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)

Cell surface hydrophobicities of Actinomyces pyogenes were determined by measuring the adherence of the bacteria to hexadecane droplets and by salt aggregation tests. Among 42 A. pyogenes cultures tested 25 (60%) adhered strongly (adherence greater than or equal to 75%) and 17 (40%) less pronounced (adherence between 25-75%) to the hexadecane droplets. Pre-treatment of the bacteria with proteolytic enzymes completely eliminated the adherence properties whereas heat treatment had no effect. The salt aggregation studies revealed that 4 (10%) cultures aggregated in ammonium sulfate solutions of a molarity of 0.05 mol/l, 5 (12%), 14 (33%) and 3 (7%) cultures in ammonium sulfate solutions with molarities of greater than or equal to 1.5 mol/l, greater than or equal to 3 mol/l and greater than or equal to 4.5 mol/l, respectively. No aggregation at all could be observed with 16 (38%) of the cultures. Pronase treatment completely eliminated the salt aggregation reactions, trypsin- and heat treatment had no effect. The results from hexadecane adherence and salt aggregation did not correspond. The differences in surface hydrophobicities, possibly related to adherence properties of A. pyogenes, could be used for epidemiological typing of individual cultures of this bacterial species.
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PMID:Cell surface hydrophobicity of Actinomyces pyogenes determined by hexadecane adherence- and salt aggregation studies. 162 75

Antibody from BALB/cByJ mice immunized against a membranous fraction of Candida albicans agglutinated whole cells as well as the membranous fraction. Hybridoma techniques were used to isolate an IgM monoclonal antibody (mAb) designated 10G which agglutinated whole cells and reacted with the subcellular fraction. Yeast cells of 15 additional C. albicans strains and isolates of C. stellatoidea, C. tropicalis, C. intermedia and C. lusitaniae were also agglutinated by mAb 10G. The antigen was not detected on other fungi, including Candida krusei, C. utilis, Cryptococcus neoformans, Cr. albidus, Torulopsis glabrata, Rhodotorula spp. and Saccharomyces cerevisiae. To determine the cellular location of the epitope to which mAb 10G is specific, freeze-substitution was compared with traditional chemical fixation methods in preparation of samples for immunocolloidal gold electron microscopy (IEM). With both fixation procedures, the antigen recognized by mAb 10G was found randomly and densely concentrated on the plasma membrane on exponential-phase yeast-form cells and had a patchy distribution on the cell wall surface. Association of the antigen with the plasma membrane was confirmed by IEM of isolated membranes. On developing hyphal cells, antigen appeared first on the plasma membrane and later on the cell wall surface. Treatment of yeast cells with beta-mercaptoethanol and Zymolyase before fixation removed the antigen from the surface but left the cytoplasmic antigen undisturbed. Treatment of yeast cells or solubilized antigen with heat or proteolytic enzyme (trypsin, Pronase B, proteinase K) did not remove or destroy the antigen, suggesting a non-protein nature of the epitope.
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PMID:A cell surface/plasma membrane antigen of Candida albicans. 170 79

Our previous studies have demonstrated the production and release of a tumor-derived factor that promoted lipolysis in normal adipocytes. We further demonstrated that this in vitro lipolysis was correlated with the in vivo loss of total carcass lipids induced by the presence of the same tumor. This study identified and isolated this "lipolysis-promoting" factor (LPF), released into the extracellular environment (conditioned media) by the human A375 melanoma cell line, which appears to be responsible for the previously demonstrated induction of in vitro and in vivo lipolytic activity. Unlike previously described non-tumor-derived molecules, such as tumor necrosis factor-alpha/cachectin, which have been implicated in cancer cachexia, the LPF induces alterations in lipid metabolism similar to those observed in cancer patients. The biochemical nature of human tumor-derived LPF appears to be a heat-stable molecule with an apparent molecular weight of approximately 6000. The lipolysis-promoting activity was trichloroacetic acid precipitable, but not precipitable with protamine sulfate or extractable with chloroform:methanol. Its activity appears to be resistant to enzymatic treatments with protease K, trypsin, Pronase, RNase, and DNase, as well as to periodate oxidation. Immunochemically, LPF appears to be distinct from tumor necrosis factor-alpha/cachectin. Furthermore, in contrast to the mechanism of action of tumor necrosis factor-alpha/cachectin, the mechanism of "lipolysis promotion" by LPF appears to be by the induction of cellular lipase activity.
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PMID:Identification of a human tumor-derived lipolysis-promoting factor. 173 44

The authors report on the extensive characterization, on normal and pathologic tissues, of the T-cell-specific monoclonal antibody (MoAb) A6, which the authors previously found to identify a fixation- and paraffin-embedding-resistant epitope. A6 reacted with most T lymphocytes, macrophages, and Langerhans' cells of normal tissues and with peripheral T-cell lymphomas (31 of 34), Ki-1+ lymphomas (12 of 18), and T-cell leukemias (1 of 5). All cases of X and non-X histiocytosis examined and monocytic leukemias with mature phenotype only were A6 positive. Three of 47 cases of B-cell lymphoma and leukemia were labeled. Hairy cell leukemias, multiple myelomas, and Hodgkin's and Reed-Sternberg cells were negative. The A6 reactivity was preserved with different fixatives (formalin, Bouin's fluid, Carnoy's fixative, and B5) and decalcification procedures and was slightly enhanced by trypsin digestion. The pattern of reactivity of A6 was similar to that obtained with MoAb UCHL-1, recognizing the CD45RO determinant of leukocyte common antigen; however, in pathologic tissues, A6 labeled a higher percentage of cells than UCHL-1. Cross-blocking and enzyme digestion studies (Pronase E [Sigma Chemical, St. Louis, MO] and neuraminidase [Sigma Chemical]) indicated that the two MoAbs may identify close epitopes on the same molecule. In conclusion, the authors' study indicates that A6 is an excellent reagent for detection of the CD45RO molecule on paraffin-embedded normal and pathologic tissues.
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PMID:A6--a new 45RO monoclonal antibody for immunostaining of paraffin-embedded tissues. 182 47

The purposes of this study were to examine the immunogenicity of the low molecular weight human salivary mucin (MG2) and determine its distribution within major and minor human salivary glands. Anti-MG2 sera were produced in Balb/c mice by a variety of immunization schedules. Chromatographically or electrophoretically purified MG2 and partially purified mucin chromatographic fractions exposed to mild denaturing conditions were not immunogenic. Only MG2 without prior exposure to urea or guanidine was able to elicit an immune response. A murine anti-MG2 monoclonal antibody (clone 1/F9) was produced and its monospecificity confirmed by immuno-dot blotting and SDS-PAGE Western transfer. Clone 1/F9 (IgG1; kappa) was of moderate affinity and was directed to a Pronase- and TPCK trypsin-sensitive but periodate-resistant epitope which was not blood group- or sialic acid-specific. Immunocytochemical studies of frozen tissue sections with clone 1/F9 using both indirect and direct methods revealed that MG2 was more heterogeneously distributed within submandibular than labial glands and was not found in parotid or palatine glands. The use of a polyclonal rabbit anti-MG2 reagent in either frozen or paraffin-embedded tissues gave the same immunocytochemical results as those obtained with the monoclonal antibody.
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PMID:Immunochemistry and immunogenicity of low molecular weight human salivary mucin. 187 31

The overproduction, purification, and determination of the active-site catalytic nucleophile of the DNA (cytosine-5)-methyltransferase (DCMtase) enzyme M.HaeIII are reported. Incubation of purified M.HaeIII with an oligodeoxynucleotide specifically modified with the mechanism-based inhibitor 5-fluoro-2'-deoxycytidine [Osterman, D. G., et al. (1988) Biochemistry 27, 5204-5210], in the presence of the cofactor S-adenosyl-L-methionine (AdoMet), resulted in the formation of a covalent DNA-M.HaeIII complex, which was purified to homogeneity. Characterization of the intact complex showed it to consist of one molecule of the FdC-containing duplex oligonucleotide, one molecule of M.HaeIII, and one methyl group derived from AdoMet. Exhaustive proteolysis, reduction, and alkylation of the DNA-M.HaeIII complex led to the isolation of two DNA-bound peptides--one each from treatment with Pronase or trypsin--which were subjected to peptide sequencing in order to identify the DNA attachment site. Both peptides were derived from the region of M.HaeIII containing a Pro-Cys sequence that is conserved in all known DCMtases. At the position of this conserved Cys residue (Cys71), in the sequence of each peptide, was found an unidentified amino acid residue; all other amino acid residues were in accord with the known sequence. It is thus concluded that Cys71 of M.HaeIII forms a covalent bond to DNA during catalytic methyl transfer. This finding represents a direct experimental verification for the hypothesis that the conserved Cys residue of DCMtases is the catalytic nucleophile [Wu, J. C., & Santi, D. V. (1987) J. Biol. Chem. 262, 4778-4786].(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Direct identification of the active-site nucleophile in a DNA (cytosine-5)-methyltransferase. 193 26

Rat lymph chylomicrons and chylomicron remnants were treated with trypsin or Pronase. The ability of the resulting apoprotein-free lipoproteins to be taken up by the isolated perfused rat liver, and to bind to isolated rat liver membranes, was examined. Compared with control lipoproteins, the apoprotein-free chylomicrons and remnants retained unaltered their capacity to be differentiated by the intact liver and by the isolated membranes. Further, control remnants and apoprotein-free remnants competed for binding to the isolated membranes. We conclude that apoproteins are not required for the hepatic differentiation between chylomicrons and remnants, and suggest that the lipoprotein phospholipids may play a direct role in this process.
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PMID:Apoprotein-independent binding of chylomicron remnants to rat liver membranes. 195 70

Cell surface glycans are believed to play a role in tumour invasion and metastasis. Yet, we have previously shown that the inhibitors of N-linked glycan processing swainsonine (SW) and 1-deoxynojirimycin (dNM) did not prevent invasion of chick heart fragments by MO4 murine fibrosarcoma cells in organ culture. We now present biochemical evidence that these and other inhibitors of processing were indeed effective in remodeling glycans, including those expressed at the cell surface. After metabolic labeling with tritiated mannose or fucose, glycosylpeptides were obtained by Pronase treatment of material released from intact cells by trypsin. Glycosylpeptides were separated by Biogel P-10 chromatography. With all drugs tested, there was a shift towards lower molecular weight of the glycan chains. There were, however, major quantitative differences between the different drugs and also, for monensin (MON; 0.1 microgram ml-1), between fucose-labeled and mannose-labeled chains. The shift in apparent molecular weight affected mainly fucose-labeled peptides after treatment of MO4 cells with SW (0.4 microgram ml-1). The shift induced by dNM (10 mM) + SW (0.4 microgram ml-1) in both fucosylated and mannosylated chains was much larger than that induced by SW given alone. 1-Deoxymannojirimycin (dMM; 1 mM) had major effects on both mannose and fucose-labeled structures and so did N-methyl-1-deoxynojirimycin (MdNM; 2 mM) and castanospermine (CS; 100 micrograms ml-1). With the latter drugs, incorporation of fucose in complex-type glycosylpeptides was dramatically reduced. The effect of SW on fucose-labeled glycosylpeptides of embryonic chick heart was similar to that observed on MO4 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of glycosylation inhibitors on N-glycosylpeptides and on invasion of malignant mouse MO4 cells in vitro. 214 90

A major problem encountered when isolating human microvascular endothelium is the presence of contaminating cells such as fibroblasts that rapidly over-grow the endothelial cells. We describe here a simple, rapid technique for purifying endothelial cells derived from the microvasculature of neonatal foreskin and osteoarthritic and rheumatoid arthritic synovium. This technique is based on the selective binding of the lectin Ulex europaeus I (UEA I) to the endothelial cell surface via fucose residues. Initially UEA I was covalently bound to tosyl-activated super-paramagnetic polystyrene beads (Dynabeads) by incubation for 24 h at room temperature. Cells were isolated by extracting microvascular segments from enzyme-treated (trypsin and Pronase) cubes of tissue. The mixed population of cells obtained were purified by incubating them at 4 degrees C for 10 min with the UEA I-coated Dynabeads. Endothelium bound to the beads whilst contaminating cells were removed by five washes with HBSS using a magnetic particle concentrator. The endothelial cells thus obtained grew to confluence as a cobblestone-like monolayer and expressed von Willebrand factor antigen. The cells were released from the Dynabeads by the competitive binding of fucose (10 min at 4 degrees C). This new method is simple and reproducible and allows pure human microvascular endothelial cells to be cultured within 2 h of obtaining a specimen.
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PMID:Binding of human endothelium to Ulex europaeus I-coated Dynabeads: application to the isolation of microvascular endothelium. 221 66

1. An anti-N lectin was extracted from Vicia unijuga leaves with phosphate-buffered saline (PBS). Purification of the lectin was achieved, after pretreatment of the PBS extract by ammonium sulfate fractionation and absorption with human M erythrocytes, by using a combination of conventional chromatographic techniques with asialoglycophorin AN-Sepharose CL-4B affinity chromatography. Purification steps were followed by increase of specific activity. 2. Homogeneity of the purified lectin was demonstrated by HPLC and SDS-PAGE. The purified lectin was a glycoprotein with 11.4% carbohydrate and relatively high percentages of serine, threonine and aspartic acid residues and had a Mw of 120,000 Da. 3. This lectin agglutinated human N and MN erythrocytes, but did not agglutinate M erythrocytes. Hemagglutination of the lectin was inhibited by glycophorin AN and N-active sialoglycopeptide released from human N erythrocytes by treatment with Pronase or trypsin. However, it was not inhibited by any of mono- and di-saccharides, ABH-active glycoproteins, glycophorin AM and M-active sialoglycopeptide liberated from human M erythrocytes by treatment with Pronase or trypsin.
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PMID:Purification and characterization of anti-N lectin from Vicia unijuga leaves. 232 20


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