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

---

Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The nephritogenic antigen that induces antiglomerular basement membrane antibody-induced glomerulonephritis (anti-GBM nephritis) in rats was isolated from collagenase-solubilized bovine renal basement membranes. Purification was achieved using antibody-coupled affinity columns which were originally used for the purification of trypsin-solubilized nephritogenic antigen (Sado et al. 1984a). The nephritogenic antigen was a heteropolymer composed of P2 (Mr 28 kDa) and P3 (Mr 30 kDa) polypeptides as monomers and their dimers in sodium-dodecyl-sulfate (SDS) polyacrylamide gel electrophoresis. The P3 polypeptide was considered to be the nephritogenic epitope, since a fraction composed of the P2 polypeptide alone was not nephritogenic. The properties of the nephritogenic epitope were the same as those of the Goodpasture epitope (M2*), which is a noncollagenous domain of the alpha 3 chain of type IV collagen (Butkowski et al. 1985; Saus et al. 1988), indicating that the nephritogenic antigen is the same as the Goodpasture antigen.
...
PMID:Properties of bovine nephritogenic antigen that induces anti-GBM nephritis in rats and its similarity to the Goodpasture antigen. 172 95

Tubular basement membrane (TBM) was prepared from normal human kidneys and solubilized with various enzymes. Collagenase digestion released antigenic moieties from the TBM. All four anti-TBM antibodies we studied, three from patients with idiopathic tubulo-interstitial nephritis (TIN) and one from a renal allograft recipient, distinctively reacted with collagenase-digested (CD) TBM during enzyme-linked immunoassay and could discriminate among sera of normal controls or of other nephritis patients, including anti-glomerular basement membrane (anti-GBM) nephritis. When digested with pronase, trypsin, or pepsin, antigenicity of the TBM decreased. We studied the TBM antigens with immunoprecipitation and immunoblotting. After incubation of radio-iodinated CDTBM with anti-TBM sera, immunoprecipitates were identified by single-dimension SDS polyacrylamide gel electrophoresis or two-dimension gel electrophoresis, followed by autoradiography. All four antibodies had identical results on immunoprecipitation; under nonreducing conditions, they gave two protein bands with m.w. of 54,000 and 48,000 and with pI 7.0 to 8.0 and 6.5 to 7.0. Electrophoresis performed under reducing conditions disclosed only one band at the m.w. of 48,000 and pI of 6.5, suggesting that the 54-kDa component is composed of peptides linked by interchain disulfide bonds. Immunoblot analysis showed that the anti-TBM antibodies were heterogeneous; three antibodies from the idiopathic TIN patients reacted with the 54-kDa band, but the one from the renal allograft recipient reacted with neither band. This finding suggests that there are two antigenic determinants on the 54-kDa component. One such determinant that was resistant to denaturation with SDS was detected by the first three antibodies, and the other that was sensitive to such denaturation bound to the last antibody. The 48-kDa component seemed not to be immunoreactive after incubation with SDS. We studied TBM antigens reactive with anti-GBM antibodies. By immunoblotting, all four sera from patients with anti-GBM nephritis stained TBM proteins of 45 to 50 kDa and 25 to 27 kDa at pH 8.0 to 9.0; this was similar to the staining pattern of CDGBM with the same sera, but the highly cationic (pH greater than 9.0) components were specifically detected in the CDGBM. By inhibition ELISA, the binding of the anti-GBM sera to denatured CDTBM decreased with preincubation of the sera with CDGBM, suggesting that the anti-GBM antibodies recognize the same epitope(s) on the GBM and the TBM.
...
PMID:Characterization of tubular basement membrane antigens in human kidney. 300 98

Incubation of glomerular homogenates (200 micrograms protein) with glomerular basement membrane (GBM, 30-35 micrograms hydroxyproline) at pH 7.5 for 36 h at 37 degrees C resulted in significant GBM degradation as measured by hydroxyproline release (40 +/- 6%, n = 17). GBM degradation increased with increasing incubation time (12-48 h) and glomerular protein concentration (50-250 micrograms). GBM degradation was not significantly decreased by inhibitors of serine or cysteine proteinases or the inhibitor of bacterial metalloproteinases, phosphoramidon. In contrast GBM degradation by glomerular homogenates was markedly inhibited by the metal chelators 10mM EDTA (-95 +/- 3%, n = 7) and 2mM 1,10-phenanthroline (-96 +/- 2%, n = 4). Preincubation of glomerular homogenates with trypsin (followed by soya bean trypsin inhibitor) markedly stimulated GBM degradation (+103 +/- 20%, n = 11). These results document the presence of a GBM-degrading, neutral metalloproteinase(s) in glomeruli suggesting an important role for this enzyme in glomerular pathophysiology.
...
PMID:Degradation of glomerular basement membrane by a neutral metalloproteinase(s) present in glomeruli isolated from normal rat kidney. 310 82

The sera of 21 patients positive for antibodies against GBM in indirect immunofluorescence tests were examined by immunoblotting. We demonstrated antibodies against 50, 48, 43 and 29 kD molecular weight peptides in 20 of 21 sera using collagenase-digested GBM, in 19 of 21 using trypsin-digested GBM, and in 10 of 21 using elastase-digested GBM. Although the spectrum of molecular weights of the antigenic proteins was similar in all three digests, they differed with respect to preservation of antigenicity upon reduction with mercaptoethanol. Many of the sera of patients and controls reacted with proteins unrelated to GBM, e.g. albumin and prealbumin. Furthermore, some control sera reacted with one single peptide of the above-mentioned specific GBM peptides. Our results suggest that the highly purified 29 kD peptide of the collagenase digest or the 50 kD peptide of the trypsin digest provide the best antigens to develop a screening test for antibodies against GBM. However, serum antibodies against these antigens will not be absolutely specific for anti-GBM antibody-mediated nephritis, as shown by the immunoblot experiments.
...
PMID:Characterisation and specificity of glomerular basement membrane antigens identified by sera of patients with anti-GBM nephritis. 311 Jun 69

Components were solubilized from human glomerular basement membrane by digestion with collagenase and pepsin or by extraction with guanidine-HCl either directly or after previous digestion with the enzyme. The diverse preparations were used as antigens in the enzyme-linked immunosorbent assay (ELISA) of antibody titers in sera from patients with Goodpasture syndrome and patients with other forms of glomerulonephritis, that is, systemic lupus erythematosus, periarteritis nodosa, and IgA-related nephropathy. Patients with Goodpasture syndrome had high titers of IgG antibodies reacting most strongly with collagenase digests. The antigen(s) was only partly solubilized by guanidine-HCl extraction, was destroyed by pepsin digestion as well as reduction, and partly destroyed by trypsin digestion. The antigen(s) is most likely noncollagenous protein. Antibodies from patients with other forms of nephritis were directed primarily against antigens in guanidine-HCl extracts, while the antigen(s) was not solubilized by collagenase digestion. Pepsin digestion destroyed the antigen(s). The antibodies were of a different class, that is, the patients with systemic lupus erythematosus had IgG and IgA as well as IgM antibodies; the patients with periarteritis nodosa had IgM or IgG and IgA antibodies, while the patients with IgA-related nephritis had the highest recorded titers of IgA but also had IgG as well as IgM antibodies. None of the patients had antibodies directed against triple helical collagen. The antibody response in anti-GBM antibody-related nephritis, then, is different both with respect to antigen and antibody class and depends on the underlying disease syndrome.
...
PMID:Antiglomerular basement membrane antibody: antibody specificity in different forms of glomerulonephritis. 613 25

Formalin is known to mask the antigenicity of immune deposits in glomeruli but not of surface immunoglobulins of isolated lymphocytes. We have shown in mice with experimental passive anti-GBM glomerulonephritis that formalin masks the antigenicity of GBM-bound immunoglobulins only if the tissue is fixed before sectioning. The presence of a high concentration of normal bovine serum during fixation of cryostat sections masks the antigenicity of immune deposits, whereas formalin alone has no obvious effect. The same results were obtained with human immunoglobulins (IgG, IgM and IgA) bound to tissue sections. Protease treatment with pepsin and trypsin restored the ability of the immunoglobulins to be stained. The masking effect seems to be due to extensive cross-linking of environmental proteins which prevents fluorescent conjugates reaching their antigens. Methods for detecting immunoglobulins in tissues must, therefore, take into consideration the influence of fixatives not only on epitopes but also on the environment in which the antigenic determinants are localised.
...
PMID:Detection of immune deposits in glomeruli: the masking effect on antigenicity of formalin in the presence of proteins. 616 39

Paraffin-embedded tissue can be used as substrate for immunohistochemistry after enzymatic treatment with proteases. The sensitivity of immunofluorescence on enzyme-treated paraffin sections and on unfixed cryostat sections is compared in this study. Pepsin was more efficient by weight than trypsin in restoring the antigenicity of immune deposits. Increased fluorescence intensity was obtained up to a pepsin concentration of 0.4%. Intensity was further increased when pepsin treated sections were treated with trypsin. Immune deposits were detected in enzyme treated, paraffin sections of kidneys of mice injected with anti-GBM diluted 1/200 or less and in cryostat sections of mice injected with anti-GBM diluted 1/400 or less. This small decrease in sensitivity is considered trivial compared with the advantage gained by the excellent preservation of the tissue.
...
PMID:Detection of immune deposits in glomeruli: a comparative study of paraffin-embedded, enzyme-treated sections and cryostat sections as substrates in immunofluorescence. 677 25

This report describes induction of nephritis, which was concurrently mediated by [anti-glomerular basement membrane antibody (anti-GBM) and anti-brush border antibody, in the Wistar rat immunized with a solubilized renal antigen (S-RA). The antigen was prepared by digestion of rat cortical tissue with trypsin and pronase. Ouchterlony test using antisera to the rat GBM and brush border showed that the S-RA contained both antigens. From the S-RA the brush border antigen was isolated by affinity chromatography. At the 8th week rats injected with the S-RA showed a linear or combined linear and granular distribution of rat IgG and C3 along the GBM in immunofluorescence. The capillary granular pattern was only observed at the 16th week. In contrast rats injected with the brush border antigen remained in a capillary granular pattern throughout the experimental course. It was suggested that the rat nephritis injected with the S-RA was mediated by the antibodies capable of reacting with at least two different antigens, namely the GBM and the brush border. The possibility was confirmed by demonstrating the coexistence of these two kinds of antibodies in the serum and kidney eluate from the nephritic rats.
...
PMID:Experimental induction of glomerulonephritis mediated by anti-glomerular basement membrane and anti-brush border antibodies in a single rat. 680 53

In our experiments, we succeeded in obtaining from human placenta a substance comparable to human renal nephritogenic glycoprotein prepared from GBM (glomerular basement membrane). The procedure was quite similar to that for preparation from the kidney. At first, placental terminal villi were isolated by the successive use of three metal sieves. Then trophoblast basement membranes (TrBM) were isolated from placental terminal villi by ultrasonic disruption. Finally TrBM were made soluble by trypsin digestion, after which they were further digested by pronase. Ouchterlony gel diffusion demonstrated the existence of a common precipitin line between antiserum to human renal nephritogenic glycoprotein prepared from GBM and antiserum to the sample prepared from human placental TrBM, and human renal nephritogenic glycoprotein prepared from GBM and the sample prepared from human placental TrBM. The monosaccharide composition of the sample prepared from human placental TrBM was rich in glucose (glucose, galactose and mannoside in the ratio of 1.00 : 0.74 : 1.00), and the amino acid composition of the sample contained no collagenous components. As a result of fractionation of the sample prepared from human placental TrBM by Bio Gel P200 column chromatography, the activity of renal nephritogenic glycoprotein was found within the void fraction.
...
PMID:[Nephritogenic glycoprotein isolated from human placenta (methods of isolation from placenta]. 687 36

Protease-Activated Receptor-2 (PAR2) has been implicated through genetic knockout mice with cytokine regulation and arthritis development. Many studies have associated PAR2 with inflammatory conditions (arthritis, airways inflammation, IBD) and key events in tumor progression (angiogenesis, metastasis), but they have relied heavily on the use of single agonists to identify physiological roles for PAR2. However such probes are now known not to be highly selective for PAR2, and thus precisely what PAR2 does and what mechanisms of downstream regulation are truly affected remain obscure. Effects of PAR2 activation on gene expression in Human Embryonic Kidney cells (HEK293), a commonly studied cell line in PAR2 research, were investigated here by comparing 19,000 human genes for intersecting up- or down-regulation by both trypsin (an endogenous protease that activates PAR2) and a PAR2 activating hexapeptide (2f-LIGRLO-NH(2)). Among 2,500 human genes regulated similarly by both agonists, there were clear associations between PAR2 activation and cellular metabolism (1,000 genes), the cell cycle, the MAPK pathway, HDAC and sirtuin enzymes, inflammatory cytokines, and anti-complement function. PAR-2 activation up-regulated four genes more than 5 fold (DUSP6, WWOX, AREG, SERPINB2) and down-regulated another six genes more than 3 fold (TXNIP, RARG, ITGB4, CTSD, MSC and TM4SF15). Both PAR2 and PAR1 activation resulted in up-regulated expression of several genes (CD44, FOSL1, TNFRSF12A, RAB3A, COPEB, CORO1C, THBS1, SDC4) known to be important in cancer. This is the first widespread profiling of specific activation of PAR2 and provides a valuable platform for better understanding key mechanistic roles of PAR2 in human physiology. Results clearly support the development of both antagonists and agonists of human PAR2 as potential disease modifying therapeutic agents.
...
PMID:Profiling gene expression induced by protease-activated receptor 2 (PAR2) activation in human kidney cells. 2107 96


1

---