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 hemagglutinin (HA) of influenza virus is a homotrimeric integral membrane glycoprotein. It is cotranslationally inserted into the endoplasmic reticulum as a precursor called HA0 and transported to the cell surface via the Golgi complex. We have, in this study, investigated the kinetics and cellular location of the assembly reaction that results in HA0 trimerization. Three independent criteria were used for determining the formation of quaternary structure: the appearance of an epitope recognized by trimer-specific monoclonal antibodies; the acquisition of trypsin resistance, a characteristic of trimers; and the formation of stable complexes which cosedimented with the mature HA0 trimer (9S20,w) in sucrose gradients containing Triton X-100. The results showed that oligomer formation is a posttranslational event, occurring with a half time of approximately 7.5 min after completion of synthesis. Assembly occurs in the endoplasmic reticulum, followed almost immediately by transport to the Golgi complex. A stabilization event in trimer structure occurs when HA0 leaves the Golgi complex or reaches the plasma membrane. Approximately 10% of the newly synthesized HA0 formed aberrant trimers which were not transported from the endoplasmic reticulum to the Golgi complex or the plasma membrane. Taken together the results suggested that formation of correctly folded quaternary structure constitutes a key event regulating the transport of the protein out of the endoplasmic reticulum. Further changes in subunit interactions occur as the trimers move along the secretory pathway.
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PMID:Assembly of influenza hemagglutinin trimers and its role in intracellular transport. 242 70

Antigen-specific cytotoxic T cells can be generated by primary in vitro stimulation of spleen cells from C57BL/6 mice with appropriate peptide fragments. This response can be elicited without prior in vivo immunization. Chicken OVA fragmented with either cyanogen bromide (CN OVA) or trypsin (T OVA) was used as a source of mixed peptides. A synthetic peptide, NP365-380, representing the sequence 365-380 from influenza virus A/PR/8 nucleoprotein, was also used, since this contains the main determinants recognized by CTL generated from H-2b mice infected with A/PR/8 virus. The primary in vitro cytotoxic T cell response was peptide specific, since targets were lysed only in the presence of appropriate peptide antigens. Native OVA could not elicit primary effectors in vitro nor could it sensitize targets for lysis by OVA digest-specific CTL. A synthetic peptide corresponding to residues 111-122 within the OVA sequence could sensitize targets for lysis by effectors induced against T OVA. Effectors generated by in vitro stimulation were CD8+, CD4-, and H-2Db-restricted for NP365-380 and T OVA recognition. CN OVA-specific effectors were also CD8+, CD4-, but surprisingly, were able to lyse a range of H-2-different targets in an antigen-specific manner. These effectors failed to lyse a tumor line that does not express class I MHC molecules. This broad MHC restriction pattern was also apparent at the clonal level. In all cases, the antipeptide CTL generated by primary in vitro stimulation were inefficient in lysing target cells expressing endogenous forms of antigens, such as influenza virus-infected cells or cells transfected with the OVA cDNA. However, cytotoxic T cell lines generated in vitro against the NP365-380 peptide did contain a minor population of virus-reactive cells that could be selectively expanded by stimulation with A/PR/8-infected spleen cells. These results are discussed in terms of class I-restricted T cell stimulation in the absence of antigen processing by high surface densities of peptide/MHC complexes.
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PMID:Induction of cytotoxic T lymphocytes by primary in vitro stimulation with peptides. 245 12

Influenza virus strain A/USSR/053/74/H3N2 was subjected to the effect of soluble and nonsoluble trypsin. Both enzymes appeared to affect the activity of neuraminidase. The viruses altered in this way did not induce interferon (IFN) in mice. Treatment of viral particles with soluble bromeline brought about almost complete inactivation of hemagglutinin and slight suppression of neuraminidase activity. This virus was capable of IFN induction. On the other hand, when nonsoluble bromeline was used as the treatment agent, complete inactivation of neuraminidase and reduction of hemagglutinin titer were observed. This virus was not capable of IFN induction. Viral preparations treated with pronase, trypsin and bromeline for a total extraction of the surface antigens, did not induce IFN. The viruses treated with these enzymes were examined under the electron microscope. Trypsin-treated viruses showed no changes on their surface while bromeline and pronase totally extracted the spikes of surface antigens.
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PMID:Ultrastructure and some biological properties of influenza A virus. IV. Effect of proteolytic enzymes on viral particle. 247 2

Sections of formalin-fixed, paraffin-embedded tissue of experimentally influenza virus-infected hamsters were treated with 0.25% trypsin and tested for virus antigen by indirect immunofluorescent staining. The results were comparable to those obtained with aceton-fixed cryo-microtome sections. As far as we know, this is the first description of influenza virus demonstration in formalin-fixed, paraffin-embedded tissue after reactivation by trypsin-treatment. This technique may be useful for influenza virus detection in human autopsy cases. It allows an etiological diagnosis even when fresh tissue for cryocut sections or virus cultivation is not available.
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PMID:Immunofluorescent staining of influenza virus antigen in fixed and paraffin-embedded tissue of experimentally infected hamsters. 249 71

We and others have recently described 9-O-acetyl-sialic acid esterase (9-O-Ac-SA esterase) activities that appear to be specific for removal of O-acetyl esters from the 9-position of naturally occurring sialic acids. We have now examined a variety of species for such enzymes and found them in vertebrates and higher invertebrates, but not in plants or in lower invertebrates. This evolutionary distribution correlates well with that of the sialic acids themselves. All of the 9-O-Ac-SA esterase activities tested were inhibited by diisopropyl fluorophosphate (DFP) in a dose-dependent fashion. This indicates that each of these enzymes has a serine active site similar to the well known serine esterases and serine proteases. Methyl esterification of the carboxyl group of 9-O-acetyl-N-acetylneuraminic acid significantly reduced the activity of all of the 9-O-Ac-SA esterases against the O-acetyl group. This indicates that each of these enzymes may recognize the negatively charged carboxyl group of the sialic acid. Enzymes that recognize anionic substrates frequently have an essential arginine residue (Riordan, J. F., McElvany, K. D., and Borders, C. L., Jr. (1977) Science 195, 884-886). We therefore studied the effects of the arginine-specific modifying reagents 2,3-butanedione and phenylglyoxal on 9-O-Ac-SA esterase activities from influenza C virus, human erythrocytes, rat liver, starfish gonads, and sea bass brain. All of these enzymes were inhibited in a dose-dependent fashion by both reagents, under conditions previously known to avoid nonspecific modification. In contrast, the typical serine proteases trypsin and kallikrein and the serine esterase acetylcholinesterase were not significantly affected, even by the highest concentrations of these reagents used. These data indicate that five 9-O-Ac-SA esterase activities from evolutionarily distinct origins all have serine active sites and essential arginine residues. We postulate that the arginine residue is involved in substrate recognition via the negatively charged carboxyl group of the sialic acids. Thus, these 9-O-Ac-SA esterase activities may be members of a previously undescribed class of serine esterase.
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PMID:O-acetylation and de-O-acetylation of sialic acids. Sialic acid esterases of diverse evolutionary origins have serine active sites and essential arginine residues. 250 78

Rimantadine prevents the conformational changes of influenza virus haemagglutinin (HA) caused by acid pH and the acquisition of sensitivity to trypsin, protects the haemolytic activity from inactivation and prevents the morphological changes of HA spikes on the virus surface.
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PMID:Action of rimantadine on the structure of influenza A virus haemagglutinin. 256 8

Haemadsorbing foci were found in human fetal lung (HFL) diploid cell cultures 12 h after inoculation with influenza viruses A and B. The size and number of the foci were maximal after 48 h of incubation, being limited by production of an unidentified inhibitor. By contrast, inoculation with parainfluenza virus type 3 led to haemadsorption which increased during 10 days of incubation. For the detection of influenza viruses A and B maximum sensitivity was achieved by changing the medium, the day before use to one that was serum free. The number of foci at 15.5 h post-infection and infectivity for primary African green monkey kidney (AGMK) cultures were similar. Virus infectivity and production of haemagglutinin in HFL cells were entirely cell-associated; they were not affected by treatment with trypsin. Nevertheless, influenza viruses A and B antigens were identified in the infected cells by means of immunofluorescence at 15.5 h and virus was recovered by passage of frozen and thawed cells in AGMK cultures. For rapid routine diagnosis of viral infections, the early haemadsorption test was shown to have the same sensitivity as immunofluorescence tests on specimens and virus detection by the shell-vial technique but was cheaper and simpler to perform.
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PMID:Rapid detection of influenza virus infections in human fetal lung diploid cell cultures. 266 98

The relative amounts of influenza C virus-specific receptors of 25 established lines of mammalian cells including four lines of human malignant melanoma origin were compared by virus binding experiments. All the human melanoma cell cultures studied possessed two to four times more receptors than were found on MDCK cells, a cell line known to be highly susceptible to influenza C virus. It may therefore be a feature common to human melanoma cells that O-acetylsialic acid, a determinant for the attachment of influenza C virus, exists in large quantities on their surface. This is not specific to melanoma cells, however, since several human cell lines derived from lung cancer, gastric cancer, and placenta specimens also exhibited high levels of virus binding. Twenty of 25 virus-binding cell cultures were further examined for their ability to support the replication of influenza C virus. In the presence of trypsin (5 to 20 micrograms/ml), the virus was found to undergo multiple cycles of replication much more efficiently in the HMV-II line of human melanoma cells than in MDCK cells. Additionally, by using HMV-II cells as a host, we succeeded in isolating two influenza C strains (C/Yamagata/1/88, C/Yamagata/2/88) from 241 throat swabs collected from patients with acute respiratory illness.
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PMID:A human melanoma cell line highly susceptible to influenza C virus. 273 78

The 76-kd human interferon (IFN)-induced MX protein is the homolog to the murine protein, which is necessary and sufficient to provide adequate resistance to influenza virus infection in murine cells and in mice. Fifty-one patients with systemic lupus erythematosus (SLE) were screened for the presence of the MX homolog in mononuclear cells and for IFN and anti-IFN antibodies in serum. In 47 of 51 patients, significant levels of the MX homolog were found, while only 15 of 51 patients had measurable alpha-IFN in their serum. The IFN activity found in these sera was characterized as a partially acid-labile alpha-IFN, by means of acid-stability cross-reactivity on heterologous cells, trypsin sensitivity, and neutralization by homologous or heterologous antisera. Four of the patients had no detectable MX homolog in their leukocytes; 3 of these 4 possessed an anti-alpha-IFN antibody that was able to neutralize both a natural alpha-IFN preparation and the acid-labile IFN in SLE sera. Also, acid-labile alpha-IFN-containing SLE sera induced the MX homolog in vitro in mononuclear cells from healthy donors. These observations suggest that endogenously produced alpha-IFN is responsible for the observed induction of the MX homolog in SLE and that the IFN system is activated in more than 90% of SLE patients.
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PMID:MX homologous protein in mononuclear cells from patients with systemic lupus erythematosus. 275 23

To evaluate the replication of a highly virulent avian influenza A virus in a potential reservoir host, mallard ducks (Anas platyrhynchos) were inoculated with the virulent strain A/Ty/Ont/7732/66 (H5N9). Viruses recovered from the ducks were analyzed by hemagglutination inhibition (HI) and enzyme-linked immunosorbent assay (ELISA) and found to possess antigenically altered viral hemagglutinins. Plaque formation on the Madin-Darby Canine Kidney (MDCK) cell line and on primary chicken embryo cells was investigated, and isolates recovered from the ducks differed from the wild type by being unable to form plaques on MDCK cells without trypsin. This phenotype did not appear to be due to inefficient cleavage of the hemagglutinin by host cell proteases since hemagglutinin immunoprecipitated from cell lysates was cleaved. Although the plaquing phenotype suggested attenuation of the isolates from the ducks, they were not significantly altered in their virulence for chickens shown by infectivity studies in vivo. These results indicate that replication of influenza A/Ty/Ont/7732/66 virus in ducks can produce antigenic and phenotypic variants which are still highly virulent for domestic poultry.
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PMID:Antigenic and phenotypic variants of a virulent avian influenza virus selected during replication in ducks. 281 May 50


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