Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.14.14.3 (luciferase)
 38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have constructed an artificial ligand for the hepatocyte-specific asialoglycoprotein receptor for the purpose of generating a synthetic delivery system for DNA. This ligand has a tetra-antennary structure, containing four terminal galactose residues on a branched carrier peptide. The carbohydrate residues of this glycopeptide were introduced by reductive coupling of lactose to the alpha- and epsilon-amino groups of the two N-terminal lysines on the carrier peptide. The C-terminus of the peptide, containing a cysteine separated from the branched N-terminus by a 10 amino acid spacer sequence, was used for conjugation to 3-(2-pyridyldithio)propionate-modified polylysine via disulfide bond formation. Complexes containing plasmid DNA bound to these galactose-polylysine conjugates have been used for asialoglycoprotein receptor-mediated transfer of a luciferase gene into human (HepG2) and murine (BNL CL.2) hepatocyte cell lines. Gene transfer was strongly promoted when amphipathic peptides with pH-controlled membrane-disruption activity, derived from the N-terminal sequence of influenza virus hemagglutinin HA-2, were also present in these DNA complexes. Thus, we have essentially borrowed the small functional domains of two large proteins, asialoglycoprotein and hemagglutinin, and assembled them into a supramolecular complex to generate an efficient gene-transfer system.
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PMID:Gene transfer into hepatocytes using asialoglycoprotein receptor mediated endocytosis of DNA complexed with an artificial tetra-antennary galactose ligand. 146 83

Complexes containing plasmid DNA, transferrin-polylysine conjugates, and polylysine-conjugated peptides derived from the N-terminal sequence of the influenza virus hemagglutinin subunit HA-2 have been used for the transfer of luciferase or beta-galactosidase marker genes to K562 cells, HeLa cells, and BNL CL.2 hepatocytes. These DNA complexes mimic the entry of viruses into cells, as they contain functions for (i) the packaging of the nucleic acid with polylysine, (ii) the attachment to the cell and receptor-mediated endocytosis with transferrin as a ligand, and (iii) the release from endosomes by using membrane-disrupting influenza peptides. The presence of these influenza peptide conjugates in the DNA complexes renders the complexes active in membrane disruption in a liposome leakage assay and results in a substantial augmentation of the transferrin-polylysine-mediated gene transfer.
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PMID:Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin-polylysine-DNA complexes: toward a synthetic virus-like gene-transfer vehicle. 151 16

A new procedure for a sialidase assay, by bioluminescence, has been developed. The substrate, N- acetylneuraminyllactose (sialyllactose), hydrolysed by the sialidase activity, releases lactose. This lactose is hydrolysed with beta-galactosidase. The released galactose is oxidized with galactose dehydrogenase and NAD. The NADH produced in the last step is measured by a luminescence system, coupling two enzymes, NAD(P)H dehydrogenase (FMN) and luciferase. This microassay, which is specific, rapid, simple and ultra-sensitive, is a measure for amounts as little as (at least) 5 pmol of N-acetylneuraminic acid (corresponding to 0.15 ng of the released sialic acid). It uses commercialized reagents (non-radioisotopic) and avoids interferences common in other procedures. This method has been used for measuring sialidase activity directly on intact virus, avoiding inconvenient modifications produced in the extraction of the enzyme. The specific activity of sialidase of influenza virus X31 (H3N2), determined by this procedure, is 0.65 U/mg of total virus protein.
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PMID:Sialidase assay by luminescence in the low picomole-range of sialic acid. Its application to the measurement of this activity in influenza virus. 673 52

We characterize a method by which the Med-E-Jet pneumatic vaccination gun can be used to propel intact, supercoiled plasmid DNA through skin and into skeletal muscles of mice. Intramuscular injection of plasmids containing the firefly luciferase gene linked to the human cytomegalovirus promoter resulted in the expression of several hundred picograms of luciferase enzyme in quadriceps muscles. Intramuscular injections of a plasmid containing the influenza A nuclear protein gene regulated by the same promoter resulted in the generation of potent and specific anti-nuclear protein humoral and cellular immune responses. This convenient and rapid injection method would be well-suited for genetic immunization of humans.
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PMID:Immunization with plasmid DNA using a pneumatic gun. 793 Jun 33

The process by which viruses destabilize endosomal membranes in an acidification-dependent manner has been mimicked with synthetic peptides that are able to disrupt liposomes, erythrocytes, or endosomes of cultured cells. Peptides containing the 20 amino-terminal amino acid sequence of influenza virus hemagglutinin as well as acidic derivatives showed erythrocyte lysis activity only when peptides were elongated by an amphipathic helix or by carboxyl-terminal dimerization. Interestingly, peptides consisting of the 23 amino-terminal amino acids of influenza virus hemagglutinin were also active in erythrocyte lysis. When peptides were incorporated into DNA complexes that utilize a receptor-mediated endocytosis pathway for uptake into cultured cells, either by ionic interaction with positively charged polylysine-DNA complexes or by a streptavidin-biotin bridge, a strong correlation between pH-specific erythrocyte disruption activity and gene transfer was observed. A high-level expression of luciferase or interleukin-2 was obtained with optimized gene transfer complexes in human melanoma cells and several cell lines.
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PMID:The influence of endosome-disruptive peptides on gene transfer using synthetic virus-like gene transfer systems. 817 9

Plasmid DNA/glycosylated polylysine complexes were used to transfer in vitro a luciferase reporter gene into human hepatoma cells by a receptor-mediated endocytosis process. HepG2 cells which express a galactose specific membrane lectin were efficiently and selectively transfected with pSV2Luc/lactosylated polylysine complexes in a sugar dependent manner: i) HepG2 cells which do not express membrane lectin specific for mannose were quite poorly transfected with pSV2Luc/mannosylated polylysine complexes, ii) HeLa cells which do not express membrane lectin specific for galactose were not transfected with pSV2Luc/lactosylated polylysine complexes. The transfection efficiency of HepG2 cells with pSV2Luc/lactosylated polylysine complexes was greatly enhanced either in the presence of chloroquine or in the presence of a fusogenic peptide. A 22-residue peptide derived from the influenza virus hemagglutinin HA2 N-terminal polypeptide that mimics the fusogenic activity of the virus, was selected. In the presence of the fusogenic peptide, the luciferase activity in HepG2 cells was 10 fold larger than that of cells transfected with pSV2Luc/lactosylated polylysine complexes in the presence of chloroquine.
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PMID:Specific gene transfer mediated by lactosylated poly-L-lysine into hepatoma cells. 838 43

Poliovirus protease 2A(pro) has been obtained in soluble form as a fusion protein with maltose binding protein (MBP). Addition of MBP-2A(pro) to rabbit reticulocyte cell-free systems gives rise to efficient cleavage of the initiation factor of translation p220 (eIF-4G). Translation of capped mRNA encoding the influenza virus NP protein is severely impaired in lysates in which p220 has been proteolytically cleaved. This inhibition is dependent on the concentration of mRNA added to the lysate. Thus, increasing the concentrations of mRNA substantially overcomes the blockade of NP synthesis after p220 cleavage. Notably, translation of uncapped NP mRNA is also compromised in p220-deficient rabbit reticulocyte lysates, suggesting that p220 participates in the translation of both capped and uncapped NP mRNAs. The effects of p220 proteolysis by poliovirus 2A(pro) have also been assayed on luciferase mRNA translation. Three types of mRNAs encoding for luciferase have been examined: capped, uncapped, and mRNA bearing the poliovirus 5' leader region (leader luc mRNA). Synthesis of luciferase directed by any of these mRNAs was inhibited after cleavage of p220 in rabbit reticulocyte lysates. Interestingly, supplementation of the lysate with HeLa cell extracts stimulates leader luc mRNA translation by poliovirus 2A(pro). These results indicate that activation of translation of mRNAs bearing the poliovirus leader region promoted by this poliovirus protease requires a factor present in HeLa cell extracts. These findings agree well with recent experiments implicating p220 not only in protein synthesis directed by capped mRNAs but also in the translation of naturally uncapped mRNAs.
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PMID:Cleavage of p220 by purified poliovirus 2A(pro) in cell-free systems: effects on translation of capped and uncapped mRNAs. 920 23

Potential problems with the use of viral vectors for gene therapy necessitate the development of efficient nonviral vectors. The association of transferrin, or the pH-sensitive peptide GALA, with cationic liposomes composed of 1,2-dioleoyl-3-(trimethylammonium) propane and its equimolar mixture with dioleoylphosphatidylethanolamine, under conditions where the liposome/DNA complex is negatively charged, drastically increased luciferase expression from pCMVluc. The percentage of cells transfected, measured by beta-galactosidase expression, was also increased by about 10-fold. The zeta potential of the ternary complexes was lower than that of the liposome/DNA complexes. Transfection activity of positively charged complexes was also enhanced by association with transferrin, GALA or the influenza hemagglutinin N terminal peptide HA-2, but to a smaller extent compared with the negatively charged complexes. The enhancement of gene delivery by transferrin or GALA was not affected significantly by the presence of serum and did not cause significant cytotoxicity. Our results indicate that negatively charged ternary complexes of cationic liposomes, DNA and transferrin, or fusigenic peptides, can facilitate efficient transfection of cultured cells, and that they may alleviate the drawbacks of the use of highly positively charged complexes for gene delivery in vivo.
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PMID:Gene delivery by negatively charged ternary complexes of DNA, cationic liposomes and transferrin or fusigenic peptides. 981 67

We have used spring powered jet injectors to deliver a solution of a naked DNA vaccine encoding the influenza hemagglutinin HA into the skin of mice and monkeys. We compared the immune responses induced by this needleless injection technique into the skin to the responses induced by a classical i.m. immunization. Both routes of immunization induced significant ELISA antibody titers and hemagglutination inhibition (HI) titers that were above the usual threshold values predictive of protection against influenza in mice and monkeys. In mice, both ways of immunization were equally efficient in inducing HA-specific CTL responses. Regarding antibody isotypes, the IgG1/IgG2a ratio was in favour of the IgG2a isotype for i.m. immunization and more balanced for i.d. immunization. The ability of the two injection techniques to induce immunity in mice did not correlate with transgene expression in the site of administration. In fact, local gene expression was 10-100 fold more important in the injected muscle as compared to the jet-injected skin when assessed by using the luciferase reporter system.
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PMID:Intradermal DNA immunization by using jet-injectors in mice and monkeys. 1006 67

Intramuscular injection of plasmid DNA results in myofiber cell expression of proteins encoded by the DNA. The preferred vehicle for plasmid DNA injections has been saline (154 mM sodium chloride) or PBS (154 mM NaCl plus 10 mM sodium phosphate). Here, it is shown that injection of luciferase or beta-galactosidase encoding plasmid DNA in a 150 mM sodium phosphate vehicle into murine muscle resulted in a two- to seven-fold increase in transgene expression compared with DNA injected in saline or PBS. When the DNA encoded secreted alkaline phosphatase, preproinsulin or interferon, sodium phosphate vehicle increased their serum levels by two- to four-fold. When the DNA encoded mouse erythropoietin, sodium phosphate vehicle increased hematocrits by two-fold compared with DNA injected in saline. When the DNA encoded influenza nucleoprotein, sodium phosphate increased anti-nucleoprotein antibody titers by two-fold. The expression of luciferase from plasmid DNA instilled into lung was increased five-fold compared with that in vehicle without sodium phosphate. Incubation of plasmid DNA with muscle extract or serum showed that sodium phosphate protected the DNA from degradation. Thus, a change from sodium chloride to sodium phosphate vehicle can enhance the expression of plasmid DNA in a tissue, possibly by inhibiting DNA degradation. Gene Therapy (2000) 7, 1171-1182.
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PMID:Sodium phosphate enhances plasmid DNA expression in vivo. 1091 85


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