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)

Primary and secondary cultures of rhesus monkey kidney cells supported multiple-cycle replication of Sendai virus, but later passages lost this ability, and this was reflected in decreased plaque formation. Multiple-cycle replication also did not occur in LLC-MK2 cells, a continuous line of RMK cells. Failure of replication in serially passed cells was correlated with a decrease in proteolytic cleavage of a viral surface glycoprotein (Fo), and the ability of cells to support multiple-cycle replication and plaque formation could be restored by the addition of trypsin (0.3 microgram/ml) to the overlay medium. The use of wild-type virus, which requires trypsin, and protease activation mutants that require chymotrypsin or elastase for activation has provided evidence that the activating protease supplied by primary or secondary cells has trypsin-like activity. Inactive virus, with uncleaved Fo glycoprotein, absorbed to primary or secondary cells but did not infect them, even though such cells possess the enzyme that is capable of cleaving the Fo glycoprotein of virus synthesized in these cells. The inability of these cells to activate adsorbed virus indicates that the activating protease that they possess is inacessible to adsorbed virus, although it can act on the Fo glycoprotein during virus maturation in these cells. These data provide a biochemical explanation for the failure of later passages of a cell strain or a continuous cell line to support the replication of a paramyxovirus.
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PMID:Loss on serial passage of rhesus monkey kidney cells of proteolytic activity required for Sendai virus activation. 20 71

Titers of bovine rotavirus in excess of 10(9) immunofluorescent infectious units per ml of culture fluids have been produced, using trypsin treatment of the virus. Infectivity of preparations of the virus can be increased with as little as 1 ng of trypsin per ml, with maximum increases of 1 to 2 log10 with 1 microgram of trypsin per ml. The virus grows to titers in excess of 10(5) immunofluorescent units per ml in MDBK, LLC-MK2, MA-104, and HeLa cells. When MDBK cells are infected with a multiplicity of infection of 20, maximum yields of cell-associated, trypsin-enhanceable virus are obtained 4 to 8 h postinfection. Maximum yields of cell-free, trypsin-enhanceable virus are produced 16 to 20 h postinfection. The results presented here indicate that trypsin can be used to produce high-titer stocks of bovine rotavirus.
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PMID:Production of high-titer bovine rotavirus with trypsin. 22 1

A sensitive, quantitative and reproducible plaque assay for the measurement of the simian rotavirus SAII is described. Plaque formation required the presence of the facilitators pancreatin or trypsin and diethylaminoethyl-dextran in the agar overlay. SAII produced plaques in three continuous primate cell lines: MA-104, CV-1 and LLC-MK2. MA-104 cells were the most sensitive.
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PMID:A plaque assay for the simian rotavirus SAII. 22 32

A solid-phase radioimmunoassay (RIA) has been developed for the detection of human rotavirus-specific IgA, IgG, and IgM antibodies. Nebraska calf diarrhea virus grown in LLC-MK2 cell cultures in the presence of trypsin was directly adsorbed onto polystyrene balls, and antibodies that attached to the virus-coated balls were detected by subsequent binding of 125I-labeled antibodies specific to human alpha, gamma or mu chains of human Iga, IgG, or IgM immunoglobulins. A total of 116 serum specimens from 58 adult patients were tested. Binding ratios between the positive and the negative serum varied between 5 and 15, occasionally being 20 or more in the IgA and IgG assays, but rarely exceeding 3 in the IgM assay. The RIA was found to be more sensitive in detecting antibodies to rotavirus than the complement fixation (CF) test, the RIA titers obtained being 50--100 times as high as the CF titers. The method described offers a possibility of evaluating the immune response to human rotavirus and of detecting recent infection.
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PMID:Solid-phase radioimmunoassay of IgA, IgG, and IgM antibodies to human rotavirus. 22 43

Primary rhesus monkey kidney (MK) cells have long been the cells of choice for isolation and propagation of the human paramyxoviruses (parainfluenza 1, 2, 3, 4A, 4B, and mumps). However, problems with the supply and cost of MK cells and the presence of endogenous viruses, including herpes B virus and SV-5, necessitated a search for an alternative cell line. Continuous cell cultures of human origin (L132, A-549, HuT-292, HEK, G-293, G-401, A-498, A-704, CAKI-1, RD) and simian origin (LLC-MK2, BSC-1, MA-104, Vero) were evaluated for their capacity to support the growth of the human paramyxoviruses, as followed by cytopathic effect, hemadsorption, hemagglutination, and EIA. NCI-H292 (HuT-292) human lung mucoepidermoid carcinoma cells (ATCC # CRL-1848) proved to be the most sensitive line for cultivating all serotypes and strains of the paramyxoviruses. These cells were also shown to be a suitable substitute for MK in primary isolation of paramyxoviruses from clinical specimens. RPMI-1640 with 1.5 micrograms/ml trypsin was the preferred maintenance medium; alternatively, Eagle's MEM supplemented with 1.5 micrograms/ml trypsin and 0.1% ITS was satisfactory. NCI-H292 cells are a continuous line with excellent growth characteristics, although the genetic polyploidy of the cells may limit the number of passages of usable cells.
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PMID:NCI-H292 as an alternative cell line for the isolation and propagation of the human paramyxoviruses. 226 Sep 24

Eight cytopathic human rotavirus strains were isolated in LLC-MK2 cells and in human embryo fibroblasts. The strains were isolated from faeces collected from pediatric and adult patients. Pretreatment of specimens with trypsin and trypsin incorporation in maintenance medium were not performed. Inoculated monolayers were not subjected to centrifugation and were incubated stationary at 36 degrees C. Viruses were identified by electron microscopy and by fluorescent antibody techniques. It is suggested that these rotaviruses are different from any previously recovered from man.
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PMID:Isolation from faecal specimens of new strains of human rotavirus primarily cytopathic for stationary cell cultures without trypsin. 298 29

1,25-Dihydroxyvitamin D3 receptors are cytosoluble proteins detectable in a variety of tissues responsive to 1,25(OH)2D3. They are DNA binding-proteins analogous to other steroid receptors and it is this functional property which is likely involved in the activation of hormone-sensitive genes. Utilizing 1,25(OH)2D3 and DNA binding assays, as well as anti-receptor monoclonal antibodies, we have probed the relationship between the 1,25(OH)2D3 receptor binding domains after selective cleavage with trypsin. These studies reveal that the hormone and DNA binding regions are separable, and are consistent with the finding that tissue resistance to 1,25(OH)2D3 is a result of structural defects in these domains. Recently, a primate model, the LLC-MK2 monkey kidney line, has been uncovered which may exemplify a hormone-binding defect. Here, 25-hydroxyvitamin D3-24-hydroxylase induction, a 1,25(OH)2D3 bioresponse, requires 100-fold higher concentrations of the hormone for maximal response. Concomitantly, this cell contains a variant receptor form which displays a correspondingly lowered apparent affinity for the hormone despite its seemingly normal DNA binding characteristics. Taken together, these studies suggest that the 1,25(OH)2D3 receptor is a macromolecule with multiple domains each of which may produce modified cellular resistance to 1,25(OH)2D3 if structurally altered.
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PMID:1,25-Dihydroxyvitamin D3 receptors: altered functional domains are associated with cellular resistance to vitamin D3. 301 80

In developing countries it is often impractical to use conventional methods to isolate and identify influenza viruses. The use of trypsin-treated LLC-MK2 cells for the isolation of myxoviruses, in conjunction with the indirect fluorescent antibody technique for identification of isolates and for direct detection of viral antigens in specimens, was an effective combination of techniques which enabled our laboratory in Papua New Guinea to participate in an influenza surveillance programme. The application of these techniques in routine respiratory virus surveillance and in the investigation of an outbreak of influenza-like illness is described.
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PMID:Influenza surveillance: alternative laboratory techniques for a developing country. 387 37

Human rotaviruses were capable of efficient multiplication in LLC-MK2 cells when the inoculum was pre-treated with trypsin, centrifuged on to the cell monolayer and the infected cells maintained in a medium containing trypsin. However, not all of the human rotavirus isolates used to infect cells resulted in efficient virus production. The ability of human isolates to multiply in cultured cells was studied by direct observation of virus in the electron microscope, by radioactive labelling with 3H-uridine of the newly synthesized virus and by electron microscopic examination of thin sectioned infected cells. With one of the specimens used (F-617) only 5 to 10% of the cells showed evidence of virus multiplication, with the great majority of the infected cells showing numerous complete (double-capsid) virus particles scattered in the cytoplasm. When cells were inoculated with another human specimen (SIB-I), infected cells were more abundant, reaching a maximum of 60%; however, a variety of particle types, probably representing different subviral structures or different steps of rotavirus morphogenesis, were commonly observed. The presence of these aberrant or incomplete virus structures may represent a manifestation of the defectiveness of this virus and may explain the difficulties encountered in its serial passage.
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PMID:Multiplication of human rotavirus in cultured cells: an electron microscopic study. 624 81

A trypsin-like protease which is responsible for activation of Sendai virus was found in the chorioallantoic fluid (CAF) of embryonated chicken eggs. Treatment of the inactive form of Sendai virus, grown in LLC-MK2 cells, with CAF enhanced both hemolytic activity and infectivity for the cells. Soybean trypsin inhibitor restrained the enhancing activity of CAF. These results indicate that CAF contains a trypsin-like protease which activates the inactive form of Sendai virus. The activation was strongly inhibited by phenylmethylsulfonylfluoride, ethylenediaminetetraacetate, antipain, and leupeptin but not by tosyllysylchloromethylketone, suggesting that the activating enzyme in CAF is a protease similar to but not identical with trypsin. The inactive form of the virion was produced in ovo when the seed virus was inoculated along with antipain or leupeptin. In deembryonated chicken eggs in which CAF was substituted for a culture medium, multiple cycle growth occurred, but not when soybean trypsin inhibitor was present. These observations indicate that some activating enzyme, possibly the same one as found in CAF, was secreted from the chorioallantoic membrane.
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PMID:Trypsin action on the growth of Sendai virus in tissue culture cells. V. An activating enzyme for Sendai virus in the chorioallantoic fluid of the embryonated chicken egg. 624 22


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