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)

A wide variety of influenza A viruses, comprising human, equine, porcine, and avian strains, grew productively in an established line of canine kidney cells (MDCK) under an overlay medium containing trypsin, and formed well-defined plaques regardless of their prior passage history. Plaquing efficiency was comparable to the efficiency of infection in fertile eggs via allantoic route. MDCK cells have also been successfully employed for the primary isolation of influenza A virus from throat washings of patients. Parallel titration of several clinical specimens showed that the inoculation into MDCK cells followed by incubation in the presence of trypsin was an isolation procedure as sensitive as the amniotic inoculation into fertile eggs.
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PMID:Plaque assay and primary isolation of influenza A viruses in an established line of canine kidney cells (MDCK) in the presence of trypsin. 121 9

A new antigenic variant of swine influenza virus was isolated from the lungs of pigs experiencing respiratory problems in 7 different swine herds in Quebec. Pigs of different ages were affected, and the main clinical signs were fever, dyspnea, and abdominal respiration. Coughing was not a constant finding of the syndrome. At necropsy, macroscopic lesions included the overall appearance of pale animals, general lymphadenopathy, hepatic congestion, and consolidation of the lungs. Histopathologic findings were mainly proliferative pneumonia with a significant macrophage invasion, necrotic inflammatory cells in the alveoli and the airways, a marked proliferation of type II pneumocytes, and thickening of the alveolar septae. Fluorescent antibody examination of lungs of sick piglets did not demonstrate porcine parvovirus, transmissible gastroenteritis virus, or encephalomyocarditis virus. However, evidence of the presence of an influenza type A infection was demonstrated by indirect immunofluorescence (IIF) staining using monoclonal antibody directed to nucleocapsid protein (NP) of human type A influenza virus. The virus was isolated either by intra-allantoic inoculation of specific-pathogen-free embryonating hens' eggs or propagation in canine kidney (MDCK) cells in the presence of trypsin. By hemagglutination inhibition tests, no cross-reactivity was demonstrated with human influenza H1N1, H2N2, and H3N2 strains, and infected MDCK cells did not react by IIF with monoclonal antibodies to NP protein of type B influenza virus. The hemagglutination activity of plaque-purified isolates was only partly inhibited by hyperimmune serum produced to subtypes A/Wisconsin/76/H1N1 and A/New Jersey/76/H1N1 of swine influenza virus. Gnotobiotic piglets that were infected intranasally with egg-adapted isolates of this new antigenic variant of swine influenza virus developed the very same type of lesions observed in field cases.
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PMID:Antigenic variant of swine influenza virus causing proliferative and necrotizing pneumonia in pigs. 133 15

The low pH-induced fusion of influenza virus with intact erythrocyte plasma membranes is preceded by a delay time following pH reduction, that is itself pH- and temperature dependent. At 37 degrees C/pH 4.8, lipid mixing between virus and target membranes begins < 2 s after pH reduction, whereas at 4 degrees C/pH 4.8, fusion does not commence until > 10 min after pH reduction. We have found that within this time period at 4 degrees C, a population of virus acquires the capacity to subsequently undergo fusion with high efficiency at elevated temperatures and pH 7.4. Both the kinetics and the extent of this pH 7.4 fusion depend upon the time of pre-incubation at pH 4.8/4 degrees C. Incubation at pH 7.4/4 degrees C, following this pre-incubation does not result in fusion, but the capacity to fuse at pH 7.4/37 degrees C is retained for a time period exceeding 1 h. The longevity of this fusion committed state makes it amenable to biochemical and immunological analysis. We have shown that it is insensitive to dithiothreitol, neuraminidase and trypsin, but is incapacitated by thermolysin or protease K. We conclude that only the HA2 sub-unit of influenza haemagglutinin is a necessary protein component of later stages of the fusion pathway.
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PMID:A long-lived state for influenza virus-erythrocyte complexes committed to fusion at neutral pH. 139 18

Different proteases from various microorganisms present in the respiratory tract were capable of enhancing influenza virus infectivity and pathogenicity in mice by proteolytic activation of hemagglutinin (HA). Aerococcus viridans, isolated from a patient with pneumonia, secreted a protease that could activate HA directly, similarly to some Staphylococcus aureus strains. The protease of Pseudomonas aeruginosa could not activate HA directly, but combined application of P. aeruginosa protease and virus into mice enhanced virus titers and pathogenicity. Generation of trypsin-like activity in bronchoalveolar lavage fluids resulting from this combination treatment may be responsible for HA activation. A similar indirect effect on HA activation was induced by streptokinase and staphylokinase, which are known to generate plasmin by plasminogen activation. It was concluded that plasminogen-activating streptococci and staphylococci facilitate viral replication and pathogenicity of plasmin-sensitive influenza virus strains by amplification of the plasminogen/plasmin system.
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PMID:Interactions between bacteria and influenza A virus in the development of influenza pneumonia. 152 12

We investigated the possible involvement of oxidative mechanisms in the pathogenesis of influenza A/PR8/34 virus infection in mice. As a biochemical marker of oxidative stress, we determined the endogenous concentrations of the antioxidants glutathione and vitamins C and E in their reduced and oxidized forms in the lungs, liver and blood plasma of control and infected animals. Following intranasal infection with 8 to 10 LD50, influenza virus was detected in the lungs, but not in the plasma, liver or other organs. Infection resulted in a decrease in the total concentration of glutathione and vitamins C and E, whereas no relevant change in the ratio of oxidized to total concentration of antioxidants was observed. Changes in the concentration of hepatic antioxidants were significant in the early stages of the infection. The results suggest that hepatic alterations may be caused indirectly by mechanisms related to the host response to virus infection. The observed general decrease in the antioxidant buffering capacity may reduce the ability of tissues to protect against potential oxidative stress. Such stress can occur during bacterial superinfections, which are common in influenza, thereby rendering the host more susceptible to the pathogenic effects of such agents. In addition, reactive oxygen species produced in the lung may inactivate protease inhibitors, resulting in increased protease activity. Using an in vitro system consisting of alpha 1-antiprotease, trypsin and HOCl as the oxidant, we have shown that the infectivity of influenza viruses can be increased up to 10,000-fold by proteolytic cleavage of haemagglutinin, leading to activation of the fusogenic properties of this protein.
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PMID:Alterations in antioxidant defences in lung and liver of mice infected with influenza A virus. 153 Sep 63

Antibody-dependent enhancement (ADE) of influenza A NWS virus infection was investigated in primary murine macrophages (M phi) using anti-hemagglutinin(HA) monoclonal antibody (mAB). Contrary to previous reports of abortive influenza virus infection in primary M phi, this study demonstrated that the NWS virus replicated productively in both resident peritoneal M phi and thioglycolate-elicited peritoneal M phi providing cleavage of the HA was achieved by trypsin; 5 micrograms/ml of trypsin was the optimum concentration for the induction of infectivity. Under multiple-cycle growth conditions in the presence of mAB at various concentrations in trypsin-containing media, ADE was demonstrated in both M phi in the presence of subneutralizing concentrations of mAB. Flow cytometric analysis showed that the mechanism of virus entry into M phi could be through HA to specific virus receptors, or HA plus antibody to Fc receptors. These results indicate that ADE of the NWS virus infection actually occurs on Fc receptor-bearing primary murine M phi depending on the concentration of antibody in the presence of the appropriate protease for cleavage of viral HA.
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PMID:Infection enhancement of influenza A NWS virus in primary murine macrophages by anti-hemagglutinin monoclonal antibody. 156 51

A novel trypsin-like protease associated with rat bronchiolar epithelial Clara cells, named Tryptase Clara, was purified to homogeneity from rat lung by a series of standard chromatographic procedures. The enzyme has apparent molecular masses of 180 +/- 16 kDa on gel filtration and 30 +/- 1.5 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Its isoelectric point is pH 4.75. Studies with model peptide substrates showed that the enzyme preferentially recognizes a single arginine cleavage site, cleaving Boc-Gln-Ala-Arg-4-methylcoumaryl-7-amide most efficiently and having a pH optimum of 7.5 with this substrate. The enzyme is strongly inhibited by aprotinin, diisopropylfluorophosphate, antipain, leupeptin, and Kunitz-type soybean trypsin inhibitor, but inhibited only slightly by Bowman-Birk soybean trypsin inhibitor, benzamidine, and alpha 1-antitrypsin. Immunohistochemical studies indicated that the enzyme is located exclusively in the bronchiolar epithelial Clara cells and colocalized with surfactant. An immunoreactive protein with a molecular mass of 28.5 kDa was also detected in airway secretions by Western blotting analyses, suggesting that the 30-kDa protease in Clara cells is processed before or after its secretion. Proteolytic cleavage of the hemagglutinin of influenza virus is a prerequisite for the virus to become infectious. Tryptase Clara was shown to cleave the hemagglutinin and activate infectivity of influenza A virus in a dose-dependent way. These results suggest that the enzyme is a possible activator of inactive viral fusion glycoprotein in the respiratory tract and thus responsible for pneumopathogenicity of the virus.
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PMID:Isolation and characterization of a novel trypsin-like protease found in rat bronchiolar epithelial Clara cells. A possible activator of the viral fusion glycoprotein. 161 59

Using immunoelectron microscopy, the distribution of influenza A virus neuraminidase (NA) glycoproteins was examined, after performing immunoreactions to virions on the grid. With polyclonal antibody, the immunolabels of the glycoproteins were found to be homogeneously distributed, whereas with monoclonal antibody they were found to be distributed in clusters. After destruction of haemagglutinin (HA) but not of NA activity with a high concentration of trypsin, the remaining visible spikes were evenly distributed. This finding was consistent with the absence of immunolabelling with anti-HA antibody, and the homogeneous pattern of immunolabels with anti-NA polyclonal antibody, but not with the clustered labelling with the anti-NA monoclonal antibody. Thus, the immunolabelling image with anti-NA polyclonal antibody was considered to reflect the true one.
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PMID:Immunoelectron microscopy of influenza A virus neuraminidase glycoprotein topography. 164 38

Revertants were isolated from the protease activation mutant of Sendai virus, F1-R, which causes a systemic infection in mice. The fusion (F) glycoprotein of F1-R is susceptible to activation cleavage by ubiquitous cellular proteases and is thus responsible for pantropism in mice (Tashiro et al., 1988. Virology 165, 577-583). The revertants regained several phenotypes of wild-type virus; they required exogenous trypsin for activation of the F protein in cell cultures and in nonpulmonary mouse tissues and they were exclusively pneumotropic in mice. On the other hand, phenotypes of F1-R that remained unchanged by the revertants were bipolar budding in polarized epithelial cells, enhanced electrophoretic migration of the matrix protein, and the lack of a glycosylation site in the F2 subunit of the F protein. Comparative RNA sequence analysis of the F gene of the revertants revealed that the reduced cleavability of the F protein of the revertants was the result of the predicted single amino acid reversion (Pro to Ser) at residue 115 adjacent to the cleavage site. Thus the sequence at the cleavage site of the revertants was Ser-Lys compared with Pro-Lys for F1-R and Ser-Arg for wild-type virus. The results indicate that enhanced cleavability of the glycoprotein, a feature often associated with multiple basic residues within the cleavage site of paramyxovirus F proteins and influenza virus hemagglutinins, can also be determined by a single basic amino acid following proline. Additionally, the revertants were less susceptible to the activator for wild-type virus present in mouse lungs and less pathogenic for this organ than wild-type virus. These results provide further evidence that proteolytic activation of the F protein by host proteases is the primary determinant for organ tropism and pathogenicity of Sendai virus in mice. One of the revertants was also temperature sensitive (ts); the ts lesion in the nucleoprotein gene was identical to that found in ts-f1, the ts host range mutant from which F1-R was derived.
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PMID:Pneumotropic revertants derived from a pantropic mutant, F1-R, of Sendai virus. 165 90

Direct virus inactivation of tachyplesin I and related isopeptides, which are antimicrobial peptides isolated from the hemocytes of the horseshoe crab (Tachypleus tridentatus and Limulus polyphemus), was examined against several viruses. Vesicular stomatitis virus (VSV) was inactivated by incubation with tachyplesin I and its isopeptides. Influenza A (H1N1) virus was slightly inactivated by tachyplesin I, whereas herpes simplex virus 1 and 2, adenovirus 1, reovirus 2 and poliovirus 1 were resistant to inactivation. The inactivation of VSV by tachyplesin I depended on the concentration, the time and the temperature of incubation. Pretreatment of tachyplesin I with trypsin or lipopolysaccharide of gram-negative bacteria entirely abolished the antiviral activity. Electron microscopy of VSV treated with tachyplesin I showed naked and damaged virions. These data suggest that tachyplesin I directly inactivates the VSV by destroying its envelope subunits.
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PMID:Direct virus inactivation of tachyplesin I and its isopeptides from horseshoe crab hemocytes. 166 45


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