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)

Successful endothelialization of vascular grafts by seeding with endothelial cells (EC) at implantation is related to the number of EC which initially adhere to the graft. Using an in vitro system we examined the initial adherence of EC from human perinephric fat capillaries to woven Dacron that was either unmodified or precoated with several substrates. We studied capillary EC because they have not been investigated as a source of EC for graft seeding, although transinterstitial capillary ingrowth is one possible mechanism for spontaneous graft endothelialization. EC were isolated using collagenase and characterized morphologically and functionally including positive factor VIII-related antigen staining. EC were studied at three phases in culture: (A) primary EC with no subcultivations (EC-0); (B) EC after two subcultivations with trypsin (EC-2); and (C) EC after 10 subcultivations with trypsin (EC-10). EC were seeded onto graft material at a density of 10(5) cells/cm2 (100% confluence) and examined for cell counts and morphology after one day in culture by light and electron microscopy. Results are as follows: (table; see text) The conclusions are: (1) All capillary EC demonstrated adherence to Dacron, but this initial adherence was strongly influenced by graft pretreatment with collagen or plasma. (2) Serially subcultivated EC (EC-2 and EC-10) had significantly higher initial adherence to pretreated Dacron compared to the primary cells (EC-0) (P less than .05). This suggests that briefly cultured and subcultivated EC have superior initial adherence characteristics to treated dacron compared to primary EC with no subcultivations. (3) Fat capillary EC are easily procured and cultured and provide a rich source of human EC for endothelializing vascular prostheses.
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PMID:Initial adherence of human capillary endothelial cells to Dacron. 295 Feb 77

F344 male rats were given 90 ppm diethylnitrosamine in their drinking water ad libitum in two cycles. Livers containing neoplastic nodules, hepatomas, and no sarcomas in the sections sampled were digested in parallel with 0.05% collagenase, 0.1% Pronase, or 0.25% trypsin. Cells were transplanted into 9- to 19-day-old F344 rats. Despite the absence of sarcomas in the sections examined microscopically from each such liver before digestion and the presence of multiple hepatomas in all sections examined, vascular sarcomas, probably angiosarcomas, were observed in a large proportion of animals injected with the suspensions of cells; hepatomas were not observed in these animals. Morphology by light microscopy, immunohistochemical demonstration of factor VIII, histochemical demonstration of alkaline phosphatase, and the presence of Weibel-Palade bodies strongly suggest that these tumors are angiosarcomas. Similar tumors developed from cells obtained in parallel with the aid of Pronase, collagenase, or trypsin. Cell suspensions obtained with Pronase yielded tumors with the shortest latent period between the injection of cells and the death of one-half of the transplant recipients. The procedure that we used provides a consistent method for the production of transplantable sarcomas. The absence of sarcomas in the single sections taken from donor livers and multiple sections of similar livers not used for transplantation suggests that transplantability of these sarcoma cells is acquired very early in this neoplasm.
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PMID:Vascular sarcomas (probably angiosarcomas) transplanted from suspensions of liver cells from diethylnitrosamine-treated rats. 299 44

A large-scale method for the isolation of von Willebrand factor (vWF) from human factor VIII concentrates was developed in order to study the structure of this protein and its platelet binding activity. vWF is composed of a number of glycoprotein subunits that are linked together by disulfide bonds to form a series of multimers. These multimers appear to contain an even number of subunits of 270K. Two minor components of Mr 140K and 120K were also identified, but these chains appear to result from minor proteolysis. The smallest multimer of vWF contained nearly equimolar amounts of the 270K, 140K, and 120K subunits, while the largest multimers contained less than 20% of the two minor components. Amino acid sequence analysis, amino acid composition, and cleavage by cyanogen bromide indicate that the 270K subunits are identical and each is a single polypeptide chain with an amino-terminal sequence of Ser-Leu-Ser-Cys-Arg-Pro-Pro-Met-Val-Lys and a carboxyl-terminal sequence of Glu-Cys-Lys-Cys-Ser-Pro-Arg-Lys-Cys-Ser-Lys. Platelet binding in the presence of ristocetin was 8-fold greater with multimers larger than five (i.e., containing more than 10 subunits of 270K) as compared to multimers less than three (containing less than six subunits of 270K). However, partially reduced vWF (Mr 500K), regardless of whether it was prepared from large or small molecular weight multimers, gave platelet binding similar to that of the smallest multimers. Likewise, partial proteolysis by elastase, thermolysin, trypsin, or chymotrypsin produced small "multimer-like" proteins with platelet binding properties similar to either partially reduced vWF or to the smallest multimers. We conclude that human vWF contains identical 270K subunits assembled into a multivalent structure. Disassembly by either partial reduction or partial proteolysis produces essentially monovalent protein with platelet binding properties similar to that of the smallest multimers. Multivalency is likely the primary factor responsible for the increase in biological activity with multimer size.
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PMID:Human von Willebrand factor: a multivalent protein composed of identical subunits. 301 99

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

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

Human coagulation factor VIII:C has been purified approximately 5000-fold from commercial preparations with an average activity yield of 35%. Proteins of 92 kD and 77-80 kD enriched during purification are precipitated by a human serum polyclonal antibody which inhibits factor VIII:C activity. Evidence suggests that these polypeptides are linked by a calcium ion bridge. Partial amino acid sequence information from these proteins has been obtained from the intact polypeptides and from products of digestion with thrombin, endoproteinase lysC, or trypsin after citraconylation. An oligonucleotide probe designed from one of the amino acid sequences was used to isolate a partial genomic clone from a human 4X chromosome library in bacteriophage lambda. The genomic segment was used to isolate two cDNA molecules encompassing the entire human kidney factor VIII:C mRNA. Biologically active factor VIII:C has been produced in a mammalian cell line utilizing a complete cDNA construction.
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PMID:Characterization of the polypeptide composition of human factor VIII:C and the nucleotide sequence and expression of the human kidney cDNA. 393

The low level of enzymatic activity of certain alpha 2-macroglobulin-proteinase complexes could be important to the function of factor VIII/von Willebrand glycoprotein since it is especially sensitive to proteolytic cleavage. To test this possibility, complexes of alpha 2-macroglobulin with plasmin, trypsin, and thrombin were formed in at least a 2:1 molar ratio of alpha 2-macroglobulin:proteinase and tested for effects on the factor VIII procoagulant activity of the factor VIII/von Willebrand glycoprotein. Neither the alpha 2-macroglobulin-trypsin complex nor the alpha 2-macroglobulin-plasmin complex affected factor VIII procoagulant activity. The behavior of the alpha 2-macroglobulin-thrombin complex was different. When alpha 2-macroglobulin and thrombin were incubated in a mole ratio of 3:1 or less, factor VIII procoagulant activity was enhanced to about the same extent as with free thrombin. Even at a 24:1 mole ratio, the mixture could produce 45% of the increase in factor VIII activity obtained with free thrombin. The isolated alpha 2-macroglobulin-thrombin complex could also activate the factor VIII procoagulant function to about 45% of the level obtained with an identical amount of uncomplexed thrombin. Analysis of the alpha 2-macroglobulin-125I-labeled thrombin complexes by rechromatography or by polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated that this activation was not due to free thrombin. We conclude that the alpha 2-macroglobulin-thrombin complex retains sufficient proteolytic activity to activate the procoagulant function of factor VIII/von Willebrand glycoprotein despite the latter being a very large substrate, having an estimated molecular weight of 1-20 million.
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PMID:Proteolytic activity of alpha 2-macroglobulin-enzyme complexes toward human factor VIII/von Willebrand factor. 618 90

Primary cultures of confluent human endothelial cells (ECM) were grown in media containing the major lipoproteins (LP) and lipoprotein deficient serum (LDS). The release of 6-keto-PGF1 alpha, von Willebrand factor (VIII RAg) and apolipoproteins (apo) A-I and A-II were investigated by radioimmunoassay. The cell-associated VIII RAg, apo A-I and apo A-II were also confirmed by fluorescein antibodies, and the synthesis of the apolipoproteins was examined by incorporation of [3H]leucine. Apo A-I and apo A-II were located and synthesized in ECM, yet only apo A-I was released into the medium. Very low density (VLDL) and low density lipoproteins (LDL) in concentrations of 50-600 micrograms/ml stimulated release of apo A-I. Stimulation of ECM for 5 min with thrombin (T) or arachidonic acid (A) did not induce apo A-I release. VIII RAg was always released into the media from ECM. The release was not affected by the lipoproteins. VIII RAg was also localized on the cell surface (VIII RAgC) and approximately 80% was released by trypsin. LDL stimulated the occurrence of factor VIII RAg on the cell surface. 6-Keto PGF1 alpha was always released into the medium and the production was stimulated by T and AA. The main lipoproteins (50-600 micrograms/ml) and apo A-I and A-II did not affect the release of 6-keto-PGF1 alpha. This study shows that endothelial cells synthesize and release proteins important for thrombogenesis and atherosclerosis. The release of apolipoproteins A-I was stimulated by VLDL and LDL, and the concentration of cell-related factor VIII RAg was stimulated by LDL.
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PMID:The effect of lipoproteins on the synthesis of prostacyclin, von Willebrand factor and apolipoproteins A-I and A-II in cultured human endothelial cells. 642 92

Thirteen adult dogs underwent thoracoabdominal bypass operations with 6-mm, double-velour Dacron grafts 25 to 30 cm long. Experimental grafts were seeded with cultured autologous endothelial cells (n = 7). Unseeded grafts served as controls (n = 6). Endothelial cells were harvested from external jugular vein segments using 0.1% trypsin and 0.5% collagenase solutions. Grafts were studied at weeks 2 and 4. Endothelial cell coverage of experimental graft surfaces after two weeks was 60% to 70%, and approximately 80% after four weeks. Immunofluorescence using factor VIII-related antigen confirmed the graft's inner surface to be endothelium. Endothelial cell coverage in control grafts occurred as pannus ingrowth, and never exceeded more than 10% of the conduit surface. Generation of an early endothelial surface in prosthetic grafts is possible in a canine model using cultured autologous cells.
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PMID:Endothelial cell seeding of prosthetic vascular grafts: early experimental studies with cultured autologous canine endothelium. 644 93

The uptake and release of factor VIII/von Willebrand's protein by cultured human umbilical vein endothelial cells have been examined using highly purified 125I-factor VIII possessing von Willebrand's factor activity. 125I-factor VIII/vWF was taken up by the cells, reaching maximum binding within 4 h with a t1-2 of binding of 15 min. Endothelial cell binding of 125I-factor VIII/vWF reached saturation at a concentration of 1.5 mg/l. Binding was inhibited by coincubation of excess unlabelled factor VIII/vWF. Most of the cell-associated radioactivity was released by treatment of the cells with trypsin. Internalization of bound protein was evidenced by the incorporation into the cells of radioactivity which could not be released by trypsin. Human vascular smooth muscle cells did not bind 125I-factor VIII/vWF. Addition of 0.1 microM epinephrine to the 125I-factor VIII/vWF labelled endothelial cultures induced the release of cell bound, protein-associated radioactivity into the medium. Propranolol inhibited completely epinephrine-induced release, whereas phenylephrine had no effect. Endothelial cells maintained in medium partially depleted of factor VIII/vWF by tricalcium citrate cellulose treatment of plasma did not release factor VIII antigen into the culture medium during subsequent incubation. Although [3H]proline was incorporated into proteins released by endothelial cells under these experimental conditions, specific incorporation of label into factor VIII/vWF antigen was not detectable by a sensitive solid-phase immunoradiometric assay. We conclude that factor VIII/vWF binds to endothelial cells and that this cell-bound protein is mobilized by epinephrine through beta-adrenergic stimulation.
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PMID:Binding and release of factor VIII/von Willebrand's factor by human endothelial cells. 677 81


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