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)

The ability of specific gangliosides to function as host cell receptors for Sendai virus was investigated by using Madin-Darby bovine kidney cells which become resistant to infection upon treatment with Vibrio cholerae sialidase. Sialidase-treated cells were incubated for 20 min at 37 degrees C with individual, highly purified gangliosides containing homogeneous carbohydrate moieties and then inoculated with virus for 10 min. Susceptibility of the cells to infection was monitored by hemagglutination titer of the virus produced 48 hr after inoculation. Incubation of the cells with gangliosides containing the sequence NeuAc alpha 2,3Gal beta 1,3GalNAc (i.e., GD1a, GT1b, and GQ1b) fully restored susceptibility to infection to the cells. However, the ganglioside GQ1b in which the sequence ends with two sialic acids in a NeuAc alpha 2,8NeuAc linkage instead of a single sialic acid as in GD1a and GT1b, was effective as a receptor at a concentration 1/100th that of any of the other gangliosides tested. Incubation with gangliosides similar in structure to GD1a, GT1b, and GQ1b but lacking the sialic acid attached to the terminal galactose (i.e., GM1 and GD1b) had no effect. The results from control experiments in which gangliosides were incubated at 0 degrees C with cells or in which trypsin was used to remove gangliosides adsorbed to cells were consistent with the premise that the gangliosides must actually insert into the cellular membrane to function as Sendai virus receptors. Addition of 4 X 10(6) molecules of 14C-labeled GD1a per cell made the cells fully susceptible to infection. Analysis of the ganglioside content of cell membranes showed that gangliosides GD1a, GT1b, and GQ1b are natural components of these cells and are present in quantities sufficient to act as receptors. These results demonstrate that gangliosides with the proper carbohydrate sequence, such as GD1a, GT1b, and GQ1b, function as natural receptors for Sendai virus in host cells.
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PMID:Specific gangliosides function as host cell receptors for Sendai virus. 627

The uptake of ganglioside analogues by a permanent mouse fibroblast cell line has been studied by radio-tracer techniques and ESR spectroscopy with 3H- and nitroxide-labeled compounds. Analogues of GM1, GM2, and GM3 monosialogangliosides and of GD1a and GD3 disialogangliosides were synthesized. The spin-label group was situated on the 5-, 9-, or 13-carbon atom of the C18 fatty acid chain, and the 3H label was in the carbohydrate moiety. Part of the ganglioside associated with the cells could be removed by trypsin treatment and was shown to consist of ganglioside micelles attached to the cell surface. The trypsin-resistant component displayed characteristic anisotropic ESR spectra which closely resembled those of the same spin-labeled analogues at low dilution in liposomes prepared from the extracted cell lipids. The flexibility gradient, polarity profile, and temperature dependence displayed by the spectra were similar to those found for fluid phospholipid bilayer model membranes, and the high effective order parameters suggested a location in the cell plasma membrane. Similar results were obtained for all the different ganglioside analogues, indicating a common anchoring region in the hydrophobic interior of the membrane. Under the incubation conditions used the amount of trypsin-resistant ganglioside analogue taken up by the cells was about 15 nmol/mg of cellular protein, irrespective of the nature of the oligosaccharide moiety. By use of the natural ganglioside [3H]GM3, the trypsin-resistant uptake was about 19 nmol/mg of cellular protein. Although these amounts are quite similar, the uptake kinetics differed between the true ganglioside GM3 and the ganglioside analogues.
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PMID:Incorporation of ganglioside analogues into fibroblast cell membranes. A spin-label study. 631 58

GM1 ganglioside has been localized on the surfaces of myelinated, peripheral nerve fibres by using immunofluorescence to detect cholera toxin receptors. Unfixed, mouse sciatic nerves were teased into individual, intact fibres in order to expose their extracellular surfaces. Cholera toxin binding sites were abundant at all nodes of Ranvier; they were scarce on the internodal fibre surfaces. The nodal receptors were resistant to various degradative enzymes, including trypsin and proteinase K. Proteases did not unmask receptors on the internodal surfaces. Exogenous GM1 successfully competed for the toxin binding sites on the fibres. From this evidence and the specificity of cholera toxin binding, we conclude that GM1 ganglioside is abundantly present on the membrane surfaces of peripheral nodes of Ranvier and is not present on the internodal Schwann cell surfaces in an appreciable amount. The patterns of fluorescence within the node suggest that the axon and Schwann cell structures are sites where GM1 is localized. Treatment of the teased fibres with Vibrio cholerae neuraminidase, which is known to reduce polysialogangliosides to the monosialoganglioside GM1, induced cholera toxin binding on the internodal Schwann cell surfaces. The induced receptors, as well as their precursors, were resistant to trypsin and proteinase K. We conclude that the internodal Schwann cell surface is rich in an unidentified polysialoganglioside(s) that can be converted to GM1 by neuraminidase.
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PMID:Ganglioside localization on myelinated nerve fibres by cholera toxin binding. 636 31

One of the earliest events in the adhesion of fibroblasts to a substratum is the recognition by the cells of macromolecular adhesive factors, such as fibronectin. This early event is followed by a complex series of cell alterations leading to adhesion and spreading. To identify cell surface components involved in the initial cell-fibronectin recognition step, we have employed an assay involving latex particles coated with radiolabelled plasma Fibronectin (Fn). In previous studies from this laboratory (Harper & Juliano , J cell biol 87 (1980) 755) [28], we demonstrated that Fn-mediated adhesion of CHO cells is temperature-dependent, cation-dependent and sensitive to cytoskeletal disrupting agents; by contrast, binding of 3H-Fn beads was unaffected by these factors, indicating that this process reflects binding and recognition events at the cell surface which are independent of cytoskeletal and metabolic activity. Biological specificity of 3H-Fn bead-to-cell binding was confirmed by the ability of anti-Fn antisera to completely block the process. To examine surface components which may mediate binding we treated Fn beads with purified glycosaminoglycans (GAGs) or glycolipids prior to incubation with cells. Among the GAGs tested, heparin, heparan sulfate and dermatan sulfate blocked bead binding in a dose-related fashion with heparin being most potent. The gangliosides GT1, and GM1, also inhibited bead binding. However, treatment of cells with neuraminidase had no effect on bead binding while subsequent analysis of treated cells by thin layer chromatography revealed a drastic reduction in the amount of GM3, the predominant CHO cell ganglioside. CHO cells were also incubated with a panel of proteolytic enzymes to study the possible role of cell surface proteins or glycoproteins in Fn bead binding. We found 3H-Fn bead binding to be quite sensitive to pretreatment with thermolysin, pronase, and papain but only moderately sensitive to treatment with trypsin. From our findings we suggest: (1) binding of Fn beads to CHO cells reflects an early step in the adhesion process; (2) glycolipids may block bead binding but are probably not the endogenous binding site for Fn; (3) protease sensitive components (glycoproteins or proteoglycans) may be more likely candidates as cell surface-binding sites for Fn.
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PMID:Interaction of fibronectin-coated beads with CHO cells. 637 25

The natural cytotoxic activity of yolk sac (YS) cells from 10-day mouse embryos against YAC-1 tumor targets was characterized for its sensitivity to modulation by a variety of factors. Experiments demonstrated the observed levels of tumor cell lysis to be unaffected by the strain of origin of the YS cells, trypsin pretreatment of the YS population, as well as a prior stimulation by environmental pathogens. Both the YS natural cytotoxic (YSNC) activity and the adult natural killer (NK) cell activity were found to be depressed when assayed in the presence of amniotic fluid. YS cells were tested for antibody-dependent cell-mediated cytotoxic activity, but were not found to generate significant levels of lysis. Experiments presented here demonstrate the YSNC activity to be dependent upon only a subpopulation of YS cells. The YSNC activity was found to be associated only with the cells exhibiting high forward angle light scatter as determined by flow cytometry. Additionally the activity was unaffected by the removal of plastic-adherent cells from the effector population. In this study, YSNC cells are also examined for the presence of a variety of lymphocyte antigens. The effector cells, as well as the total YS population, were found to lack Lyt-1, Lyt-2, Thy-1, Ly-5, NK-1, Qa-5, asialo GM1, and H-2 antigens. While these experiments demonstrate the YSNC cells to be distinct from the NK cells of the adult, they do not rule out a potential common lineage for the two natural cytotoxic cell types.
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PMID:Natural cytotoxicity in the mouse embryo: characterization of yolk sac-associated natural cytotoxic cells and their activity. 661 32

A nonganglioside factor(s) present in Sigma types II and III mixed bovine brain ganglioside preparations synergises with suboptimal amounts of serum to induce proliferation specifically in nondividing B 103 neuroblastoma cultures. The active substance is nondialysable and soluble in water as well as in chloroform-methanol mixtures of 1:1-4:1 (vol/vol). It is completely insoluble in ether and acetone at room temperature. Biological activity survives heating to 70 degrees C in the presence of 0.1 M HCl for 1 h as well as boiling at neutral pH. Loss of activity occurs on heating to 70 degrees C for 1 h with 1 M HCl or 1 M NaOH. The activity is insensitive to digestion with neuraminidase, trypsin, pronase, and phospholipases A2 and C. The factor cochromatographs with gangliosides on Dowex AG 50W and Sephadex G100 and is partially recovered with GM1 on DEAE-Sepharose, but may be isolated in a ganglioside-free fraction by sequential chromatography on Sephadex LH20 and silicic acid columns. The substance(s) has the properties of a water-soluble proteolipid protein, the amino acid composition being reported. It is not immunologically cross-reactive with antibodies to GM1 ganglioside or the major proteolipid protein of myelin.
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PMID:Characterization and partial purification of a ganglioside-associated mitogen. 661 41

Ganglioside GM1 promoted neuritogenesis of neuroblastoma cells, neuro-2a clone, in monolayer culture. GM1 bound to neuro-2a cells in three distinct forms, one removable by treatment with serum-containing solutions, one serum-resistant and labile to trypsin treatment, and one resistant to serum and trypsin treatments. The proportions among the three forms of cell-associated GM1 varied in relation to duration of exposure to ganglioside, ganglioside concentration in the medium, and number of cells in culture. The form removable by serum was predominant at the initial stages of association and at the highest ganglioside concentrations (over 10(-6)M); the trypsin-labile and -stable forms tended to increase with increasing cell number and decreasing ganglioside concentration. The neuritogenic effect of GM1 was higher when neuro-2a cells were incubated for 24 h in the presence of GM1 and fetal calf serum. Under this condition the percentage of neurite-bearing cells increased from 11% of control to 62% at the optimal ganglioside concentration of 10-4M. The effect was still present, although to a lower extent (from 11% to 28% of neurite-bearing cells), when cells were first exposed for only 2 h to GM1, then washed and incubated for 24 h in the presence of fetal calf serum. The trypsin-labile and -stable forms of cell-associated GM1 had a fundamental role in the effect, whereas the form removable by serum was not involved. The preparation of GM1 used was extremely pure (99%) and, in particular, had a peptide contamination, if any, less than 1:20,000-1:50,000.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Promotion of neuritogenesis in mouse neuroblastoma cells by exogenous gangliosides. Relationship between the effect and the cell association of ganglioside GM1. 669 71

Somatic neurohybrid SB21B1 cells grown in serum exhibit limited capacity to bind 125I-labeled tetanus toxin and cannot synthesize gangliosides higher than GM2. By 6 h after supplementing the culture medium with pure or mixtures of brain gangliosides, binding of 125I-labeled tetanus toxin to cells increases approximately 8-fold compared to that of nonsupplemented cells. The uptake of added gangliosides is a saturable process and is facilitated by serum removal (2.1-fold) or substitution of growth factors for serum (3.8-fold). Enhancement of tetanus toxin binding to cells depends on the ganglioside species and concentration; GT1b (25 micrograms/ml) is, respectively, two and three times as effective as GD1b and GM1 in increasing toxin binding. Reconstitution of ganglioside-mediated tetanus toxin binding activity is a reversible phenomenon; removal of medium gangliosides causes a 3-fold drop in toxin binding by 24 h, after which an apparent plateau for at least 3 days above the basal level is established. As in cerebral cultures, binding of toxin to ganglioside-supplemented neurohybrid cells exhibits salt and sialidase sensitivity and is enhanced 2.6-fold at 37 degrees C compared to 0-4 degrees C. The resultant temperature-dependent toxin-cell association is sialidase insensitive. Fixation of cells by formaldehyde or treatment of ganglioside-supplemented cells with trypsin has no substantial effect on ganglioside-mediated binding of the toxin. Methanol/chloroform treatment of cells causes a 91.4% loss of binding activity.
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PMID:Gangliosides mediate association of tetanus toxin with neural cells in culture. 671 26

Extensive immunohistochemical and thin-layer chromatogram-immunostain analyses were carried out to establish whether asialo GM1, a glycolipid which contains binding sites for Pseudomonas aeruginosa, is present in corneal epithelium. The data suggest that rabbit corneal epithelium does not contain detectable levels of asialo GM1 even after corneas are scarified and incubated with trypsin, P. aeruginosa, or P. aeruginosa exoproducts to expose potential cryptic sites. Preliminary immunohistochemical analyses indicated that asialo GM1 is also not found in human corneas.
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PMID:Pseudomonas aeruginosa infection of the cornea and asialo GM1. 764 20

The effects of exogenous ganglioside GM1 (1 microM) from bovine brain on the morphological state and biochemical parameters (creatine kinase, acetylcholinesterase and adenylate cyclase activities as well as the protein, phospholipid and ganglioside content) have been studied in primary cultures of trypsin-treated dissociated cells of chicken embryonic brain. Ganglioside GM1 accelerated the growth and differentiation of cultured cells, increased the phospho- and glycolipid content and stimulated the activity of the enzymes.
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PMID:[Modification of biochemical changes in developing cultures of chick embryo nerve tissue cultures]. 781 90


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