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)

Arterial endothelial cells were obtained from bovine aortae by mild treatment with collagenase and medium perfusion. These cells were cultured in RPMI-1640 medium containing 15mM Hepes buffer and 35% fetal calf serum at pH 7.35. Essentially all (90-95%) the effluent cells were viable and 80% of these cells attached to the substratum within 1 hour. Small patches of attached cells coalesced to form confluent monolayers in 3-5 days. Confluent monolayers of endothelial cells consisted of a homogeneous population of tightly packed, polygonal cells. Selected cultures were serially subcultured (trypsin-EDTA) for 12-14 months (30-35 passages) without any apparent change in morphology or loss of growth characteristics. Primary and three-month old (15 passages) cultures had population doubling times of 32-34 hours and 29-31 hours, respectively. These cells (primary and subcultures) did not require a minimum cell number to become established in culture. Bovine endothelial cells (primary, first, fifth and thirteenth passages) were characterized ultrastructurally by the presence of Weibel-Palade bodies, pinocytotic vesicles and microfilaments and immunologically by the presence of thrombosthenin-like contractile proteins and Factor VIII antigen. The intercellular junctions of post-confluenct cultures stained specifically with silver nitrate. From these data, we concluded that identifiable endothelial cells could be obtained from bovine aortae and cultured and maintained for prolonged periods of time.
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PMID:Culture of arterial endothelial cells: characterization and growth of bovine aortic cells. 17 37

A decreased platelet adhesion to rabbit aorta subendothelium (Baumgartner technique) is confirmed in the Bernard Soulier (giant platelet) syndrome. Electron microscope techniques using a purified antibody against Factor VIII/von Willebrand protein, revealed an apparently normal presence of the Factor VIII/von Willebrand protein on the Bernard Soulier platelets. Electrophoretic characterization of the major protein and glycoprotein components of the Bernard Soulier platelets following sodium dodecyl sulfate solubilization indicated a relatively normal protein content but suggested a reduced content of the 155,000 molecular weight major platelet glycoprotein. This was confirmed by a reduced release of high molecular weight acidic glycopeptides following incubation of washed Bernard Soulier platelets with trypsin. It is proposed that this abnormality may be related to the previously reported reduced sialic acid content and the reduced electrophoretic mobility of the Bernard Soulier platelets and that a glycoprotein reduced or abnormal in the Bernard Soulier platelets is necessary for the normal adhesion of platelets to subendothelium.
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PMID:Bernard-Soulier syndrome: a new platelet glycoprotein abnormality. Its relationship with platelet adhesion to subendothelium and with the factor VIII von Willebrand protein. 108 83

A binding domain for Factor VIII (F.VIII) has been previously identified on the N-terminal portion of human von Willebrand Factor (vWF) subunit [amino acids (AA) 1-272]. In order to characterize other possible structures of vWF involved in its capacity to bind and to protect F.VIII against human activated protein C (APC), we used a series of purified vWF fragments overlapping the whole sequence of the subunit. Among those were fragments SpIII (dimer; AA 1-1365), SpII (dimer; AA 1366-2050) and SpI (monomer; AA 911-1365) generated by Staphylococcus aureus V8 proteinase, a P34 species (monomer; AA 1-272) obtained with plasmin, a monomeric 39/34 kDa dispase fragment (AA 480-718) and a tetrameric III-T2 fragment (AA 273-511/674-728) produced from SpIII by trypsin. Three other fragments without precise extremities were located using selected monoclonal antibodies to vWF. Two C-terminal fragments of 270 and 260 kDa, overlapping SpI and SpII, were respectively generated from vWF with trypsin and protease 1 from Crotalus atrox venom. An N-terminal 120 kDa fragment, overlapping P34 and 39/34 kDa fragments, was produced by protease 1. Our results show that vWF bound to F.VIII and protected it from degradation by APC in a dose-dependent way. Among the C-terminal and central vWF fragments (SpII, tryptic 270 kDa, 260 kDa, SpI, 39/34 kDa and III-T2), none had the capacity to bind or to protect F.VIII, even at high concentrations. The three N-terminal fragments (SpIII, 120 kDa and P34) bound to F.VIII in a dose-dependent and saturable fashion. SpIII and the 120 kDa fragment had the capacity to protect F.VIII in a dose-dependent way. In contrast, the P34 species did not significantly protect F.VIII, even when using high concentrations of the fragment. In conclusion, the N-terminal end of vWF subunit (AA 1-272) plays a crucial role in binding to F.VIII, but requires additional structures of the 120 kDa fragment to protect it against APC. In addition, the presence of a secondary binding and/or protecting domain on other portions of the vWF subunit (potentially destroyed during the proteolysis of vWF) is highly unlikely.
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PMID:Evidence that a secondary binding and protecting site for factor VIII on von Willebrand factor is highly unlikely. 153 49

A number of fixation and decalcification procedures were evaluated to determine their suitability for immunohistochemistry on trephine samples of bone marrow after paraffin embedding. In particular, the immunoreactivity of antigens characteristic for various hematopoietic cell lines (immunoglobulin heavy and light chains for plasmacytoid cells; elastase for neutrophil myeloid cells; lysozyme, alpha-1-antitrypsin and alpha-1-antichymotrypsin for hystiocytic cells; leukocyte common antigen for lymphocytes; hemoglobin and glycophorin A for erythroid cells; Factor VIII-related antigen for thrombocytoid cells) as well as some antigens specific for epithelial tumors (CEA, 115D8, and keratin) were investigated. Fixation in a mercuric chloride-formaldehyde mixture followed by decalcification in acetic acid-formaldehyde-saline proved to be the best procedure for antigen preservation and retention of morphologic detail. Moreover, there is no need of trypsinization when using this procedure. The only exception was Factor VIII-related antigen in megakaryocytes, which was best demonstrated in trypsin-digested sections of formalin-fixed and acetic acid-decalcified biopsies.
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PMID:Influence of fixation and decalcification on the immunohistochemical staining of cell-specific markers in paraffin-embedded human bone biopsies. 241 61

Recent studies indicate that intramammary lymphatic invasion represents an important prognostic parameter in breast carcinomas. However, the identification of intramammary lymphatic invasion in tissue sections is a subjective procedure, frequently hampered by factors such as fixation artefacts and interobserver variations. In this study, monoclonal antibodies to ABH isoantigens were applied on formalin-fixed, paraffin-embedded breast carcinoma tissue by using the avidin-biotin-peroxidase complex technique. In addition, the H antigen was localized using the Ulex europeus agglutinin I lectin binding technique. Isoantigen localization provided excellent delineation of lymphatics and blood vessels, in general unhampered by the retention of isoantigen expression in some breast carcinomas. In comparison, Factor VIII-related antigen localization required prior trypsin enhancement and was less sensitive and less consistent. The staining for isoantigens was more intense in vascular than in lymphatic endothelium. ABH isoantigen localization of lymphatic channels identified lymphatic tumor emboli peripheral to and within the carcinomas, and distinguished bona fide intramammary lymphatic invasion from tissue shrinkage artefacts. The applicability to routinely processed tissue permits retrospective studies and renders the identification of intramammary lymphatic invasion a more objective procedure. Further studies are needed to assess the role of this technique in evaluating the prognostic value of intramammary lymphatic invasion; the technique may be extended also to the study of other neoplasms.
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PMID:Intramammary lymphatic invasion in breast carcinomas. Evaluation using ABH isoantigens as endothelial markers. 242 60

Endothelium provides a specific binding site for Factor IX/IXa which can propagate activation of coagulation by promoting Factor IXa-VIII-mediated activation of Factor X. In this report the endothelial cell Factor IX/IXa binding site has been identified and the coagulant function of the receptor blocked. Studies using [3H]Factor IX derivatized with the photoaffinity labeling agent N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH) and cultured bovine endothelial cells demonstrated cross-linking to a trypsin-sensitive cell surface protein of Mr approximately equal to 140,000. Immunoprecipitation of metabolically labeled endothelium with Factor IX derivatized with the cleavable cross-linking agent N-succinimidyl(4-azidophenyl)-1,3'-dithiopropionate and antibody to Factor IX demonstrated the endothelial cell origin of the Mr 140,000 cell surface protein. Blockade of the Factor IX/IXa binding protein by covalently linking SANPAH-5-dimethylaminonaphthalene-1-sulfonyl-Glu-Gly-Arg-Factor IXa or SANPAH-Factor IX prevented both specific Factor IXa binding and effective Factor IXa-VIII-mediated activation of Factor X on endothelium. Following extraction of endothelium with detergents, Factor IX/IXa binding activity was solubilized and could be assayed using a polyvinyl chloride plate binding assay. Western blots of cell extracts demonstrated binding of 125I-Factor IX at Mr approximately equal to 140,000 which was blocked by excess Factor IX, but not antisera to Factor VIII, von Willebrand factor, alpha 2-macroglobulin, or epidermal growth factor receptor. These data indicate that endothelium provides a distinct binding site for Factor IX/IXa consisting, at least in part, of a membrane protein which can modulate the coagulant activity of Factor IXa on the cell surface.
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PMID:Identification of a factor IX/IXa binding protein on the endothelial cell surface. 303 54

Human fetal lung homogenates contain an inactive form of renin which may be revealed by trypsin treatment. When activated, this form of renin has some biochemical similarities with fetal kidney renin: the pH optimum of fetal lung renin is approximately 6.5; it is bound by Affigel Blue affinity chromatography resin; and is inhibited by a monoclonal antibody (R-3-36-16) raised to human kidney renin. Inactive renin, partially-purified from both fetal kidney and lung, differs from this in that the renal form is of low-molecular weight (LMW, 45,000 daltons), whereas that from fetal lung is of high molecular weight (HMW, 58,000 daltons). Using a sensitive alkaline phosphatase-anti-alkaline phosphatase (APAAP) procedure with a polyclonal anti-renin antibody (R-15), immunoreactive renin in fetal lung was found in vessels in mesenchyme between airways. The pattern of staining was distributed similarly to Factor VIII-related antigen, suggesting localization in endothelial cells.
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PMID:Renin in human fetal lung--a biochemical and immunohistochemical study. 305 97

Factor VIII activation by thrombin is the result of a proteolytic cleavage of the procoagulant component. These studies examine the effect of human antibody on this activation step in a solid phase immunoadsorbent assay system. Radiolabeled factor VIII antibody: factor VIII protein immune complexes were bound to agarose beads by mouse monoclonal antifactor VIII R:Ag antibody. The incubation of these bound labeled immune complexes with high ionic strength buffers (1 M NaCl, 0.24 M CaCl2), or with acidic buffers (0.01 M glycine-0.1 M NaCl, pH 3.0 or 3.5), or with trypsin (1, 5, and 20 mg per ml) dissociated 14 to 62 percent of the bound radiolabel. Thrombin at a concentration of 0.05 U per ml, however, only dissociated 2.9 percent of the label, an amount not significantly different than borate buffered saline control. It is concluded that inactivation of factor VIII is the result of human antibody inhibition of thrombin-induced proteolysis of factor VIII procoagulant protein.
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PMID:Mechanism of factor VIII inactivation by human antibodies. IV. Antibody binding prevents factor VIII proteolysis by thrombin. 309 25

Cultured microvascular endothelial cells (MEC) have become a valuable model for studies of microvascular physiology and pathology. Most current techniques involve manual removal of undesirable cell types or cloning, require one to several months, and yield high population doubling level cultures derived from a relatively small sample of the original population. We have devised a technique to more rapidly produce larger numbers of MEC. This method provided primary cultures consisting predominantly of MEC within 1 wk. The technique involves selective aspiration of gray matter from the bovine cerebral cortex followed by homogenization, sieving, enzymatic dissociation, and then dense plating (10(4) to 10(5) vessel fragments/cm2) onto gelatin- or fibronectin-coated plastic. Typical yields were 0.1 to 0.5 X 10(6) fragments/g of aspirated gray matter. The optimal culture medium for these cells was 15% equine plasma derived serum, 20% conditioned medium, 2% retinal extract, 60% fresh medium, and 500 micrograms/ml heparin. Cells attached within 24 h, well-spread colonies were present within 1 to 2 d, and cultures approached confluence within 2 to 3 d. Alkaline phosphatase staining confirmed the microvascular origin of the material plated. Morphology, Factor VIII-related antigen staining and 1,1'-dioctacecyl-3,3,3'3,-tetramethyl-indocarbocyanine perchlorate acetylated low density lipoprotein uptake suggested that MEC predominated. Cultures could be passaged and additionally purified by sequential exposure to pancreatin and trypsin-EDTA. Pancreatin selectively removed MEC colonies leaving a relatively homogeneous pericyte population. The relative ease with which such cultures can be produced should facilitate the in vitro study of brain microvascular function and may also provide insights useful for growing MEC from other vascular beds.
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PMID:Microvascular endothelium and pericytes: high yield, low passage cultures. 375 90

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


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