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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Purified influenza virus (A/FPV/Rostock/34; H7N1) was reacted with one of three chemical crosslinking reagents [dimethylsuberimidate (DMS), tartryl diazide (TDA) and formaldehyde] under conditions designed to give a ladder of crosslinked polypeptides (putative homo- and heteropolymers) when analysed by SDS-polyacrylamide gel electrophoresis under reducing conditions. The different virion polypeptides were identified by Western blotting with monospecific antisera against HA1, HA2, NP, and M1. When reacted with any crosslinker NP preferentially formed 2mer and 4mer homopolymers while M1 formed 2mers, 4mers, 6mers, and 8mers. 2mers and 3mers of HA1 were detected after crosslinking with TDA and DMS but homopolymers of HA2 could not be identified with certainty due to comigrating M1. One heteropolymer was clearly identified as 1NP:1M1 (with DMS and TDA) and others, as expected, as components of the haemagglutinin spike 1HA1:1HA2, 2HA1:2HA2, and 3HA1:3HA2. Formaldehyde gave rise only to HA1:HA2 polymers. The presence of other heteropolymers containing NP in conjunction with HA2 and HA1 seemed likely. Whenever HA2 ran with an Mr of about 50k it comigrated with M1 suggesting it may have formed (with DMS or TDA) a 1HA2:1M1 heterodimer. However it is possible that this band consisted of HA2 homodimers comigrating with M1 homodimers. Patterns of crosslinking with DMS and TDA were similar although not identical, but those obtained with formaldehyde were markedly different. All patterns were highly reproducible.
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PMID:Chemical crosslinking of proteins of the influenza virion. 1. Interrelationships. 260 45

Purified influenza virus (A/FPV/Rostock/34;H7N1) was exposed briefly to pH 5 before returning to an alkaline pH. Virus was then reacted with one of three chemical cross-linking reagents [dimethyl suberimidate (DMS), tartryl diazide (TDA), or formaldehyde which span 11, 6, and 2A, respectively]. Cross-linked polypeptides were analysed by SDS-polyacrylamide gel electrophoresis under reducing conditions and identified with monospecific antisera against HA1, HA2, NP and M1. Acidification resulted in changes in the cross-linking patterns for both HA1 and HA2 which could be detected with all three reagents. Most notable were the data with formaldehyde: under alkaline conditions cross-linking gave only HA1:HA2 heteropolymers but after brief acidification none of these were formed and in their place was a novel HA1 homodimer, an HA2 homotrimer and an HA2 of Mr 50k cross-linked to form a homodimer with another HA2 or to a heterodimer with M1. Although cross-linking by formaldehyde was much more affected by acidification of the virus than cross-linking by DMS or TDA, over half the polymers cross-linked by DMS were no longer formed after acidification. The patterns of cross-linking of NP and M1 were unchanged by low pH treatment.
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PMID:Chemical cross-linking of proteins of the influenza virion. 2. Acid-induced irreversible conformational changes in HA1 and HA2. 260 46

When polymorphonuclear leukocytes (PMN) are exposed to most harvests of influenza A virus (depressing virus, DV) for 20 min, chemotactic, secretory, and oxidative functions are depressed upon subsequent exposure to soluble or particulate stimuli. Other harvests of influenza A virus (non-DV) do not alter these activities. The DV-induced changes in multiple functions suggest the virus may interfere with steps involved in PMN activation. Because some of these steps may be regulated by protein phosphorylation, we examined the effect of non-DV and DV on cellular protein phosphorylation. PMN loaded with 32P-labeled inorganic orthophosphate were exposed to non-DV, DV, or buffer for 30 min; cells were then treated with buffer, FMLP (10(-6) M), or PMA (100 ng/ml) for 30 s. Samples were sonicated and centrifuged; cytosolic and particulate fractions were analyzed by SDS-PAGE and autoradiography. Exposure of PMN to either non-DV or DV caused phosphorylation of several cell proteins. However, when DV-treated PMN were then stimulated with FMLP or PMA, further phosphorylation was inhibited compared to non-DV- or buffer-treated cells. This suggests that DV-induced depression of PMN end-stage functions may be due to changes in cell protein phosphorylation. DV could interfere with phosphorylation of PMN proteins by altering protein kinase activity. We therefore examined the influence of non-DV and DV on some parameters that could affect kinase function. PMN intracellular [Ca2+] was monitored by using the fluorescent Ca2+ indicator, Indo 1, and cAMP levels were measured by RIA. PMN treated with DV alone or DV plus FMLP had higher intracellular [CA2+] than PMN similarly treated with non-DV or buffer. Exposure of PMN to non-DV, DV, or buffer caused minimal changes in cAMP levels, and similar increases occurred in cAMP levels upon FMLP stimulation. To determine whether DV interferes with transmembrane signaling, the effect of influenza virus on PMN transmembrane potential was studied by using a fluorescent cyanine dye. Transmembrane potential changes were greater in PMN exposed to DV than to non-DV or buffer; however, subsequent stimulation with FMLP caused equivalent changes in transmembrane potential. Our data show that protein phosphorylation in PMN is induced by DV and non-DV infection; upon subsequent stimulation with FMLP or PMA, there is inhibited cellular phosphorylation only in PMN previously exposed to DV.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Alterations in cell protein phosphorylation in human neutrophils exposed to influenza A virus. A possible mechanism for depressed cellular end-stage functions. 283 42

Membranes isolated from chick embryo cells (CEC) were found to contain an endogenous protein kinase that phosphorylated endogenous proteins. 32P incorporation into membrane proteins was analysed by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and detected by autoradiography. Membrane phosphorylation in the presence of physiological saline (PM-phys) and in the presence of influenza virus (PM-V) were compared. Under short-time incubation (less than 1 min) with gamma-32P ATP there was practically no difference between PM-phys and PM-V in 32P incorporation. Under prolonged incubation (3-15 min), gradual dephosphorylation of the phosphoprotein moving in SDS-PAGE in the zone of relative molecular mass (Mr) of 60 kDa (phosphoprotein P60) was observed in PM-phys whereas in PM-V, phosphorylation of P60 increased with the time of incubation with gamma-32P ATP. Dephosphorylation of P60 in PM-phys was inhibited by 2.5 mmol/l EGTA as well as by 100 mumol/l chlorpromazine (CPZ) and stimulated by calmodulin (CaM) in the presence of Ca2+. Responsible for the dephosphorylation was probably the endogenous Ca2+ and/or CaM-dependent membrane protein phosphatase. It was inhibited by influenza virus (even in the presence of Ca2+ and CaM). The possible role of P60 phosphorylation in the first step of virus infection is discussed.
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PMID:Effect of influenza virus on protein phosphorylation in isolated membranes of chick embryo cells. 285 56

Anti-haemagglutinin monoclonal antibodies were prepared and their HA1 or HA2 specificity was determined by solid phase radioimmunoassay (RIA) using purified viral haemagglutinin (HA) and haemagglutinin glycopolypeptides HA1 and HA2, by radioimmunoprecipitation followed with SDS-PAGE, by immunoblotting and by inhibition of virus-induced haemagglutination. The capacity of these methods to estimate HA1 or HA2 specificity of anti-HA monoclonal antibodies (MoAb) was compared. HA1 specificity was demonstrated for all hybridomas originating from lymphocytes of mice immunized with complete influenza virus, except IIF4 hybridoma which was HA2-specific. All hybridomas obtained with lymphocytes from mice immunized with HA glycopolypeptide HA2 were HA2-specific. Anti-HA2 MoAb neither inhibit haemagglutination induced by the virus or by HA subunits nor neutralized viral infectivity, either alone or in mixture. As expected, all anti-HA1 MoAb were H3 subtype-specific, showing usually good reactivity only with viruses close to the virus strain used for immunization. Two anti-HA1 MoAb (IVA1 and IVG6) showed unusual cross-reactivity within the H3 subtype. All anti-HA2 MoAb were broadly cross-reactive within the H3 subtype. Moreover, a half of them showed high cross-reactivity with influenza viruses of the H7 HA subtype. But the same antibodies did not react with HA of H1, H2 and H8 subtypes.
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PMID:Monoclonal antibodies to glycopolypeptides HA1 and HA2 of influenza virus haemagglutinin. 289 Dec 76

The polymerase proteins (PB1, PB2, PA) of the influenza virus strain A/PR/8/34 (H1N1) were isolated from whole virion or ribonucleoprotein (RNP) fractions by electrophoresis on polyacrylamide gel and electroelution or Sepharose CL-6B chromatography in the presence of SDS. Antisera to polymerase proteins (P proteins) were raised in rabbits; the immunoglobulins (Ig) were purified by affinity chromatography. Characterization of the antibody fraction by Western blot analysis showed a highly monospecific reaction with the three polymerase proteins. Spot immunobinding assay was used to compare the immunoreactivity of the monospecific polymerase antibodies with the P proteins of other influenza A subtypes and influenza B strains, revealing high immunoreactivity with the components of all influenza A strains and only insignificant reactivity with the components of the influenza B strains tested.
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PMID:Antibodies to polymerase proteins of influenza virus A/PR/8/34 (H1N1): comparison on the immunoreactivity with polymerase proteins of other influenza A and B virus strains. 290 33

Influenza undergoes extensive antigenic variation in nature. These new antigenic variants invariably supplant the preceding antigenic type that then disappears. In apparent contrast to this, two H3N2 strains that were forwarded to the Laboratory Centre for Disease Control, Ottawa, in 1984 from a Canadian public health laboratory for reference analysis were shown to be most closely related to 1973 and 1974 strains. Laboratory contamination on isolation by the regional public health laboratory was the most likely explanation for the occurrence of these strains, since one of the isolates (RV/76/84) was identical by T1 mapping to an A/Eng-like isolate being grown in the laboratory of isolation. The two isolates, RV/74/84 and RV/76/84, were shown to be distinct from each other and from prototype H3N2 strains by RNase T1 oligonucleotide mapping, SDS-PAGE, peptide mapping of hemagglutinin, and hemagglutination inhibition assay. RV/74 and RV/76 appeared to have been genetically stable for the 10 to 11 years preceding 1984; this is consistent with laboratory frozen storage for this period of time. This paper demonstrates the utility of RNase T1 mapping for the characterization of novel or spurious influenza isolates.
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PMID:The use of T1 oligonucleotide mapping to determine that the emergence of Canadian 1973-like and 1974-like H3N2 strains in 1984 was the result of laboratory contamination. 312 Nov 60

The acetylesterase of influenza C virus has been reported recently to be inhibited by diisopropylfluorophosphate (DFP) [Muchmore EA, Varki A (1987) Science 236: 1293-1295]. As this inhibitor is known to bind covalently to the serine in the active site of serine esterases, we attempted to determine the serine in the active site of the influenza C acetylesterase. Incubation of purified influenza C virus with 3H-DFP resulted in the selective labelling of the influenza C glycoprotein HEF. The labelled glycoprotein was isolated from a SDS-polyacrylamide gel. Following reduction and carboxymethylation, tryptic peptides of HEF were prepared and analyzed by reversed phase HPLC. The peptide containing the 3H-DFP was subjected to sequence analysis. The amino acids determined from the NH2-terminus were used to locate the peptide on the HEF polypeptide. Radiosequencing revealed that 3H-DFP is attached to amino acid 17 of the tryptic peptide. These results indicate that serine 71 is the active-site serine of the acetylesterase of influenza C virus.
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PMID:Serine 71 of the glycoprotein HEF is located at the active site of the acetylesterase of influenza C virus. 314 64

Three slightly different procedures for the preparation of influenza B virus reassortants from B/Lee/40 and B/Lyon/79, and B/Johannesburg/58 and B/Hong Kong/82 parental viruses are described. Following cloning procedures in eggs or allantois-on-shell cultures in the presence of antisera to B/Lee or B/Johannesburg, twenty-eight putative reassortants were obtained. Using SDS-PAGE to determine the migration rates of virion polypeptides, eighteen isolates from mixed infections of B/Lee with B/Lyon, and three isolates from mixed infections of B/Johannesburg with B/Hong Kong, were found to be reassortants. All reassortants possessed surface proteins derived from either B/Lyon or B/Hong Kong and one to three internal polypeptides from either B/Lee or B/Johannesburg. None of the reassortants showed a growth capacity in embryonated eggs as high as that of their B/Lee or B/Johannesburg parent viruses. The procedures used, and the influenza B reassortants thus generated, would appear to offer no major advantages over the existing methods and influenza B strains at present employed for the preparation of inactivated influenza virus vaccines.
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PMID:Reassortants of influenza B viruses for use in vaccines: an evaluation. 315 42

The order and relative mobility of proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is affected by unknown components that are differentially present in SDS preparations obtained from different sources [J.B. Swaney, G.F. Vande Woude, and H.L. Bachrach (1974) Anal. Biochem. 58, 337-346]. The modified separation capabilities of such SDS preparations are useful but the use of this phenomenon in a controlled manner requires that the components responsible for the altered separation be identified. Accordingly, this paper describes a polyacrylamide gel electrophoresis system [mixed alcohol/detergent-polyacrylamide gel electrophoresis (MAD-PAGE)] that employs a mixture of alcohol and detergent instead of SDS alone to modify and enhance protein separation relative to conventional SDS-PAGE. A defined mixture consisting of four sulfated alkyl detergents (dodecyl sulfate, tetradecyl sulfate, hexadecyl sulfate, octadecyl sulfate) as well as the four alcohols of corresponding aliphatic chain length was found to be effective at duplicating the electrophoretic effect of USP-grade SDS and thus changed the relative order and position of polypeptides on electrophoresis relative to conventional SDS-PAGE. This method serves as an adjunct to conventional SDS-PAGE by providing another means of resolving proteins that are not normally resolved by SDS-PAGE. Further, it was found that MAD-PAGE is capable of resolving the NS1 protein of influenza virus into three fractions, whereas conventional SDS-PAGE yields one electrophoretic species. Reelectrophoresis of these novel NS1 bands by conventional SDS-PAGE indicated that they were not modified during MAD-PAGE and probably represented distinct molecular forms present in infected cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mixed anionic detergent/aliphatic alcohol-polyacrylamide gel electrophoresis alters the separation of proteins relative to conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 321 45


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