Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The objective of this study was to investigate the comparative immune response following administration of biodegradable microparticles loaded with influenza viral vaccine using subcutaneous and oral routes. Influenza viral vaccine was entrapped in poly(d,l-lactide-co-glycolide) (PLG) and poly(isobutylcyanoacrylate) (PIBCA) microparticles. Stability and immunogenicity of entrapped antigen were retained, as evaluated by SDS-PAGE and immunoblot. Microparticles in the size range of <11 microm were evaluated for protein loading and in vitro antigen release. The mice were immunized with microparticle loaded antigen and IgG levels in blood and IgA levels in saliva and gastric secretions were monitored by ELISA method. When the mice were immunized with microparticle suspensions, IgG levels were higher if administered by subcutaneous primed by oral route compared to oral primed by subcutaneous route or subcutaneous or oral route. The IgA level in saliva and gastric secretions were also found to be higher when subcutaneous immunization was given followed by oral booster than oral priming followed by subcutaneous booster. The polymer types of the microparticles had effects on both IgG and IgA levels. This study provided insights into the design of microparticles of influenza vaccine for subcutaneous administration followed by an unlimited oral boosting, which will have high cost-effectiveness and patient compliance.
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PMID:Biodegradable microparticles of influenza viral vaccine: comparison of the effects of routes of administration on the in vivo immune response in mice. 1005 95

The nature and role of glycosylation in AT1 angiotensin receptor (AT1-R) function were investigated by expressing glycosylation-deficient influenza hemagglutinin (HA) epitope-tagged rat AT1a-Rs (HA-AT1a-Rs) in COS-7 cells. All three asparagine residues (Asn4, Asn176, Asn188) contained within consensus sites for N-linked glycosylation could be glycosylated in Cos-7 cells and appeared to be glycosylated on the endogenous AT1-R in bovine adrenal glomerulosa cells. Heterogeneity of glycosylation at each site accounted for the broad migration pattern of the AT1-R in SDS-PAGE. Mutation at each glycosylation site, either alone or in combination, had little effect on ligand binding parameters (although the N4K mutant had higher affinity) or signaling activity. However, an increasing number of mutated glycosylation sites was associated with decreasing cell surface receptor expression, which was minimal for the unglycosylated N4K/N176Q/N188Q receptor. Decreased surface expression of mutant HA-AT1a-Rs was correlated with decreased total cell receptor content as revealed by immunoblotting with an anti-HA antibody. These findings suggest that glycosylation enhances receptor stability, possibly by protecting nascent receptors from proteolytic degradation.
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PMID:N-linked glycosylation is required for optimal AT1a angiotensin receptor expression in COS-7 cells. 1021 49

The paramyxovirus fusion (F) protein mediates membrane fusion. The biologically active F protein consists of a membrane distal subunit F2 and a membrane anchored subunit F1. A highly stable structure has been identified comprised of peptides derived from the simian virus 5 (SV5) F1 heptad repeat A, which abuts the hydrophobic fusion peptide (peptide N-1), and the SV5 F1 heptad repeat B, located 270 residues downstream and adjacent to the transmembrane domain (peptides C-1 and C-2). In isolation, peptide N-1 is 47% alpha-helical and peptide C-1 and C-2 are unfolded. When mixed together, peptides N1 + C1 form a thermostable (Tm > 90 degrees C), 82% alpha-helical, discrete trimer of heterodimers (mass 31,300 M(r)) that is resistant to denaturation by 2% SDS at 40 degrees C. The authors suggest that this alpha-helical trimeric complex represents the core most stable form of the F protein that is either fusion competent or forms after fusion has occurred. Peptide C-1 is a potent inhibitor of both the lipid mixing and aqueous content mixing fusion activity of the SV5 F protein. In contrast, peptide N-1 inhibits cytoplasmic content mixing but not lipid mixing, leading to a stable hemifusion state. Thus, these peptides define functionally different steps in the fusion process. The parallels among both the fusion processes and the protein structures of paramyxovirus F proteins, HIV gp41 and influenza virus haemagglutinin are discussed, as the analogies are indicative of a conserved paradigm for fusion promotion among fusion proteins from widely disparate viruses.
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PMID:The paramyxovirus fusion protein forms an extremely stable core trimer: structural parallels to influenza virus haemagglutinin and HIV-1 gp41. 1033 33

Influenza virus hemagglutinin (HA), the viral envelope glycoprotein that mediates fusion between the viral and cellular membranes, is a homotrimer of three subunits, each containing two disulfide-linked polypeptide chains, HA(1) and HA(2). Each HA(2) chain spans the viral membrane with a single putative transmembrane alpha-helix near its C-terminus. Fusion experiments with recombinant HAs suggest that this sequence is required for a late step of membrane fusion, as a glycosylphosphatidylinositol-anchored analogue of HA only mediates "hemifusion" of membranes, i.e., the merging of the proximal, but not distal, leaflets of the two juxtaposed lipid bilayers [Kemble et al. (1994) Cell 76, 383-391]. To find a structural explanation for the function of the transmembrane domain of HA(2) in membrane fusion, we have studied the secondary structure, orientation, oligomerization, and lipid interactions of a synthetic peptide representing the transmembrane segment of X:31 HA (TMX31) by circular dichroism and attenuated total reflection Fourier transform infrared spectroscopy and by gel electrophoresis. The peptide was predominantly alpha-helical in detergent micelles and in phospholipid bilayers. The helicity was increased in lipid bilayers composed of acidic lipids compared to pure phosphatidylcholine bilayers. In planar lipid bilayers, the helices were oriented close to the membrane normal. TMX31 aggregated into small heat-resistant oligomers composed of two to five subunits in SDS micelles. Amide hydrogen exchange experiments indicated that a large fraction of the helical residues were accessible to water, suggesting the possibility that TMX31 forms pores in lipid bilayers. Finally, the peptide increased the acyl chain order in lipid bilayers, which may be related to the preferential association of HA with lipid "rafts" in the cell surface and which may be an important prerequisite for complete membrane fusion.
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PMID:Secondary structure, orientation, oligomerization, and lipid interactions of the transmembrane domain of influenza hemagglutinin. 1064 74

Protein folding in the living cell begins cotranslationally. To analyze how it is influenced by the ribosome and by the translocon complex during translocation into the endoplasmic reticulum, we expressed a mutant influenza hemagglutinin (a type I membrane glycoprotein) with a C-terminal extension. Analysis of the nascent chains by two-dimensional SDS-PAGE showed that ribosome attachment as such had little effect on ectodomain folding or trimer assembly. However, as long as the chains were ribosome bound and inside the translocon complex, formation of disulfides was partially suppressed, trimerization was inhibited, and the protein protected against aggregation.
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PMID:Role of ribosome and translocon complex during folding of influenza hemagglutinin in the endoplasmic reticulum of living cells. 1067 29

Agonist-induced phosphorylation of the human corticotropin-releasing factor type 1 receptor (hCRF(1)-R) was investigated using an influenza hemagglutinin (HA) epitope-tagged receptor transiently expressed in COS-7 cells. The HA-hCRF(1)-R migrated as a broad band (M(r) 60,000-70,000) in SDS-PAGE and showed increased mobility (M(r) approximately 48,000) after enzymatic deglycosylation with peptide-N-glycosidase F, consistent with the predicted size (47 kDa) of the nonglycosylated HA-hCRF(1)-R protein. A marked increase in HA-hCRF(1)-R phosphorylation was observed in HA-hCRF(1)-R-expressing COS-7 cells exposed to 1 microM ovine CRF for 5 min, whereas activation of protein kinase A (PKA) by 50 microM forskolin, or of Ca(2+)/calmodulin (CaM)-dependent kinases by 10 microM ionomycin, had little effect. These findings are consistent with preliminary data suggesting that CRF(1)-R phosphorylation mediated by G protein receptor kinase 3 (GRK3), but not by PKA or CaM-dependent kinases, has an important role in the homologous desensitization of brain CRF(1)-Rs.
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PMID:Rapid agonist-induced phosphorylation of the human CRF receptor, type 1: a potential mechanism for homologous desensitization. 1067 45

The conformation and interactions with membrane mimics of the NH(2)-terminal fragment 1-25 of HA2, HA2-(1-25), of influenza virus were studied by spectroscopic methods. Secondary structure analysis of circular dichroism data revealed 45% helix for the peptide at pH 5.0. Tryptophan fluorescence quenching by acrylamide and NMR experiments established that the Trp(14) is inside the vesicular interior and residues 16-18 are at the micellar aqueous boundary. NBD fluorescence enhancement of the NH(2)-terminal labeled fluorophore on the vesicle-bound peptide indicated that the NH(2) terminus of the fusion peptide was located in the hydrophobic region of the lipid bilayer. No significant change in insertion depth was observed between pH 5.0 and 7.4. Collectively, these spectroscopic measurements pointed to an equilibrium between helix and non-helix conformations, with helix being the dominant form, for the segment in the micellar interior. The conformational transition may be facilitated by the high content of glycine, a conformationally flexible amino acid, within the fusion peptide sequence. Self-association of the 25-mer peptide was observed in the N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine SDS-gel electrophoresis experiments. Incorporating the NMR signal attenuation, fluorescence, and gel electrophoresis data, a working model for the organization of the fusion peptide in membrane bilayers was proposed.
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PMID:The amino-terminal region of the fusion peptide of influenza virus hemagglutinin HA2 inserts into sodium dodecyl sulfate micelle with residues 16-18 at the aqueous boundary at acidic pH. Oligomerization and the conformational flexibility. 1076 1

A novel trypsin-type serine proteinase, which processes the precursors of the envelope fusion glycoproteins of pneumotropic Sendai and human influenza A viruses, was purified to homogeneity from pig lungs. On SDS/PAGE, the purified enzyme gave a protein band corresponding to about 32 kDa, and has an apparent molecular mass of 120 kDa, as determined by gel permeation chromatography. Immunohistochemical staining with antibodies against this enzyme revealed that the enzyme is located in pig lung mast cells. The N-terminal 44-amino-acid sequence of the enzyme exhibits about 80% identity with those of mast cell tryptases from other species. Of the inhibitors tested, di-isopropyl fluorophosphate, antipain, leupeptin, benzamidine and a few proteinaceous inhibitors, such as mucus protease inhibitor and aprotinin, inhibited this enzyme activity. Heparin stabilized the enzyme, but high-ionic-strength conditions did not, unlike for human mast cell tryptase. The purified enzyme efficiently processed the fusion glycoprotein precursor of Sendai virus and slowly processed hemagglutinin of human influenza A virus, and triggered the infectivity of Sendai virus in a dose-dependent manner, although human mast cell tryptase beta and rat mast cell tryptase (rat MCP-7) from lungs did not process these fusion glycoproteins at all. These results suggest that mast cell tryptase in pig lungs is the possible trigger of the pneumotropic virus infections.
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PMID:Mast cell tryptase from pig lungs triggers infection by pneumotropic Sendai and influenza A viruses. Purification and characterization. 1082 3

Epidemiological evidence points to prenatal viral infection being responsible for some forms of schizophrenia and autism. We hypothesized that prenatal human influenza viral infection in day 9 pregnant mice may cause changes in the levels of neuronal nitric oxide synthase (nNOS), an important molecule involved in synaptogenesis and excitotoxicity, in neonatal brains. Brains from 35- and 56-day-old mice were prepared for SDS-gel electrophoresis and Western blotting using polyclonal anti nNOS antibody. Quantification of nNOS showed time and region-dependent changes in the levels of nNOS protein. Mean rostral brain area value from prenatally infected animals showed a significant (p=0.067) increase of 147% in nNOS levels at 35 days postnatally, with an eventual 29% decrease on day 56. Middle and caudal brain areas showed reductions in nNOS in experimental mice at 35 and 56 days, with a significant 27% decrease in nNOS in the middle segment of day 56 brains (p=0.016). Significant interactions were found between group membership and brain area (Wilks lambda=0.440, F(2.9)=5.72, p=0.025); there was also a significant interaction between brain area, group and age (Wilks lambda=0.437, F(2.9)=5.79, p=0.024). These results provide further support for the notion that prenatal viral infection affects brain development adversely via the pathological involvement of nNOS expression.
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PMID:Prenatal viral infection causes alterations in nNOS expression in developing mouse brains. 1084 64

Photoaffinity and fluorescent analogues of the 70-amino acid chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) were designed, synthesized, characterized, and applied to probe MIP-1alpha interactions with the chemokine receptors CCR1 and CCR5. The photoactivatable MIP-1alpha ligand, BP-MIP-1alpha, and the fluorescent ligand, Flu-MIP-1alpha were prepared by selective chemical coupling of p-benzoylphenylthiocarbamyl or fluoresceinthiocarbamyl, respectively, at the N-terminus of MIP-1alpha. Both ligands BP-MIP-1alpha and Flu-MIP-1alpha retained high binding affinity and agonist potency at CCR1 and CCR5. Photoaffinity labeling of CCR1 and CCR5 receptors stably expressed in CHO cells resulted in specific covalent attachment of [(125)I]BP-MIP-1alpha and production of protein complexes of 54 and 48 kDa, respectively, on SDS-PAGE. This represents the first photo-cross-linking between a chemokine and its receptor. Flu-MIP-1alpha selectively labeled CCR1 or CCR5 receptors expressed in CHO cells and was used to characterize receptor binding domains. When bound to CCR1 or CCR5 receptors, the fluorescence signal of Flu-MIP-1alpha was quenched by collision with iodide indicating that the N-terminal end of MIP-1alpha is accessible to the solvent. These data strongly suggest that the N-terminal end of MIP-1alpha interacts with domains of CCR1 or CCR5 receptors located at the extracellular surface. The photoactivatable BP-MIP-1alpha described here should prove valuable for the identification of contact sites on receptors by photoaffinity labeling experiments.
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PMID:Synthesis and characterization of fluorescent and photoactivatable MIP-1alpha ligands and interactions with chemokine receptors CCR1 and CCR5. 1117 Jun 31


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