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)

CD spectra of reduced and S-3-(trimethylated amino) propylated lysozyme (TMAP lysozyme) have been measured in various solutions containing guanidine hydrochloride or trifluoroethanol (TFE). The CD spectra indicate that there remain residual secondary structures in protein in aqueous solution. The addition of TFE further promotes the formation of secondary structures. In order to examine whether secondary structures are evenly induced over all the polypeptide chain, or locally at particular segments, the limited proteolysis of TMAP lysozyme by trypsin has been performed, and the CD spectra of all the final and intermediate products have been observed in solutions containing TFE. As a result, the fragments vary in a helix-forming propensity. The CD spectra of peptide fragments T5, T7, T9T10, T12T13, T14T15T16, and T17T18 are not significantly affected by the addition of TFE, where T refers to the nomenclature of R.E. Canfield [(1963), Journal of Biological Chemistry, Vol. 238, pp. 2691-2697]. They are fragments of a helix-breaking propensity. On the other hand, fragment I2 composed of T1-T4, and fragments T6T7, T8, and T11, attain secondary structures with the addition of TFE. They are fragments of a helix-forming propensity. Further, it is found that the fragments of a helix-forming propensity just correspond to the helical segments in native lysozyme. We examine the interactions between neighboring fragments, which contribute to the stabilization of local structures along the polypeptide chain.
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PMID:Local structures in unfolded lysozyme and correlation with secondary structures in the native conformation: helix-forming or -breaking propensity of peptide segments. 186 65

Whereas by indirect immunofluorescence approximately 80% of freshly isolated human peripheral blood lymphocytes bound T3 (Leu 4)-specific and T11-specific monoclonal antibodies, by a more sensitive technique--the indirect antiglobulin rosetting reaction--more than 90% of these lymphocytes were T3+ (Leu 4+) and T11+; this finding suggested that some non-T cells may also express T3 and T11 determinants. In double labelling experiments most normal surface membrane immunoglobulin (smIg)-bearing lymphocytes, ie, B cells, were shown to express T3 and T11 determinants at low density. The determinants were re-expressed after stripping with trypsin. Moreover, greater than 90% of neoplastic lymphoid cells expressing monotypic smIg from six donors were T3+ and/or T11+. Together these results provide new data on the cell distribution of 'T cell markers'. These findings also indicate that the rosette test is too sensitive to distinguish between biologically distinct lymphocyte population, ie between T and B cells.
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PMID:Expression of T3 and T11 determinants on human peripheral lymphocytes as analysed by the indirect antiglobulin rosetting reaction (IARR) and indirect immunofluorescence. 241 98

Immunoconjugates consisting of mAb covalently coupled to plant or bacterial toxins or to cytotoxic drugs have provided novel experimental reagents for the treatment of malignancies and autoimmune diseases. In this report, we analyzed the efficacy of three ricin A chain-containing immunotoxins (IT-A) which recognize different epitopes on the CD2 molecule (E rosette receptor) on human T cells. Although all IT-A had similar binding avidities and A-chain activities, one (RFT11-A) was 100-1000-fold more effective in killing normal and malignant T cells than the others (35.1-A, 9.6-A). Immunoprecipitation experiments confirmed that all IT-A bound to the CD2 molecule. However, cross-blocking experiments, differential proteolysis with trypsin, and T cell co-activation experiments showed that the less effective IT-A, 35.1-A and 9.6-A, bound to an epitope far from the cell membrane (region I), whereas the more effective IT-A, RFT11-A bound to an epitope closer to the membrane (region II). Using cellular RIA and immunoelectron microscopy, it was shown that both RFT11-A and 35.1-A were rapidly internalized by T cells, but that their intracellular fates differed. The more toxic IT-A, RFT11-A, was retained for longer periods of time inside the cells and was more slowly degraded than the less effective IT-A, 35.1-A, which was rapidly transported to lysosomes, digested, and expelled. These results demonstrate that different IT-A targeting the same surface molecule can differ markedly in potency, and that the epitope recognized by an IT-A may have a significant impact on the ability of the IT-A to insert into cell membranes, translocate to the cytosol, and kill cells.
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PMID:Ricin A-chain containing immunotoxins directed against different epitopes on the CD2 molecule differ in their ability to kill normal and malignant T cells. 246 93

A large fraction of colonic epithelial cells (45 +/- 15%) formed rosettes with sheep red blood cells (RBC) but not with bovine or human RBC. This interaction was independent of the T11 receptor since it was not inhibited by anti-T11 antibody or trypsin treatment of the epithelial cells. The binding was also not due to surface immunoglobulin reactive with sheep RBC since antibodies to immunoglobulin light chains did not block the interaction. Sheep RBC rosettes formed around colonic and jejunal epithelial cells, a few percent of colonic adenocarcinoma cells, but not around uterine epithelial cells. Sheep RBC rosette formation should not be used to quantitate or isolate intestinal mucosal T cells if epithelial cells are present.
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PMID:Intestinal epithelial cells rosette with sheep red blood cells. 308 24

We asked whether we could distinguish the roles of the human lymphocyte membrane proteins LFA-1, LFA-2, and LFA-3 in the function of CTL-mediated killing. Little is known about the functions of these molecularly distinct proteins beyond the facts that i) binding of a monoclonal antibody (MAb) to any one of them is sufficient to inhibit killing, ii) that in each case inhibition involves prevention of CTL-target cell conjugate formation, and iii) that MAb to LFA-1 and LFA-2 inhibit best when bound to the CTL, whereas anti-LFA-3 inhibits only when bound to the target cell. This latter is despite the fact that (in our test system) LFA-1 and LFA-3 are expressed both on the CTL and on the target. When the target cells were pretreated with trypsin, the sensitivity of CTL-mediated killing was affected in a different way for each site. Inhibition of anti-LFA-1 was increased by approximately 20-fold. Inhibition by anti-LFA-2 was unaffected. Inhibition by anti-LFA-3 was abolished. Trypsin did not remove the specific antigens recognized by the various CTL, HLA-A,B,C or HLA-DR. Nor did it remove LFA-1 from the target cell. It did, however, selectively remove LFA-3 from the target cell. These results indicate, for the first time, that LFA-1 and LFA-2 have functionally distinct roles. They suggest that an unidentified trypsin-sensitive target cell molecule, operationally designated the "trypsin-sensitive counter blocker" (TSCB), plays an important role in the function of LFA-1, possibly by providing a target cell binding site for LFA-1 on the CTL. The hypothesis that this TSCB is identical to LFA-3 (and the related possibility that LFA-1 and LFA-3 are mutual ligands) is not favored by our data, but is not excluded. Finally, the data indicate that the mechanisms by which MAb inhibit killing differ at the LFA-1 and LFA-3 sites. They are consistent with LFA-1 providing adhesion strengthening by binding to another site (the TSCB?) and with LFA-3 delivering an inhibitory signal when provoked with MAb.
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PMID:Functional distinctions between the LFA-1, LFA-2, and LFA-3 membrane proteins on human CTL are revealed with trypsin-pretreated target cells. 387 Nov 2

We have isolated and characterized the human cardiac mast cell (CMC) and compared this novel mast cell (MC type with MC obtained from uterus, skin, and lung. Heart tissue was obtained from 14 patients with cardiomyopathy (CMP, heart transplantation). CMC were isolated by enzymatic digestion using collagenase, pronase-E, hyaluronidase, and DNAse. Substantial amounts of CMC (0.5% to 1.5% of isolated cells) were found in the atrial appendages but not in ventricular digests or other sites of the heart (< 0.1%). In situ staining of atrial tissue revealed the presence of CMC in the myocardium (2.16 +/- 0.7 MC/mm2), endocardium (2.24 +/- 0.9 MC/mm2), and epicardium. As assessed by combined toluidine blue/immunofluorescence staining with monoclonal antibodies (MoAbs), isolated CMC expressed surface IgE, the receptor for stem cell factor (c-kit receptor/CD117), the p24 antigen (CD9), the Pgp-1 homing receptor (CD44), the pan leukocyte antigen (CD45), and the ICAM-1 antigen (CD54). CMC were not recognized by MoAbs to lymphocyte function associated antigen 2 (LFA-2; CD2), T-cell receptor (TcR; CD3), T4 antigen (CD4), LFA-1 alpha-chain (CD11a), C3biR alpha-chain (CD11b), CR4 alpha-chain (CD11c), LPS-R related Ag (CD14), 3-FAL/x-hapten (CD15), Fc gamma RIII (CD16), lactosylceramid (CDw17), the B-cell antigen CD19, or CR1 (CD35). In situ expression of leukocyte antigens on CMC was demonstrable by indirect immunoperoxidase staining technique and double-labeling immunohistochemistry. Almost all CMC (90%) reacted with MoAbs against tryptase and chymase and thus were MCTC. Cardiac mast cells were also stained by the heparin-binding dye Berberine sulfate and expressed measurable amounts of histamine (4.6 +/- 1.4 pg per cell). Cross linking of either IgE receptor or SCF receptor (c-kit) on CMC resulted in histamine secretion (non-specific release: < 6% of total histamine, alpha IgE induced: 12% to 52%; SCF-induced release: 9% to 18%), whereas neither substance P (a skin MC agonist) nor the basophil agonist FMLP showed an effect on CMC. Together, the CMC is an MCTC primarily located in the appendage of the atrium. This novel type of MC exhibits surface membrane antigen and functional properties similar to those of lung and uterus MC.
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PMID:The human cardiac mast cell: localization, isolation, phenotype, and functional characterization. 752 50

Zona pellucida glycoproteins play an important role in fertilization. In this study, attempts have been made to identify and define epitopes of monoclonal antibodies (mAbs) possessing contraceptive efficacy in vitro. The porcine zona glycoprotein pZPC, a homologue of mouse/human ZP3, was reduced and alkylated and subsequently digested with trypsin. Reverse-phase HPLC of the tryptic digest yielded twenty two peaks (T1-T22). When tested against mAbs reactive against sequential determinants on pZPC, T11 was immunoreactive with two mAbs, mAb-455 and mAb-467, as shown by antigen inhibition ELISA. IC50 values of 3.1 nM and 8.6 nM were recorded versus mAb-455 and mAb-467 respectively, and approximated the IC50 values obtained with intact pZPC. Amino acid analysis, Edman degradation, and FAB-MS identified T11 as the N-blocked decapeptide pyro-Gln-Pro-Val-Trp-Gln-Asp-Glu-Gly-Gln-Arg derived from the N-terminus of pZPC. Synthesis of overlapping octapeptides further identified VWQDE and WQDE as the minimum motifs with antigenic activity for mAb-455 and mAb-467, respectively. Glycine replacement peptides confirmed residues W,Q,E as critical for binding mAb-455 and W,Q,D,E as critical for binding mAb-467. Both mAbs inhibited binding of boar sperm to zona-encased porcine oocytes. These results, the first to define peptide epitopes of porcine zona glycoprotein, will assist in the design of an immunocontraceptive vaccine based on synthetic peptides corresponding to pZPC or its homologues in other species.
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PMID:Localization of epitopes for monoclonal antibodies at the N-terminus of the porcine zona pellucida glycoprotein pZPC. 856 67

In this paper we describe the use of tandem mass spectrometry to identify modified sites in human hemoglobin after in vitro exposure to bis(2-chloroethyl) sulfide (sulfur mustard). Globin isolated from human whole blood which had been exposed to sulfur mustard was degraded with trypsin, and the digests were analyzed by micro LC/MS. Alkylated tryptic fragments (alpha-T1, alpha-T4, alpha-T6, alpha-T9, beta-T1, beta-T9, beta-T10, beta-T11, and beta-T10-S-S-beta-T12) could be tentatively assigned upon comparison with a digest from nonexposed globin. Subsequent tandem mass spectrometry of these peptides allowed unambiguous assignment of 5 specific modified residues: alpha-Val-1, alpha-His-20, beta-Val-1, beta-His-77, and beta-His-97. The results demonstrate the usefulness of microbore LC in combination with tandem mass spectrometry for the structural determination of chemically modified peptides and proteins.
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PMID:Characterization of sulfur mustard induced structural modifications in human hemoglobin by liquid chromatography--tandem mass spectrometry. 883 23

The term mastocytosis denotes a heterogenous group of disorders characterized by abnormal growth and accumulation of mast cells in one or more organs. Cutaneous and systemic variants of the disease have been described. Mast cell disorders have also been categorized according to other aspects, such as family history, age, course of disease, or presence of a concomitant myeloid neoplasm. However, so far, generally accepted disease criteria are missing. Recently, a number of diagnostic (disease-related) markers have been identified in mastocytosis research. These include the mast cell enzyme tryptase, CD2, and mast cell growth factor receptor c-kit (CD117). Several gain-of-function-mutations in the kinase domain of c-kit appear to occur in mastocytosis supporting the clonal (neoplastic) nature of the disease. Also, certain point mutations appear to be associated with distinct variants of mastocytosis, i.e. Asp-816-->Val with a subset of sporadic persistent (systemic) mastocytosis (mostly adults), and Gly-839-->Lys with (a subset of) typical pediatric (mostly cutaneous) mastocytosis. Another potential indicator of mast cell neoplasm is the T-/NK-cell-associated marker CD2. This antigen (LFA-2) is abnormally expressed on neoplastic mast cells in cases of systemic mastocytosis or mast cell leukemia, but not found on normal mast cells. The mast cell enzyme tryptase is increasingly used as a serum- and immunohistochemical marker to estimate the actual spread of disease (burden of neoplastic mast cells). The clinical significance of novel mastocytosis markers is currently under investigation. First results indicate that they may be useful to define reliable criteria for the delineation of the disease.
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PMID:Recent advances in mastocytosis research. Summary of the Vienna Mastocytosis Meeting 1998. 1052 83

The protein toxin ricin, which originates from the seeds of Ricinus communis plants, has been the subject of increased interest, due to its potential terrorist use. Exceptionally, this toxin is also subject to the Chemical Weapons Convention. In this paper, it is shown that mass spectrometry can be used to unambiguously verify the presence of ricin in crude toxin preparations. It is demonstrated that MALDI MS can be used for screening, either by direct analysis or by trypsin digestion and peptide mapping. Purified ricin from several varieties of R. communis was characterized by LC-ES MS(/MS). A crude ricin preparation from a single bean was similarly characterized. An LC method was set up with product ion MS/MS detection of selected marker peptides specific for ricin: T5, T7, T11, T12, and T13 from the A-chain and T3, T5, T14, T19, and T20 from the B-chain. This method was then used to unambiguously identify ricin in a crude preparation of ricin. The MALDI MS molecular weight analysis and the marker peptides LC-ES MS/MS analysis give a forensic level of identification of ricin when combined with activity testing.
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PMID:Forensic identification of neat ricin and of ricin from crude castor bean extracts by mass spectrometry. 1576 56


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