Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q9NRP7 (fused)
 58,367 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Oral administration of lactobacilli evokes mucosal and systemic immune responses against epitopes associated with these organisms (Gerritse et al., 1990, 1991). The adjuvant function of different Lactobacillus species was investigated under the conditions of intraperitoneal (i.p.) injection or oral administration. After i.p. injection of trinitrophenylated chicken gamma-globulin, high DTH responses were observed with Lactobacillus casei and Lactobacillus plantarum, but low responses with Lactobacillus fermentum and Lactobacillus delbrueckii subsp. bulgaricus. In different experimental model systems L. casei and L. plantarum consistently showed significant adjuvanticity. A series of expression and expression-secretion vectors containing the strong constitutive promoter of the L. casei L-ldh gene or the regulatable promoter of the Lactobacillus amylovorus amy gene (Pouwels and Leer, 1995) was used for the intracellular, extracellular and surface-bound expression of an influenza virus antigenic determinant fused to Escherichia coli beta-glucuronidase. Intracellular expression of the fusion protein amounted to 1-2% of total soluble protein. Lactobacilli synthesizing the fusion protein intracellularly evoked an oral immune response after subcutaneous priming.
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PMID:The potential of Lactobacillus as a carrier for oral immunization: development and preliminary characterization of vector systems for targeted delivery of antigens. 871 2

We have studied the in vitro activation of murine lymphocytes with LPS incorporated in the membranes of both phospholipid vesicles (liposomes) and vesicles composed of fusogenic, reconstituted influenza virus envelopes (virosomes). The incorporation of Salmonella minnesota rough-LPS in liposomes reduced the potency of LPS to stimulate splenocyte proliferation and cell surface kappa-light chain expression on 70 Z/3 pre-B cells by over 100-fold. Salmonella minnesota rough-LPS inserted into virosomes was at least 10-fold more potent than free LPS, both when prebound virosomes were allowed to be taken up by the cells at neutral pH and when the virosomes were fused into the plasma membrane by low pH treatment. Inactivation of the virosomes by low pH pretreatment reduced the potency of the virosomal LPS to the level of liposome-incorporated LPS. The association of the various LPS forms with the cells was quantitated using radio-iodinated LPS. Correcting for uptake, virosomal LPS remained 2- to 10-fold more potent than free LPS in stimulating B lymphocytes and at least 100-fold more active than liposomal LPS or fusion-inactivated virosomes. After low pH-induced fusion with the plasma membrane, the majority (80%) of the prebound virosomes had fused with the cells, compared with about 8% after neutral uptake. From these results we conclude that LPS inserted into the plasma or endosomal membranes efficiently activates murine lymphocytes. The fusion data suggest that the incorporation into endosomal membranes might be a more effective stimulus.
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PMID:Activation of murine lymphocytes by lipopolysaccharide incorporated in fusogenic, reconstituted influenza virus envelopes (virosomes). 875 6

The putative envelope glycoproteins of hepatitis C virus (HCV), E1 and E2, were expressed as recombinant, secretory proteins in Sf9 insect cells through infection with recombinant baculoviruses. The influenza virus hemagglutinin signal sequence (HASS) was inserted upstream of the HCV-cDNAs in order to effect secretion. Furthermore, a hexa-histidine tag for purification on a Ni(2+)-nitrilotriacetic acid (Ni(2+)-NTA) column and a protein kinase A (PKA) recognition sequence for in vitro-phospholabeling were fused upstream of the HCV-cDNA. E1- and E2 proteins lacking their carboxy-terminal, hydrophobic sequence were produced by baculovirus-infected insect cells in bioreactors of 23 1. The medium was concentrated and proteins were purified under native conditions on Ni(2+)-NTA columns. Purified proteins could be phospholabeled in vitro using the catalytic subunit of protein kinase. A isolated from bovine heart and gamma-[32P]ATP. Labeled E1 and E2 proteins expressed in insect cells could be immunoprecipitated with sera from HCV-infected patients. Co-expression of these E1 and E2 proteins led to the formation of E1-E2 complexes within the insect cell and to secretion of these complexes into the medium.
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PMID:Purification and in vitro-phospholabeling of secretory envelope proteins E1 and E2 of hepatitis C virus expressed in insect cells. 889 40

Mutants of human neurofibromin and c-Raf-1 genes were fused to the 3' end of the hemagglutinin (HA) gene of influenza A virus by oligonucleotide-directed polymerase chain reaction (PCR). The two resulting chimeric genes, HA (1-534)/NF1 (1441-1518) and HA (1-534)/Raf-1 (51-132) which we designated HN and HR, respectively, were cloned in a vaccinia virus expression vector (pTMI) under the control of a T7 RNA polymerase promoter. The clones were expressed in a monkey cell line (CV-1) and the resulting chimeric proteins analysed. We found that expression levels of the chimeric proteins were similar to that of wild-type HA protein. Comparative endoglycosidase treatment revealed that the expressed chimeric proteins HN and HR were processed as wild-type HA, and FACS-analysis showed that both chimeric expression products localised in the cell membrane as the wild-type control. HN and HR expressing cells showed similar fusogenic activity as CV-1 cells transfected with wild-type HA indicating the correct topology of the fusion inducing portion (HA) of these chimera in the membrane. These findings show that the influenza virus hemagglutinin (HA) is a suitable vehicle to target foreign proteins with therapeutical potential into the cell membrane. In this respect HN and HR could potentially be used to block the abnormal signals generated by particular proteins in the cell membrane that lead to cell transformation.
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PMID:Targeted delivery of human neurofibromin and c-Raf-1 mutants to the cytoplasmic membrane by use of the influenza virus hemagglutinin. 912 41

The orphan receptor ARP-1/COUP-TFII, a member of the chicken ovalbumin upstream promoter transcription factor (COUP-TF) subfamily of nuclear receptors, strongly represses transcriptional activity of numerous genes, including several apolipoprotein-encoding genes. Recently it has been demonstrated that the mechanism by which COUP-TFs reduce transcriptional activity involves active repression and transrepression. To map the domains of ARP-1/COUP-TFII required for repressor activity, a detailed deletion analysis of the protein was performed. Chimeric proteins in which various segments of the ARP-1/COUP-TFII carboxy terminus were fused to the GAL4 DNA binding domain were used to characterize its active repression domain. The smallest segment confering active repressor activity to a heterologous DNA binding domain was found to comprise residues 210 to 414. This domain encompasses the region of ARP-1/COUP-TFII corresponding to helices 3 to 12 in the recently published crystal structure of other members of the nuclear receptor superfamily. It includes the AF-2 AD core domain formed by helix 12 but not the hinge region, which is essential for interaction with a corepressor in the case of the thyroid hormone and retinoic acid receptor. Attachment of the nuclear localization signal from the simian virus 40 large T antigen (Flu tag) to the amino terminus of ARP-1/COUP-TFII abolished its ability to bind to DNA without affecting its repressor activity. By using a series of Flu-tagged mutants, the domains required for transrepressor activity of the protein were mapped. They include the DNA binding domain and the segment spanning residues 193 to 399. Transcriptional activity induced by liver-enriched transactivators such as hepatocyte nuclear factor 3 (HNF-3), C/EBP, or HNF-4 was repressed by ARP-1/COUP-TFII independent of the presence of its cognate binding site, while basal transcription or transcriptional activity induced by ATF or Sp1 was not perturbed by the protein. In conclusion, our results demonstrate that the domains of ARP-1/COUP-TFII required for active repression and transrepression do not coincide. Moreover, they strongly suggest that transrepression is the predominant mechanism underlying repressor activity of ARP-1/COUP-TFII. This mechanism most likely involves interaction of the protein with one or several transcriptional coactivator proteins which are employed by various liver-enriched transactivators but not by ubiquitous factors such as Sp1 or ATF.
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PMID:Functional domains of the human orphan receptor ARP-1/COUP-TFII involved in active repression and transrepression. 927 71

The effect of the cytoplasmic tail of influenza hemagglutinin (HA) (H3 subtype) on fusion kinetics and pore growth was examined An SV40 recombinant virus was used to express wild-type (WT) HA and HA mutants containing changes in the HA cytoplasmic tail. HA and its mutants were expressed in CV-1 cells and the ability of these cells to fuse to either red blood cells (RBCs) or planar bilayer membranes was determined quantitatively. The percentage of cells expressing HA and the levels of expression were the same for WT HA or HA lacking its cytoplasmic tail (CT-), and for a mutant, MAY, in which the three HA C-terminal cysteine residues were replaced to block the addition of palmitate. When RBCs were colabeled with large and small aqueous dyes and fused to CV-1 cells expressing WT HA, transfer of the large dye was significantly slower and extent of transfer was lower than that of the small dye, indicating that pores did not expand quickly to large diameters. An absence of the HA cytoplasmic tail did not alter the time course of spread for either dye. When CV-1 cells expressing WT HA were fused to planar membranes, small pores tended to open and close repetitively ("flicker") before a pore would continue to either grow irreversibly to large conductances or grow to intermediate sizes and then contract. For HA mutants CT- and MAY, flickering was less likely to occur, but these pores did evolve in a manner identical to WT HA postflicker pores. We conclude that palmitate covalently linked to cysteine residues of the HA cytoplasmic tail is required for pore flickering, but that the tail does not play an important role in subsequent pore enlargement.
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PMID:The role of the cytoplasmic tail region of influenza virus hemagglutinin in formation and growth of fusion pores. 930 43

Two new fluorescent lysophosphatidylcholine probes have been synthesized for use as a donor-acceptor pair in fluorescence resonance energy transfer (FRET): 9-anthrylvinyl (LAPC) as donor and 3-perylenoyl (LPPC) as acceptor. The partition coefficients between membrane and aqueous phases were 8.3 x 10(5) and 10.5 x 10(5) for LAPC and LPPC, respectively. The inner leaflets of unilamellar lipid vesicles were labeled with these probes to assess conservation of membrane sidedness after membrane fusion. After medium-sized unilamellar vesicles (MUV) were prepared with a probe in both leaflets, probe in the outer leaflet was removed by repeatedly washing with an excess of unlabeled giant unilamellar vesicles (GUV). MUV and GUV were separated by centrifugation. The probes did not flip-flop across bilayers at 25 degrees C for at least 12 h. MUV containing the ganglioside GT1b were labeled with the LAPC/LPPC pair in the inner leaflet and incubated for 30 min at neutral pH with influenza virus. Fusion was triggered by acidification to pH 5.0 and was monitored by an increase in donor fluorescence in a FRET assay. When the inner leaflets of MUV were labeled by LAPC only, its fluorescence did not change after fusion. However, the fluorescence decreased by 60% when the LAPC was removed from the outer leaflets of the fused membranes by repeated washings with GUV. We conclude that the lipids of the inner and outer leaflets of the fused MUV/virus complexes intermixed.
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PMID:New fluorescent lysolipids: preparation and selective labeling of inner liposome leaflet. 937 Feb 52

Influenza virus nucleoprotein (NP) shuttles between the nucleus and the cytoplasm. A nuclear localization signal (NLS) has been identified in NP at amino acids 327 to 345 (J. Davey et al., Cell 40:667-675, 1985). However, some NP mutants that lack this region still localize to the nucleus, suggesting an additional NLS in NP. We therefore investigated the nucleocytoplasmic transport of NP from influenza virus A/WSN/33 (H1N1). NP deletion constructs lacking the 38 N-terminal amino acids, as well as those lacking the 38 N-terminal amino acids and the previously identified NLS, localized to both the cytoplasm and the nucleus. Nuclear localization of a protein containing amino acids 1 to 38 of NP fused to LacZ proved that these 38 amino acids function as an NLS. Within this region, we identified two basic amino acids, Lys7 and Arg8, that are crucial for NP nuclear import. After being imported into the nucleus, the wild-type NP and the NP-LacZ fusion construct containing amino acids 1 to 38 of NP were both transported back to the cytoplasm, where they accumulated. These data indicate that NP has intrinsic structural features that allow nuclear import, nuclear export, and cytoplasmic accumulation in the absence of any other viral proteins. Further, the information required for nuclear import and export is located in the 38 N-terminal amino acids of NP, although other NP nuclear export signals may exist. Treatment of cells with a protein kinase C inhibitor increased the amounts of nuclear NP, whereas treatment of cells with a phosphorylation stimulator increased the amounts of cytoplasmic NP. These findings suggest a role of phosphorylation in nucleocytoplasmic transport of NP.
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PMID:Nuclear import and export of influenza virus nucleoprotein. 937 35

In polarized Madin-Darby canine kidney (MDCK) cells, sorting of membrane proteins in the trans-Golgi network for basolateral delivery depends on the presence of cytoplasmic determinants that are related or unrelated to clathrin-coated pit localization signals. Whether these signals mediate basolateral protein sorting through common or distinct pathways is unknown. The cytoplasmic domain of the cation-dependent mannose 6-phosphate receptor (CD-MPR) contains clathrin-coated pit localization signals that are necessary for endocytosis and lysosomal enzyme targeting. In this study, we have addressed the function of these signals in polarized sorting of the CD-MPR. A chimeric protein, made of the luminal domain of the influenza virus hemagglutinin fused to the transmembrane and cytoplasmic domains of the CD-MPR was stably expressed in MDCK cells. This chimera (HCD) is able to interact with the AP-1 Golgi-specific assembly proteins and is detected on the basolateral plasma membrane of MDCK cells where it is endocytosed. Deletion analysis and site-directed mutagenesis of the cytoplasmic domain of the CD-MPR indicate that HCD chimeras devoid of clathrin-coated pit localization signals are still transported to the basolateral membrane where they accumulate. A HCD chimera containing only the transmembrane domain and the 12 membrane-proximal amino acids of the CD-MPR cytoplasmic tail is also found on the basolateral membrane but is unable to interact with the AP-1 assembly proteins. However, the overexpression of this mutant results in partial apical delivery. It is concluded, therefore, that the basolateral transport of this chimera requires a saturable sorting machinery distinct from AP-1.
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PMID:Basolateral sorting of the cation-dependent mannose 6-phosphate receptor in Madin-Darby canine kidney cells. Identification of a basolateral determinant unrelated to clathrin-coated pit localization signals. 941 63

The inner leaflet of unilamellar lipid vesicles was labeled with fluorescent lysophosphatidylcholines. The probes make a donor-acceptor pair in resonance energy transfer (RET), being labeled with 9-anthrylvinyl (L-APC, donor) and 3-perylenoyl (L-PPC, acceptor) fluorophores. They migrate rapidly between bilayers through the water phase: tau 1/2 of equilibration is approximately 5 min at 37 degrees C. The probe(s) can be removed from the outer leaflet of uniformly labeled medium-size unilamellar vesicles (MUV) by repeated washings with excess unlabeled large unilamellar vesicles (LUV) (separation by centrifugation). The probes flip-flop across bilayers rather slowly. MUV containing the ganglioside GT1b and labeled with the L-APC/L-PPC pair in the inner leaflet were fused with an equal amount of influenza virus; the process was monitored by an increase of the donor fluorescence in RET assay. If inner MUV leaflet was labeled with the anthrylvinyl probe only, the probe fluorescence decreased by half when the probe was removed from the outer leaflets of the fused membranes. This shows that the lipids of the inner and outer leaflets of the MUV randomize in the process of fusion.
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PMID:Selective labeling of the inner liposome leaflet by fluorescent lipid probes, and studies of liposome fusion with influenza virus. 955 39


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