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)

Physiologic and pathologic events associated with cutaneous differentiation and repair are the result of a concerted action of various types of resident tissue cells. In vitro models simulating this complex in vivo situation are therefore needed to clarify the specific contribution and relevant interaction of, for example, dermal mast cells with other major cutaneous cells. The aim of this study was to establish a long-term coculture model that includes dermal mast cells, dermal fibroblasts, and keratinocytes in a human skin equivalent organotypic setting. Normal dermal mast cells and fibroblasts (1:4) were enclosed in collagen gel and normal keratinocytes were grown on top with exposure to the air interface. Under these conditions, mast cell integrity and functionality was preserved even after 4 wk of culture, as shown by electron microscopy and immunohistochemistry using antibodies against the mast-cell-specific granule enzyme tryptase and the receptors for stem cell factor and IgE. Mast cells also released histamine on stimulation with anti-IgE, and on ultrastructure were found to degranulate, with decrease of granule matrix density and formation of cell-cell contacts with fibroblasts. After 2 wk of culture, keratinocytes had formed an epidermis-like multilayer and were able to proliferate and differentiate, as shown by bromodeoxyuridine incorporation of basal cells and immunohistochemical staining for transglutaminase and cytokeratins 1 and 10. The model presented here thus provides a potentially relevant tool to further clarify the interaction of dermal mast cells with major other skin cells and their contribution to cutaneous physiology, repair processes, and pathology.
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PMID:A long-term coculture model for the study of mast cell-keratinocyte interactions. 1219 Aug 64

Bovine pancreatic trypsin was modified by the mono-6-amino-6-deoxy derivatives of alpha-, beta-, and gamma-cyclodextrin through a transglutaminase-catalyzed reaction. The trypsin-cyclodextrin conjugates, containing about 3 mol of oligosaccharide per mole of protein, were tested for their catalytic and stability properties. The specific esterolytic activity and the kinetics constants of trypsin were significantly improved following the transglutaminase-induced structural modifications. Trypsin-cyclodextrin conjugates were also found markedly (sixfold) more resistant to autolytic degradation at alkaline pH, and their thermal stability profile was improved by about 16 degrees C. Moreover, they were particularly resistant to heat inactivation when treated at different temperatures ranging from 45 degrees C to 70 degrees C for different periods of time.
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PMID:Transglutaminase-catalyzed synthesis of trypsin-cyclodextrin conjugates: kinetics and stability properties. 1252 88

Streptoverticillum sp. transglutaminase was used as catalyst for the attachment of several beta-cyclodextrin derivatives to the glutamine residues in bovine pancreatic trypsin. The modifying agents used were mono-6-ethylenediamino-6-deoxy-beta-cyclodextrin, mono-6-propylenediamino-6-deoxy-beta-cyclodextrin, mono-6-butylenediamino-6-deoxy-beta-cyclodextrin and mono-6-hexylenediamino-6-deoxy-beta-cyclodextrin. The transformed trypsin preparations contained about 3 mol of oligosaccharides/mol of protein. The specific esterolytic activity of trypsin was increased by about 4-21% after conjugation. The K (m) values for cyclodextrin-trypsin complexes represented about 58-87% of that corresponding to the native enzyme. The optimum temperature for esterolytic activity of trypsin was increased by about 5-10 degrees C after enzymic modification with the cyclodextrin derivatives. The thermostability was increased by 16 degrees C for the modified trypsin. Thermal inactivation at different temperatures ranging from 45 to 60 degrees C was markedly increased for the oligosaccharide-trypsin complexes. This modification also protected the enzyme against autolysis at alkaline pH.
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PMID:Thermal stabilization of trypsin by enzymic modification with beta-cyclodextrin derivatives. 1259 75

A peptidomics approach was developed to identify transglutaminase-susceptible Q residues within a pepsin-trypsin gliadin digest. Based on tagging with a monodansylcadaverine fluorescent probe, six alpha/beta-, gamma-gliadin, and low molecular weight glutenin peptides were identified by nanospray tandem mass spectrometry. In functioning as an acyl acceptor, tissue transglutaminase was able to form complexes with the glutamine-rich gliadin peptides, whereas by lowering pH, the peptides were deamidated by transglutaminase at the same Q residues, which were previously transamidated. The main common feature shared by the peptides was the consensus sequence Q-X-P. Our findings offer relevant information for the understanding of how dietary peptides interact with the host organism in celiac disease.
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PMID:Susceptibility to transglutaminase of gliadin peptides predicted by a mass spectrometry-based assay. 1504 21

Staphylococcal fibronectin-binding protein (FnbA) is a surface-associated receptor responsible for the reversible binding of bacteria to human fibronectin and fibrin(ogen). Recently we have shown that FnbA serves as a substrate for coagulation factor XIIIa and undergoes covalent cross-linking to its ligands, resulting in the formation of heteropolymers (Matsuka, Y. V., Anderson, E. T., Milner-Fish, T., Ooi, P., and Baker, S. (2003) Staphylococcus aureus fibronectin-binding protein serves as a substrate for coagulation factor XIIIa: Evidence for factor XIIIa-catalyzed covalent cross-linking to fibronectin and fibrin, Biochemistry 42, 14643-14652). Factor XIIIa also catalyzes the incorporation in FnbA of fluorescent probes dansylcadaverine and glutamine-containing synthetic peptide patterned on the NH(2)-terminal segment of fibronectin. In this study, the above probes were utilized for site-specific labeling and identification of reactive Gln and Lys residues targeted by factor XIIIa in rFnbA. Probe-decorated rFnbA samples were subjected to trypsin or Glu-C digestion, followed by separation of labeled peptides using reversed phase HPLC. Sequencing and mass spectral analyses of isolated probe-modified peptides have been employed for the identification of factor XIIIa-reactive Gln and Lys residues. Analysis of dansylcadaverine-labeled peptides resulted in the identification of one major, Gln103, and three minor, Gln105, Gln783, and Gln830, amine acceptor sites. The labeling procedure with dansyl-PGGQQIV probe revealed that Lys157, Lys503, Lys620, and Lys762 serve as amine donor sites. The identified reactive glutamine acceptor and lysine donor sites of FnbA may participate in transglutaminase-mediated cross-linking reactions resulting in the covalent attachment of pathogenic Staphylococcus aureus to human host proteins.
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PMID:Identification of factor XIIIA-reactive glutamine acceptor and lysine donor sites within fibronectin-binding protein (FnbA) from Staphylococcus aureus. 1536 70

Among the major proteins in the rat seminal vesicle secretion, transglutaminase catalyzed the cross-links among RSVS I-III. Six peptide sequences determined from the trypsin digests of RSVS III were confirmed in the protein sequence derived from two paralogs, RSVS III(alpha) and RSVS III(beta) gene, in rat 3q42. Their transcription units are organized with the first exon encoding a signal peptide, and the second a secreted protein, whereas the third encompasses a 3'-non-translated nucleotide that shares common features of rapidly evolving substrates of transglutaminase (REST) gene family. These two genes have 99% identity in their coding region and both express in adult rats with the same transglutaminase cross-linking site, manifesting functional preservation. All of REST genes reported thus far for human and Muridae were mapped in a chromosomal locus between KCNS1 and SLPI suggesting the locus as an active evolving region. The molecular evolution of this gene family is discussed.
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PMID:Functional preservation of duplicated pair for RSVS III gene in the REST locus of rat 3q42. 1558 86

An enzymatic approach, based on a transglutaminase-catalyzed coupling reaction, was investigated to modify bovine liver catalase with an end-group aminated dextran derivative. We demonstrated that catalase activity increased after enzymatic glycosidation and that the conjugate was 3.8-fold more stable to thermal inactivation at 55 degrees C and 2-fold more resistant to proteolytic degradation by trypsin. Moreover, the transglutaminase-mediated modification also improved the pharmacokinetics behavior of catalase, increasing 2.5-fold its plasma half-life time and reducing 3-fold the total clearance after its i.v. administration in rats.
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PMID:Transglutaminase-catalyzed site-specific glycosidation of catalase with aminated dextran. 1644 4

In the present study, we have utilized the transglutaminase (TGase) enzyme to modify the primary structure of VIP with diaminopropane (DAP) at the level of the Gln16. We have investigated the conformational stability of VIP and VIP-DAP in solution by limited proteolysis experiments. The VIP-DAP appears to be more resistant to the proteolytic attack of trypsin, thus indicating that the derivatization in position 16 is able to stabilize the structure of the peptide. However, we have studied their role in cell cycle modulation and antioxidant activity in the oropharyngeal epidermoid carcinoma KB cells.
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PMID:Effects of VIP and VIP-DAP on proliferation and lipid peroxidation metabolism in human KB cells. 1688 60

Microbial transglutaminase (mTG) mediated modification of bovine beta-lactoglobulin (bLG) at ambient and high hydrostatic pressure was investigated in order to characterize preferred sites of the crosslinking reaction by identifying reactive glutamine residues. bLG was labeled with triglycine (GGG) by incubation with mTG at ambient pressure or at 400 MPa, respectively, and was subjected to an enzymatic digestion with trypsin. The resulting peptides were separated and those containing glutamine residues modified with GGG were unambiguously identified using RP-HPLC with ESI-TOF-MS. For bLG treated with mTG at ambient pressure for 1 h at 40 degrees C, no labeling was observed, thus confirming that the native protein is no substrate for mTG. After incubation of the protein with mTG at 400 MPa for 1 h at 40 degrees C, four out of nine glutamine residues, namely at positions 5, 13, 35, and 59 were identified as accessible for the mTG catalyzed reaction, indicating partial unfolding of bLG under pressure and exposure of previously unaccesible glutamine residues. Thus, only a limited number of glutamine residues were substrates for mTG, which points to a pronounced substrate specificity of mTG toward individual glutamine residues within a protein.
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PMID:Modification of beta-lactoglobulin by microbial transglutaminase under high hydrostatic pressure: localization of reactive glutamine residues. 1737 48

After trauma, articular cartilage often does not heal due to incomplete bonding of the fractured surfaces. In this study we investigated the ability of chemical cross-linkers to facilitate bonding of articular cartilage, either alone or in combination with a pre-treatment with surface-degrading agents. Articular cartilage blocks were harvested from the femoropatellar groove of bovine calves. Two cartilage blocks, either after pre-treatment or without, were assembled in a custom-designed chamber in partial apposition and subjected to cross-linking treatment. Subsequently, bonding of cartilage was measured as adhesive strength, that is, the maximum force at rupture of bonded cartilage blocks divided by the overlap area. In a first approach, bonding was investigated after treatment with cross-linking reagents only, employing glutaraldehyde, 1-ethyl-3-diaminopropyl-carbodiimide (EDC)/N-hydroxysuccinimide (NHS), genipin, or transglutaminase. Experiments were conducted with or without compression of the opposing surfaces. Compression during cross-linking strongly enhanced bonding, especially when applying EDC/NHS and glutaraldehyde. Therefore, all further experiments were performed under compressive conditions. Combinations of each of the four cross-linking agents with the degrading pre-treatments, pepsin, trypsin, and guanidine, led to distinct improvements in bonding compared to the use of cross-linkers alone. The highest values of adhesive strength were achieved employing combinations of pepsin or guanidine with EDC/NHS, and guanidine with glutaraldehyde. The release of extracellular matrix components, that is, glycosaminoglycans and total collagen, from cartilage blocks after pre-treatment was measured, but could not be directly correlated to the determined adhesive strength. Cytotoxicity was determined for all substances employed, that is, surface degrading agents and cross-linkers, using the resazurin assay. Taking the favourable cell vitality after treatment with pepsin and EDC/NHS and the cytotoxic effects of guanidine and glutaraldehyde into account, the combination of pepsin and EDC/NHS appeared to be the most advantageous treatment in this study. In conclusion, bonding of articular cartilage blocks was achieved by chemical fixation of their surface components using cross-linking reagents. Application of compressive forces and prior modulation of surface structures enhanced cartilage bonding significantly. Enzymatic treatment in combination with cross-linkers may represent a promising addition to current techniques for articular cartilage repair.
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PMID:Bonding of articular cartilage using a combination of biochemical degradation and surface cross-linking. 1750 33


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