antibody recognizing the members of the epidermal-type suprabasal keratin pair and in-situ-hybridization experiments using specific cDNA probes for the members of the internal-type keratin pair showed that the two keratin pairs are uniformly coexpressed in living suprabasal forestomach keratinocytes. Furthermore, it could be shown that distinct cells in the basal cell layer acquire the ability to express both the 67,000/59,000 dalton and the 57,000/47,000 dalton keratin pair and that some basal cells apparently lose the ability to synthesize mRNAs for basal keratins.
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PMID:The intermediate filament system of the keratinizing mouse forestomach epithelium: coexpression of keratins of internal squamous epithelia and of epidermal keratins in differentiating cells. 245 87

Cod Gadus morhua and bovine trypsin degraded human epidermal keratin with similar efficacies in vitro around optimal pH, which was at pH 8.4 for cod trypsin and at pH 9.5 for bovine trypsin. Extract of intestines of cod, Atlantic herring Clupea harengus, Atlantic salmon Salmo salar, and redfish Sebastes marinus degraded keratin with similar efficacies with pH optima between 8.5 and 9.5. Sheets of plantar callus were degraded with somewhat lower efficacy than keratin. The keratin-degrading activity of extract of cod intestines had a temperature optimum around 45 degrees C. Inhibition with benzamidine and 4-phenylbutylamine showed that trypsin amounted to more than 2/3 of the keratin-degrading activity in all extracts of fish intestines. Apart from cod intestines, which had the lowest chymotrypsin content, chymotrypsin made a smaller but significant contribution to the keratin-degrading activity. The present investigation demonstrates that fish trypsin and extract of fish intestines are effective in degrading human epidermal keratin in vitro, and in a recent investigation the same was shown with fish pepsin. This may suggest a possible mechanism for the development of irritative contact eczema caused by exposure to fish.
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PMID:Degradation of human epidermal keratin by cod trypsin and extracts of fish intestines. 246 41

A culture of cells derived from the endometrium was established to study in vitro the effects of hormone on the endometrium. Endometrial tissue was cut into small pieces and suspended in trypsin and collagenase-containing medium. The primary cell culture was obtained by six passages. The following observations appeared to indicate that the cultured cells consisted of endometrial epithelium-derived adenocytes, endometrial stromal cells and fibroblasts: 1. The epithelium-derived adenocytes and the endometrial stromal cells were positive and the fibroblasts were negative for staining with keratin by the enzyme-labelled antibody method. 2. Transmission electron microscopic observation revealed microvilli on the surface of the adenocytes, abundant free ribosomes in the cytoplasm, and nucleoli in distinct nuclei whose margin was deeply stained. Junction complexes among the cells were also observed. There were no microvilli on the surface of the endometrial stromal cell. Filaments, mitochondria and secreting granules were distinctly seen in the cytoplasm. The adenocytes on the margin of the nucleus itself were more deeply stained, and the margin was notched. The fibroblasts were spindle-shaped, and there no basement membrane structure laterally along the surface of the fibroblast. There were no intercellular junction complexes but there were filaments in the intercellular borders and in the cytoplasm. Chromatin in the large oval nuclei was dispersed.
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PMID:[Establishment of primary culture of cells derived from human uterine endometrium]. 246 25

In order to gain insight into the metabolism of keratin intermediate filaments (KIF) as well as the ability of KIF degradation products to interact with the immune system, we performed enzymatic degradation of purified KIF and examined their interaction with anti-KIF autoantibodies and their ability to act as immunogens. Aliquots of KIF aggregates were exposed to 3 different enzymes, that is, alpha-chymotrypsin, plasmin, and trypsin, in dose- and time-dependent experiments. The effect of the digestion was monitored sequentially by polyacrylamide gel electrophoresis and simultaneously by transmission electron microscopy. Furthermore, the KIF degradation proteins were then examined for their ability to bind anti-KIF autoantibodies by immunoblot and for their immunogenicity. In addition, preincubation of KIF with anti-KIF autoantibodies prior to the digestion procedure was performed to investigate a possible protective effect of this treatment against proteolytic degradation. The experiments demonstrated that: (1) KIF are degraded by serine proteinases, (2) with prolonged incubation time intact KIF are progressively replaced by more granular-amorphous material in transmission electron microscopy, (3) anti-KIF autoantibodies bind to KIF degradation proteins, (4) preincubation of KIF with anti-KIF autoantibodies does not exert any major protective effect for KIF against proteolytic degradation, and (5) the enzymatic degradation products of KIF can function as effective immunogens causing the formation of high-titer anti-KIF antibodies.
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PMID:Immunologic properties of enzymatically degraded human keratin intermediate filaments. 257 28

The effect of preliminary trypsinization on the immunoreactivity of keratin proteins in formalin-fixed, paraffin-embedded tissues of a variety of tumors (squamous cell carcinomas, adenocarcinomas, mesotheliomas, and transitional cell carcinomas) was evaluated. Three types of trypsin (Type II and Type IX porcine trypsin and Type III bovine trypsin) and varying concentrations of trypsin were assessed. Immunoreactivity of keratin proteins was determined using rabbit anti-keratin antibodies and monoclonal antibodies (combination of AE1 and AE3) and immunoperoxidase techniques. Preliminary trypsinization was mandatory for optimal immunoreactivity of keratin proteins using either polyclonal or monoclonal antibodies. Excellent results were obtained using Type II porcine trypsin at concentrations of 25 mg/dl for 30-45 min or 50 mg/dl for 20 min, at 37 degrees C. Trypsin treatment with excessive concentrations of enzyme and/or extended incubation times promoted tissue digestion and in some cases, yielded decreased immunoreactivity and altered staining patterns.
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PMID:Optimal immunoreactivity of keratin proteins in formalin-fixed, paraffin-embedded tissue requires preliminary trypsinization. An immunoperoxidase study of various tumours using polyclonal and monoclonal antibodies. 258 Aug 83

The development of the pleural inflammatory response to asbestos remains poorly defined. Importantly, the role of the pleural mesothelial cell in recruitment of neutrophils to the pleural space is not known. We hypothesized that rabbit pleural mesothelial cells stimulated by asbestos fibers release chemotactic factor(s) for neutrophils. Primary cultures of rabbit pleural mesothelial cells were established, and their purity verified by the presence of keratin and hyaluronic acid mucin. Mesothelial cells in serum-free media, in the presence of 30 micrograms/ml of crocidolite asbestos, released chemotaxins for neutrophils. This activity was not dependent on the type of asbestos fiber or fiber length. It was dose-dependent until 30 micrograms/ml of asbestos. The chemotactic fractions had the ability to increase both directed and random migration of neutrophils. The chemotactic activity was not present in sonicated fractions of unstimulated mesothelial cells, nor in supernates of asbestos fibers alone. Characterization of the chemotactic activity showed that it was heat stable (56 degrees C per 30 min) and sensitive to digestion with trypsin and papain. On Sephadex G-50 chromatography, it had a molecular weight between 6,000 and 9,000. Production of the chemotactic activity was inhibitable by cycloheximide. These results demonstrate that pleural mesothelial cells can actively synthesize a protein fraction with chemotactic activity for neutrophils. Production of this mesothelial cell-derived chemotactic activity for neutrophils may play an important role in the initiation of the inflammatory response of the pleura to asbestos.
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PMID:Pleural mesothelial cells stimulated by asbestos release chemotactic activity for neutrophils in vitro. 264 74


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