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)

Two murine monoclonal antibodies, SK-930 (isotype IgG2a) and SK-117 (isotype IgG1), were produced from spleen cells of mice immunized against human pancreatic carcinoma cell lines, MIA-PaCa 2 and Panc-1. With the use of the avidin-biotin-immunoperoxidase technique, the SK-930 and SK-117 antibodies detected an antigen found in 24 and 23 formalin-fixed tissue sections, respectively, of tumors obtained from 30 different patients with pancreatic carcinoma. Reactivity was also frequently found with tumors of the gallbladder, bile duct, stomach, colon and esophagus, while a large panel of normal human tissues, including normal pancreatic tissues, displayed little reactivity. These observations suggest that SK-930 and SK-117 are of value in identifying tumor-associated antigen (TAA) expressed in pancreatic carcinoma and other carcinomas of the digestive system. SK-930 antibody immunoprecipitated a 134 kilodalton molecule from extracts of 125I- or [35S]methionine- or [3H]glucosamine-labeled tumor cells. The SK-117-defined antigen corresponds to 152/137 kilodalton molecules. Moreover, cytofluorometric analyses showed that cells treated with periodic acid exhibited greatly decreased reactivity to the two antibodies, but cells treated with neuraminidase, trypsin or pronase showed unchanged reactivity. The findings suggest that the epitopes of the novel TAA expressed on pancreatic carcinoma cells are carbohydrate moieties.
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PMID:Immunochemical and immunohistological analyses of tumor-associated antigens defined by murine monoclonal antibodies against human pancreatic carcinoma cells. 245 17

Peanut lectin (PNA) is known to bind more strongly to keratinocytes that are undergoing terminal differentiation than to proliferating keratinocytes, both in intact epidermis and in culture. In order to investigate the significance of this change in cell-surface carbohydrate we have identified the PNA-binding glycoproteins of cultured human keratinocytes and raised antibodies against them. Two heavily glycosylated bands of 110 and 250 kDa were resolved by PAGE of [14C]galactose- or [14C]mannose- and [14C]glucosamine-labeled cell extracts eluted with galactose from PNA affinity columns. The higher molecular weight band was also detected on PNA blots of unlabeled cell extracts transferred to nitrocellulose. Both bands were sensitive to Pronase digestion, but only the 250-kDa band was digested with trypsin. A rabbit antiserum that we prepared (anti-PNA-gp) immunoprecipitated both bands from cell extracts. In contrast to PNA, anti-PNA-gp bound equally to proliferating and terminally differentiating cells, indicating that some epitope(s) of the PNA-binding glycoproteins is present on the cell surface prior to terminal differentiation. When keratinocytes grown as a monolayer in low-calcium medium (0.1 mM calcium ions) were switched to medium containing 2 mM calcium ions in order to induce desmosome formation and stratification, there was a dramatic redistribution of the PNA-binding glycoproteins, which became concentrated at the boundaries between cells. This may suggest a role for the glycoproteins in cell-cell interactions during stratification.
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PMID:The peanut lectin-binding glycoproteins of human epidermal keratinocytes. 245 53

The predicted cytoplasmic orientation and two-domain structure of the multidrug efflux pump P-glycoprotein were demonstrated with sequence-specific antibodies. We synthesized peptides corresponding to amino acid residues, Glu393-Lys408 (anti-P) and Leu1206-Thr1226 (anti-C) in P-glycoprotein from human mdr1 cDNA and used these peptides to produce polyclonal antibodies. From the primary structure of P-glycoprotein, and anti-C antibody is expected to recognize another position, Leu561-Thr581, in the duplicate structure of P-glycoprotein, but anti-P recognizes only one site. These antibodies bind to multidrug-resistant cells (KB-C2) with permeabilized plasma membrane but do not bind to nonpermeabilized KB-C2 cells or parental KB cells, supporting the predicted cytoplasmic orientation of these sequences. With immunoblotting of the membrane fractions from KB-C2 cells, a major 140-kDa polypeptide of the P-glycoprotein was detected with both anti-P and anti-C. Two minor polypeptides with molecular mass of 95 and 55 kDa were also detected. When membrane vesicles were digested mildly with trypsin, the amount of these two polypeptides increased. Anti-P detected only the 95-kDa polypeptide, and anti-C detected both 95- and 55-kDa polypeptides. Achromobacter lyticus protease I (lysyl endopeptidase) and Staphylococcus aureus V8 protease also produced two polypeptides with similar molecular weights. Absorption into lectin-agarose beads and labeling with [3H]glucosamine indicated that the 95-kDa polypeptide was glycosylated but that the 55-kDa polypeptide was not. These two polypeptides as well as P-glycoprotein were photoaffinity-labeled with a calcium channel blocker, [3H]azidopine, but most of the label was found in the 55-kDa polypeptide. The yield of labeled fragments from membrane vesicles photolabeled after digestion with trypsin was similar to that from membrane vesicles digested with trypsin after photolabeling. These data indicate 1) that the 95-kDa polypeptide is the fragment corresponding to the amino-terminal half of P-glycoprotein containing sugar chains; 2) that the 55-kDa polypeptide is the carboxyl-terminal half which was mainly labeled with [3H]azidopine; and 3) that P-glycoprotein has a relatively rigid structure with a small number of protease-sensitive sites and its global structure is not destroyed by tryptic cleavage.
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PMID:Cytoplasmic orientation and two-domain structure of the multidrug transporter, P-glycoprotein, demonstrated with sequence-specific antibodies. 247 41

The chemical structure and immunomodulating activities of the cell wall peptidoglycans isolated from Actinobacillus actinomycetemcomitans were investigated. Peptidoglycans were isolated from A. actinomycetemcomitans strains Y4 and ATCC 29522 by boiling in 4% sodium dodecyl sulfate and by digestion with pronase, trypsin and alpha-amylase. Analysis of amino acids and amino sugars of the peptidoglycans revealed that glucosamine, muramic acid, D-glutamic acid, D-alanine, and meso-2, 6-diaminopimelic acid (A2pm) were the principal components. Serine and glycine were not found. Dinitrophenylation method revealed that about half of A2pm residue had a free aminogroup, and analysis by hydrazinolysis showed that a small part of alanine and A2pm located at the C-terminal. The above results indicate that one of the amino groups of A2pm residue at one strand of the stem peptide subunit crosslinked to the carboxyl group of alanine of the neighboring strand. It was thus revealed that the peptidoglycans of A. actinomycetemcomitans belonged to the Al gamma type of the classification by Schleifer and Kandler. Peptidoglycans isolated from A. actinomycetemcmitans strain Y4 and ATCC 29522 were found to be definitely adjuvant-active in induction of delayed type hypersensitivity against ovalbumin when administered to guinea pigs as water-in oil emulsion and stimulation of increase serum antibody levels was found in both peptidoglycans. Regarding mitogenicity on splenocytes of BALB/c and BALB/c nu/nu mice, peptidoglycans from two strains of A. actinomycetemcomitans were markedly enhanced the uptake [3H] thymidine in dose of 10 micrograms/10(5) cells, however thymocytes were not reactive. Stimulation effects on peritoneal macrophages from a guinea pig to incorporation of 14C-glucosamin were not exhibited on addition of 100 micrograms of both peptidoglycans. These findings indicate that peptidoglycan of A. actinomycetemcomitans might eventually be responsible for destruction of periodontal tissue by host mediated activities.
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PMID:[Chemical structure and immunomodulating activities of peptidoglycan from Actinobacillus actinomycetemcomitans]. 248 65

The effect of all-trans retinoic acid on metastatic B16 melanoma lung colonization and synthesis and properties of glycosaminoglycans was examined. Injection of tumour cells, pretreated with 10(-6) M-retinoic acid or grown to low density, into the tail vein of syngeneic C57 mice produced significantly fewer pulmonary tumours compared to subconfluent control cells. By cochromatography of glycosaminoglycans isolated from control ([14C]glucosamine-labelled) and 10(-6) M-retinoic acid-treated ([3H]glucosamine-labelled) cells on DEAE ion-exchange columns, differences in elution profiles were detected. Chondroitin sulphates isolated from retinoic acid-treated cells eluted at a lower salt concentration than those from control cells, while retinoic acid-treated cells synthesised heparan sulphates of a higher charge density than heparans from control cultures. These changes were apparent in both medium and trypsin-releasable fractions. Retinoic acid-treated cultures were seeded so that they were of a similar density to control cultures when harvested, as cell density was shown to affect glycosaminoglycan synthesis, the glycosaminoglycans from low-density cultures having similar properties to those isolated from retinoic acid-treated cultures. Retinoic acid treatment also reduced the overall synthesis of glycosaminoglycans while having little effect on the composition or distribution between medium, trypsin-releasable and cell-associated fractions. These observed changes in glycosaminoglycans may, in part, be responsible for retinoic acid-induced inhibition of lung colonization, and reduced adhesion to basement membrane components, which we have previously demonstrated.
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PMID:Retinoic acid-induced inhibition of lung colonization and changes in the synthesis and properties of glycosaminoglycans of metastatic B16 melanoma cells. 251 93

The expression of heparan sulfate proteoglycans (HSPGs) by human glioma cells was examined by biochemical and immunological methods in vitro and in vivo. Chondroitin sulfate was shown to represent the major [3H]glucosamine-labeled glycosaminoglycan synthesized by cultured normal brain cells. However, high-grade glioma-derived cells were shown to express significantly increased quantities of hyaluronic acid and heparan sulfate and approximately equal amounts of chondroitin sulfate compared with normal glial cells. To investigate further the differential expression of HSPGs, proteoglycans were isolated from glioma cells and were used as an immunogen to generate monoclonal antibodies (MAbs). One of these MAbs, 39H (an IgM), was shown to bind more to high-grade glioma-derived cells then to low-grade glioma or normal brain cells in vitro. MAb 39H was also observed to bind to isolated HSPGs but not to heparan sulfate glycosaminoglycan chains or trypsin-treated cells. Immunofluorescence staining of the cultured high-grade glioma cells revealed an intense diffuse cell surface staining pattern over the entire cell and also isolated footpads. In contrast, the low-grade tumor or normal glial cells showed a distinctive punctated staining. A similar differential staining of MAb 39H was most prominent between tissue sections of glioblastoma multiforme and anaplastic astrocytomas versus low-grade astrocytomas and normal brain. The low grade gliomas exhibited a weak punctated staining, whereas the high-grade gliomas showed significantly more intense staining, particularly along the apical regions of the cells. These results suggest that altered expression of HSPGs may be related to the malignant transformation or growth potential of glial-derived cells.
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PMID:Altered expression and distribution of heparan sulfate proteoglycans in human gliomas. 252 16

The Minnesota strain of turkey enteric coronavirus (TCV) was grown on a human rectal tumor (HRT-18) cell line in the presence of radiolabeled amino acids and glucosamine to analyse virion structural proteins. In addition to the 52,000 unglycosylated nucleocapsid protein, three major glycoprotein species were found to be associated with the viral envelope. A predominant glycosylated protein with a molecular weight of 22-24,000 represented the transmembrane matrix protein. Larger glycoproteins with apparent molecular weights of 180-200,000 (gp 200), 120-125,000 (gp 120) and 95-100,000 (gp 100) were associated to the characteristic large bulbous projections (peplomers) located at the surface of the virion. The gp 100 and gp 120 species apparently arose from a proteolytic cleavage of gp 200, as suggested by digestion studies with trypsin and chymotrypsin. An additional large glycoprotein with mol. wt. of 140,000 (gp 140), that behaved as a disulfide-linked dimer of a 66,000 molecule, was found to be associated to granular projections located near the base of the large peplomers. Digestion studies with trypsin, bromelain and pronase demonstrated that gp 140 was related to the hemagglutinating activity of the virus. An inner membranous sac or tongue-shaped structure could be visualized in the interior of the viral particles following treatment with pronase. In contrast, trypsin or chymotrypsin treatments resulted in evaginations ("budding") on the virus surface. Progeny viral particles produced in TCV-infected cell cultures in the presence of tunicamycin lacked both types of surface projections, as demonstrated by electron microscopy and electrophoresis. The matrix protein also appeared to be reduced to its unglycosylated form, concomitant with a considerable loss of its antigenicity. Thus, with respect to its morphological and biochemical characteristics, TCV resembles viruses belonging to the group of mammalian hemagglutinating coronaviruses, but differs in that both types of envelope glycoproteins are N-glycosylated as in case of the avian infectious bronchitis virus.
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PMID:Identification and location of the structural glycoproteins of a tissue culture-adapted turkey enteric coronavirus. 267 55

Synthesis of fibronectin and glycosamingoglycans (GAGs) was studied in fibroblasts from pigs with post-irradiation subcutaneous fibrosis. Fibrosis was developed in the femoral muscle by local gamma irradiation with a dose of 60 Gy. Normal fibroblasts were obtained from the healthy skin of the same animal. To measure GAG and fibronectin synthesis fibrotic and normal fibroblasts were labeled with 3H-glucosamine, 35S-sulfate and 35S-methionine. Fibrotic fibroblasts synthesized 2.5 times as much fibronectin as normal skin fibroblasts but total protein synthesis did not change. Parallel enhanced secretion of hyaluronic acid and dermatan sulfate into the cell culture medium were also observed. GAGs from the pericellular layer of trypsin-digested fibrotic fibroblasts exhibited increased 3H incorporation, but reduced 35S-sulfate incorporation. The largest reduction in the latter was observed for heparan sulfate. These results indicate that the fibroblasts from the well developed fibrotic tissue maintain enhanced synthesis of matrix macromolecules in primary cultures. Structural and/or metabolic changes in secreted GAGs, combined with the stimulation of tissue repair by growth factors may be responsible for the excessive deposition of collagen in post-irradiation fibrosis.
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PMID:Fibronectin and glycosaminoglycan synthesis by fibrotic pig fibroblasts in primary culture. 269 15

Cystic fibrosis (CF) respiratory epithelia exhibit abnormal anion transport that may be linked to abnormal lung defense. In these studies, we investigated whether primary cultures of CF respiratory epithelial cells regulate abnormally the sulfate content of high molecular weight glycoconjugates (HMG) participating in airways' mucosal defense. HMG, including glycosaminoglycans and mucin-type glycoproteins released spontaneously into medium and HMG released from cell surfaces by trypsin, were metabolically labeled with 35SO4- and [6-3H]-glucosamine (GlcN) or 35SO4- and [3H]serine. All three classes of HMG from CF cells exhibited 35S/3H labeling ratios 1.5-4-fold greater than HMG from normal or disease control cells. Differences for labeling ratios of HMG from CF cells were shown to be the consequence of increased 35SO4- incorporation rather than decreased peptide synthesis and release or HMG glycosylation. The buoyant density of CF mucin-type HMG also was increased, consistent with increased sulfation. These observations suggest that oversulfation of a spectrum of HMG is a genetically determined characteristic of CF epithelial cells and may play an important pathophysiological role by altering the properties of mucous secretions and/or the interactions between selected bacteria and HMG at the airways' surface.
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PMID:Increased sulfation of glycoconjugates by cultured nasal epithelial cells from patients with cystic fibrosis. 273 59

The number of free cysteines in each polypeptide of acetylcholine receptor from the electric organ of Torpedo californica has been assessed by alkylating the native protein with N-ethylmaleimide and iodoacetamide during homogenization of the tissue and alkylating the polypeptides with N-ethylmaleimide as they were unfolded in solutions of dodecyl sulfate. The cysteines unavailable for alkylation could be accounted for as specific cystines, connecting positions in the amino acid sequences of the individual polypeptides. Unreduced, alkylated polypeptides of acetylcholine receptor were digested with thermolysin or trypsin. Cystine-containing peptides in the chromatograms of the digests were identified electrochemically by the use of a dual gold/mercury electrode. Three thermolytic peptides and three tryptic peptides have been isolated from these digests and shown to contain intact cystines that were originally present in the native protein. The majority of these peptides contained an intact, intramolecular cystine connecting two cysteines in locations homologous to cysteines 128 and 142 from the alpha polypeptide. Each of these cystines from each of the polypeptides of acetylcholine receptor was isolated in at least one peptide, respectively. Each of these cystine-containing peptides also contained glucosamine. It can be concluded that each asparagine in the sequence Asn-Cys-Thr/Ser, which occurs in the respective, homologous location in every polypeptide, is glycosylated even though a cystine sits between the asparagine and the threonine or serine. In addition, the existence of the cystine connecting the adjacent cysteines, alpha 192 and alpha 193, in the alpha subunit of acetylcholine receptor [Kao, P. N., & Karlin, A. (1986) J. Biol. Chem. 261, 8085-8088] has been confirmed.
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PMID:Assessment of the number of free cysteines and isolation and identification of cystine-containing peptides from acetylcholine receptor. 274 50


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