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)

Immunohistochemical techniques have been used to demonstrate Factor VIII related antigen (FVIIIRA) in endothelial cells and to study a variety of tumours including Kaposi's sarcoma. Conflicting reports on the presence of this antigen within the spindle cells of Kaposi's sarcoma have been made. These differences may be due to variations in techniques, in the immunoreagents used and in the method of fixation and handling of the specimen. A major factor may also be the lack of a uniform interpretation of positive peroxidase labelling. This study was confined to paraffin-embedded tissues and compared the results and distribution of FVIIIRA labelling in Kaposi's sarcoma using two commercially available polyclonal antibodies and three monoclonal antibodies. The effects of predigesting the sections with trypsin and protease were also evaluated. Positive labelling of tumour spindle cells was accepted only when it matched that seen in labelled endothelial cells. The polyclonal antibodies provided satisfactory localization of FVIIIRA and gave positive results more frequently than the monoclonal antibodies both with and without enzyme digestion. Endothelial labelling was evident in vessels both within and around the tumour but none was seen in the tumour spindle cells. Most previous studies gave no definition of positive labelling but spindle cells were reported as labelled. However, in those few reports where a definition was given the findings are analogous to those in this report, demonstrating the importance of defining positive labelling.
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PMID:An immunohistological study of factor VIII related antigen and Kaposi's sarcoma using polyclonal and monoclonal antibodies. 392 7

A short method is described for obtaining a large number of pure vascular smooth muscle cells in culture. The smooth muscle cells were isolated from human umbilical cord arteries digested twice by an enzyme mixture of collagenase, trypsin, elastase, and DNAase with addition of alpha-tosyl-lysyl chloromethane. Primary cell culture and first subculture were not contaminated by endothelial cells, no Factor VIII being produced. The cultures consisted of smooth muscle cells as appeared from phase contrast and electron microscopy.
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PMID:Isolation and culture of smooth muscle cells from human umbilical cord arteries. 408 21

The effects of collagenase on the immunohistochemical demonstrability of laminin, fibronectin and Factor VIII/RAg in human nervous tissue have been studied. The influence of this, and other proteolytic enzymes such as pepsin and trypsin, has been investigated in relation to different fixatives. Collagenase gave better results with Carnoy fixed material than after formalin fixation; unlike trypsin and pepsin, it did not produce tissue digestion.
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PMID:Collagenase in the immunohistochemical demonstration of laminin, fibronectin and factor VIII/RAg in nervous tissue after fixation. 632 75

We have identified two functional domains on the von Willebrand factor (VWF) moiety of the Factor VIII-von Willebrand factor complex (FVIII-VWF), one interacting with blood platelets, and one interacting with vessel wall collagens, by means of two monoclonal antibodies directed against the VWF molecule, CLB-RAg 35 and CLB-RAg 201. The monoclonal antibody CLB-RAg 35 inhibited virtually all platelet adherence to artery subendothelium and to purified vessel wall collagen type III, at relatively high wall shear rates. CLB-RAg 35 also inhibited the ristocetin-induced platelet aggregation and the binding of FVIII-VWF to the platelet in the presence of ristocetin but did not affect the binding of FVIII-VWF to collagen. The monoclonal antibody CLB-RAg 201 inhibited the binding of FVIII-VWF to purified vessel wall collagen type I and III and all platelet adherence to collagen type III and the platelet adherence to subendothelium that was mediated by FVIII-VWF in plasma. The two functional domains on FVIII-VWF that were recognized by CLB-RAg 35 and CLB-RAg 201 were identified by means of immunoprecipitation studies of trypsin-digested FVIII-VWF. The domains resided on different polypeptide fragments, with a Mr of 48,000 for the collagen binding domain and a Mr of 116,000 for the platelet binding domain. The 116,000-mol wt fragment consisted of subunits of 52,000/56,000 mol wt and 14,000 mol wt after reduction. The 52,000/56,000-mol wt subunits possessed the epitope for CLB-RAg 35.
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PMID:Functional domains on von Willebrand factor. Recognition of discrete tryptic fragments by monoclonal antibodies that inhibit interaction of von Willebrand factor with platelets and with collagen. 633 19

Cells derived from isolated glomerular tufts of rats were studied in primary tissue culture after the removal of epithelial cells by collagenase treatment. The cultured cells, fusiform or stellate in shape, grew readily over a 12-day period. Immunofluorescence staining was positive for myosin and fibronectin, while negative for Factor VIII, suggesting that the outgrowing cells were derived from the glomerular mesangium. In serum-free culture, these cells produced neutral proteinase activity that occurred as a latent trypsin-activable form (apparent molecular weight range, 78,000 to 100,000 daltons) and in an active form (44,000 to 58,000 daltons). Neutral proteinase activity was inhibited by EDTA and by cysteine, and exhibited a pH optimum of 7.2 to 7.8, characteristic of an extracellularly active metalloendopeptidase. The culture supernate which contained the neutral proteinase activity was capable of degrading purified rat glomerular basement membrane. The release of hydroxyproline-containing fragments from the basement membrane indicated that degradation of the type IV collagen component of the basement membrane was occurring. These findings suggest that the neutral proteinase activity generated by mesangium-derived cells may play a role in the physiologic turnover of glomerular structural proteins in vivo.
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PMID:Neutral proteinase activity produced in vitro by cells of the glomerular mesangium. 640 73

A method to isolate and maintain microvascular endothelial cells from the cutaneous vessels of adult human skin in long-term culture has been developed. Endothelial cells lining the microvessels of the papillary dermis are released from surrounding tissue during a brief trypsin incubation (0.3% trypsin, 1% EDTA). Cells are plated onto a fibronectin substrate and maintained in Leibovitz (L15) culture medium containing pooled human serum (50%) and antibiotics. Proliferation is dependent upon the presence of several additional growth factors, cholera enterotoxin (1 X 10(-9) M), isobutyl methylxanthine (3.3 X 10(-5) M), and medium conditioned by explant culture of the mouse EHS sarcoma. Using this supplemented medium, cells proliferate readily and can be cultivated serially for more than 6 passages (3 months in vitro). These cells retain their characteristic endothelial cell morphology, stain positively for Factor VIII antigen, and contain Weibel-Palade bodies.
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PMID:Isolation and long-term serial cultivation of endothelial cells from the microvessels of the adult human dermis. 642 Mar 32

We describe here a modified nonenzymatic method for the isolation of rat aortic endothelial cells with vasoformative properties. Aortic rings placed on plastic or gelatin-coated surfaces generated outgrowths primarily composed of endothelial cells. Prompt removal of aortic explants after endothelial migration minimized fibroblast contamination. However, fibroblasts, because of their high proliferative rate tended to overgrow the endothelial cells even when present in small numbers. This potential pitfall was avoided by weeding out fibroblasts with the rounded tip of a bent glass pipette. Primary endothelial colonies free of fibroblasts were segregated in cloning rings, trypsin-treated, and transferred to gelatin-coated dishes. Endothelial cells were cultured in MCDB 131 growth medium containing 10% fetal bovine serum, endothelial cell growth supplement, and heparin. Using this technique, pure endothelial cell strains were obtained from single aortic rings. Confluent endothelial cells formed a contact-inhibited monolayer with typical cobblestone pattern. The endothelial cells were positive for Factor VIII-related antigen, took up DiI-Ac-LDL, and bound the Griffonia Simplicifolia-isolectin-B4. Endothelial cells cultured on collagen gel formed a polarized monolayer, produced basement membrane, displayed Weibel-Palade bodies and caveolae, and were connected by tight junctions. In addition, they reorganized into a network of microvascular cords and tubes when overlaid with a second layer of collagen and formed microvascular sprouts in response to fibroblast-conditioned medium. This isolation procedure yields stable strains of vasoformative endothelial cells, which can be used to study aortic endothelium-related angiogenesis and its mechanisms.
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PMID:Isolation and characterization of vasoformative endothelial cells from the rat aorta. 752 1

Two recombinant expression libraries containing small (300-600 base pairs) cDNA fragments of von Willebrand Factor (vWF) were screened in order to map the epitope of monoclonal antibodies (MAbs) to vWF. Among eleven MAbs tested, seven were effectively mapped. The epitopes of MAbs 418 and 522, which inhibit the binding of vWF to Factor VIII (FVIII), were localized between Leu 2 and Arg 53 and between Glu 35 and Ile 81 of the vWF subunit respectively, within the N-terminal trypsin fragment called SpIII-T4 [amino acids (aa) 1-272] which contains a binding domain for FVIII. The epitope of MAb 710, which inhibits the binding of vWF to glycoprotein Ib (GPIb), was identified between Ser 593 and Ser 678 on the tryptic 52/48 kDa fragment (aa 449-728) which contains binding domains for GPIb, collagen, heparin, sulfatides and subendothelium extracellular matrices. The epitope of MAb 723, which does not interfere with any known function of vWF, was localized between Ser 523 and Gly 588. The epitopes of MAb 505 and MAb 400, which inhibit the binding of vWF to collagen, were identified between Leu 927 and Arg 1114 within the SPI fragment (aa 911-1365) corresponding to the central part of the vWF subunit. The epitope of MAb 9, which inhibits the binding of vWF to GPIIb/IIIa, was identified in the C-terminal part of the vWF subunit between Gln 1704 and Asp 1746, the latter being the third aa of the RGD sequence common to adhesive proteins and serving as a recognition site for integrin receptors.
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PMID:Epitope mapping of inhibitory monoclonal antibodies to human von Willebrand factor by using recombinant cDNA libraries. 797 49

Using a collagenase trypsin-EDTA treatment, we have been able to successfully isolate and grow primary cultures of the lymphatic endothelium (LEC) that were subcultured, frozen for storage, subsequently thawed with good recovery and growth, and serially subcultured. The morphological features of cultured LEC were consistent with that observed for the endothelium of intact lymphatic vessels. A prominent feature of growing cultures was the appearance of large vacuoles in the perinuclear region of the cytoplasm, which became filled with fluid and cell debris engulfed from the culture medium. The basal cell surface lacked a well defined basal lamina and anchoring filaments were observed extending from the basal plasmalemmal surface into the underlying substratum. LEC in cultures were also positive for Factor VIII-related antigen. However, specific granules, characteristic of Weibel-Palade bodies were not observed in ultrathin sections of confluent cultures. F-actin was identified in LEC cultures using fluorescein phalloidin, and in confluent cultures actin filaments were located at the periphery of the cell as a continuous circumferential thin band and short filamentous bundles in the central part of the cell. By using heparin and endothelial cell growth supplement in the culture medium we have been able to grow stable cultures of lymphatic endothelial cells that could be maintained when serially subcultured for over two years. These LEC cultures provide an in vitro model for investigating the function and biochemical properties of the lymphatic endothelium.
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PMID:Lymphatic endothelium isolation, characterization and long-term culture. 837 89

Ten mongrel dogs underwent carotid artery bypass 17 times with dacron grafts, 4mm in inner diameter and 5cm in length. The grafts in experimental group A were immediately seeded with autologous endothelial cells harvested by 0.25% trypsin solution (n = 7). The grafts in experimental group B were seeded with autologous endothelial cells cryopreserved in liquid nitrogen for 1 week (n = 3). The grafts in contrast group was not seeded with any endothelial cells implanted in the contralateral carotid arteries in group A (n = 7). The number of endothelial cells harvested in the two groups was 0.9 +/- 0.3 x 10. Factor VIII related antigen stain method confirmed the cells harvested by 0.25% trypsin solution to be endothelial cells. The grafts were removed and studied at the end of 2, 4, and 6 week after implantation. The total patency rate of group A was 85.7% (6/7), and that of the contrast group 57% (4/7). The weight of thrombus in group A was 40.6 +/- 36.9mg and the contrast group 85.9 +/- 26.3mg (P < 0.01). Scanning electron microscopy revealed that the cells at the middle segment of the grafts in the group A and B had the characteristics of ellipse, and tight intercellular junction, while the middle segment grafts in contrast group were covered by platelets, erythrocytes, leukocytes and fibrin. Transmission electron microscopy, factor VIII related antigen stain method, and vimentin stain method demonstrated that the cells in the group A and B showed characteristics of endothelial cells. It was concluded that endothelialization of the dacron grafts could be accelerated and the quality could be improved by immediate seeding with endothelial cells or by seeding with endothelial cells cryopreserved in liquid nitrogen for 1 week.
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PMID:[Experimental study of implantation of autologous vascular endothelial cells on dacron grafts]. 873


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