Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UMLS:C0021051 (immunodeficiency)
71,517 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The infectivity of the human immunodeficiency virus (HIV) depends upon correct proteolytic processing of viral polyprotein precursors, the Pr55gag and Pr160gag-pol polyproteins. The processing is mediated spontaneously by the viral protease unit (PR) contained within the Pr160gag-pol precursor. However, little is known about the mechanism of this process. The expression in Escherichia coli and the isolation of a 14-kDa HIV-1 PR "miniprecursor" with Ala28 mutated to serine has permitted study of the mechanism for cleavage at the N-terminus of the protease. The miniprecursor is active against a synthetic peptide substrate, and its specific activity is near that of the mutant mature protease. The rate of conversion of radiolabeled precursor to mature protease is quantitated by measuring the amounts of the two radiolabeled proteins separated by SDS-PAGE. The apparent first-order conversion rate constant, kapp, is dependent on miniprecursor concentration indicating a second-order reaction and suggesting an interdimeric processing mechanism. A significant first-order rate constant is observed when the plot of kapp versus initial precursor concentration is extrapolated to zero. This observation suggests the presence of an alternative processing mechanism involving a single active precursor dimer. The presence of both mechanisms is an advantage for the virus to ensure processing under various conditions.
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PMID:Proteolytic processing mechanisms of a miniprecursor of the aspartic protease of human immunodeficiency virus type 1. 811 Jul 58

A large-scale immunoaffinity (IA) purification process was developed for the isolation of recombinant soluble antigen CD4 (sCD4) from Escherichia coli fermentations. The monoclonal antibody used for IA purification of sCD4 recognized a conformation-dependent epitope on the surface of domain 1 of CD4. IA chromatography was used to purify both sCD4-183, consisting of the N-terminal 183 amino acids of human CD4, and sCD4-PE40, a fusion protein consisting of the N-terminal 178 amino acids of CD4 and amino acids 1-3 and 253-613 of Pseudomonas exotoxin A (PE40). sCD4-183 was purified from E. coli cell pellets using cell disruption, protein solubilization, oxidation, Q-Sepharose anion-exchange and IA chromatography steps. sCD4-PE40 was purified from cell pellets using cell disruption, protein solubilization, oxidation, Cu(2+)-immobilized metal-affinity chromatography, anion-exchange and IA chromatography steps. The IA-purified sCD4 analogues demonstrated the correct apparent molecular masses on SDS/PAGE. The immobilized monoclonal antibody appeared to select for correctly folded CD4 protein, since sCD4-183 and sCD4-PE40 purified by the IA method bound human-immunodeficiency-virus glycoprotein gp120 (HIV gp120) in vitro. sCD4-PE40 purified by IA chromatography also inhibited protein synthesis in CV-1 cells expressing HIV gp120/160 at the cell surface. Relatively high recoveries of sCD4-183 and sCD4-PE40 were observed in the IA step of the purification process (71 and 79% recovery respectively). The results demonstrate that immobilized monoclonal antibodies directed against conformational epitopes may be used for rapid purification of gram amounts of correctly folded protein from mixtures of oxidized E. coli proteins.
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PMID:Large-scale immunoaffinity purification of recombinant soluble human antigen CD4 from Escherichia coli cells. 829 11

A DNA binding assay was developed for the human immunodeficiency virus type 1 (HIV-1) integrase. The assay was capable of defining discrete complexes between the enzyme and the viral long terminal repeat (LTR) substrate. DNA binding reflected the sequence requirements previously demonstrated for the enzyme's 3'-end processing activity. Binding exhibited a nonlinear dependence on integrase concentration, suggesting that the enzyme functions as a multimer. The oligomeric state was investigated by UV-photo-cross-linking of integrase-LTR oligonucleotide complexes using DNA substrates substituted with 5-bromo-2'-deoxycytidine within the integrase recognition sequence. In the absence of divalent cation, integrase cross-linked to the LTR oligonucleotide as a single species whose mobility by SDS-polyacrylamide gel electrophoresis was consistent with the formation of tetramers. Using these techniques, analysis of the binding properties of integrase mutants demonstrated that the catalytic and sequence-specific DNA binding activities of the enzyme are distinct, involving residues within the conserved "DD(35)E" and zinc finger motifs, respectively.
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PMID:Viral long terminal repeat substrate binding characteristics of the human immunodeficiency virus type 1 integrase. 830 56

Putative cell surface human immunodeficiency virus (HIV) gp41 receptor proteins of 45 and 80 kDa (p45 and p80, respectively) were identified on human cells using a 17-amino acid peptide, referred to as CS3. In contrast, murine P815 cells expressed a peptide binding protein of 80 kDa only. A segment of 8 amino acids within CS3 contains the minimum sequence able to inhibit binding of radiolabeled CS3 to p80 and p45, as shown by competitive binding studies. Human p45 was purified from CD4+ RH9 cells by CS3 peptide affinity chromatography. Human p80 was partially purified from RH9 cell lysates by size exclusion chromatography followed by SDS-polyacrylamide gel electrophoresis; a rabbit polyclonal antibody was raised against this preparation. Anti-p80 antibody inhibited HIV infection in a dose-dependent manner. The CS3 region of gp41 has been been shown previously to be exposed on viral particles and envelope-expressing cells predominately after conformational changes in the HIV envelope occur due to the interaction of CD4 with gp120. These results, together with those from previous studies, suggest that following the interaction of gp120 with CD4, there may be a second receptor interaction necessary for virus entry/fusion.
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PMID:A peptide inhibitor of human immunodeficiency virus infection binds to novel human cell surface polypeptides. 832 99

The single-stranded nucleocapsid protein that coats the RNA genome of human immunodeficiency virus within the virion core has been produced in Escherichia coli and purified to homogeneity. The mature 55-amino acid protein, normally generated from the gag polyprotein precursor by HIV protease-catalyzed processing of both its amino and carboxyl termini, was produced in E. coli with authentic termini directly, without the need for processing. The protein was purified 30-fold to apparent homogeneity, as determined by both amino acid analysis and SDS-polyacrylamide gel electrophoresis. Sequencing of each terminus of the purified protein indicated that no proteolytic degradation occurred. A molar extinction coefficient (epsilon 280 = 8350 cm-1 M-1) was determined. The purified nucleocapsid protein binds tightly to single-stranded RNA as judged by a nitrocellulose filter binding assay. A binding constant (Kw) of 1 x 10(8) M-1 was calculated. Using fluorescence quenching of nucleocapsid protein upon RNA binding as an assay, a binding site size of seven nucleotides was determined. These results contrast to a larger 15-nucleotide site measured by others for a larger form of nucleocapsid protein-containing sequences from its immature precursor. The possible relevance of these findings are discussed.
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PMID:HIV nucleocapsid protein. Expression in Escherichia coli, purification, and characterization. 834 33

The protease encoded by the human immunodeficiency virus type 1 (HIV-1) was engineered in Escherichia coli as a construct in which the natural 99-residue polypeptide was preceded by an NH2-terminal methionine initiator. Inclusion bodies harboring the recombinant HIV-1 protease were dissolved in 50% acetic acid and the solution was subjected to gel filtration on a column of Sephadex G-75. The protein, eluted in the second of two peaks, migrated in SDS-PAGE as a single sharp band of M(r) approximately 10,000. The purified HIV-1 protease was refolded into an active enzyme by diluting a solution of the protein in 50% acetic acid with 25 volumes of buffer at pH 5.5. This method of purification, which has also been applied to the purification of HIV-2 protease, provides a single-step procedure to produce 100 mg quantities of fully active enzyme.
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PMID:Large scale purification and refolding of HIV-1 protease from Escherichia coli inclusion bodies. 839 90

A principal neutralizing determinant of human immunodeficiency virus type 1 (HIV-1) lies within the V3 loop of gp120, the external major envelope glycoprotein. V3 loop peptides derived from two HIV-1 strains, HTLV-III BH-10 (V3-BH10) and LAVELI (V3-ELI), were synthesized and biotinylated. The binding of both biotinylated V3-BH10 and V3-ELI to the surfaces of MOLT-4 clone 8 cells was demonstrated by flow cytometric analyses. Both the peptides (more than 2 microM) bound to the cells (2 x 10(5) in a dose-dependent manner. The binding of biotinylated V3-BH10 was specifically inhibited by a neutralizing monoclonal antibody (0.5 beta). The binding of both of the biotinylated V3 loop peptides was enhanced by the addition of unlabeled V3-BH10. In addition, the peptides were employed as ligands on affinity columns. A major V3 loop binding protein (V3BP) was purified from the membrane soluble fraction of MOLT-4 cells by successive application to two different V3 loop columns. V3BP consisted of two major polypeptides (32 and 33 kDa). The SDS-PAGE profile of V3BP did not change under non-reducing conditions, but only a single band was observed after analysis on native PAGE. The major peak of the eluate as determined by size exclusion chromatography was broad and the estimated relative molecular mass was much larger than 33 kDa, suggesting that V3BP comprises several subunits. Taken together, we confirmed that the V3 loop peptides are useful in the characterization of V3BP(s) of which they are conformational ligands.
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PMID:Applications of biotinylated V3 loop peptides of human immunodeficiency virus type 1 to flow cytometric analyses and affinity chromatographic techniques. 848 4

Translational incorporation of the unusual amino acid selenocysteine in eukaryotes requires a coding region UGA codon (which otherwise serves as a termination signal), a selenocysteine insertion sequence (SECIS) in the 3'-untranslated region of the mRNA, and selenocysteyl-tRNA. The mechanisms involved in SECIS recognition by the eukaryotic translational machinery remain unknown. We report the detection of RNA-binding proteins that specifically recognize the SECIS from human cellular glutathione peroxidase (GPX1) transcripts. RNA gel shift assays showed three retarded bands after incubation with COS-1 whole cell lysate or S-100 cytosol fraction or with extracts from hepatoma cell lines HepG2 and Hep3B. The specificity of the binding was demonstrated by competition by cold unlabeled SECIS RNA and by lack of competition by other RNA species with similar stem-loop secondary structures, such as the human immunodeficiency virus (HIV) transactivation-response region of HIV mRNA element, and mutated SECIS constructs. UV cross-linking and SDS-polyacrylamide gel electrophoresis revealed at least two proteins, with estimated molecular masses of 55,000 and 65,000 Da, that bind to the SECIS. Examination of a series of insertion and deletion SECIS mutants indicated recognition of the SECIS primarily through the basal stem region, although the upper stem, loop, and two of three short conserved sequences also appear to contribute to the affinity of the binding.
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PMID:RNA-binding proteins that specifically recognize the selenocysteine insertion sequence of human cellular glutathione peroxidase mRNA. 853 Apr 73

Previous reports have shown that cyclophilin A (CyPA) is found to be specifically associated with human immunodeficiency virus type-1 (HIV-1) virions and is required for infectivity (Franke et al. Nature 372:359; Thali et al. Nature 372:363). We have examined CyPA associated with HIV-1MN virions. Virions from infected human lymphoid cells were analyzed by high-pressure liquid chromatography (HPLC), protein sequence, and immunoblot analysis. At least three forms of CyPA were found: an unmodified form, an N-terminally modified form, and an N-terminally modified form that migrates as a larger isoform on a reducing-SDS polyacrylamide gel. Using a protease digestion procedure, CyPA that is associated with virions was found to be located inside the viral membrane. Similar examination of SIVMne produced by HUT-78 human T cells did not detect specific incorporation of CyPA into SIV virions. Our results are consistent with the role of CyPA acting early in the infectious process of HIV-1.
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PMID:Analysis and localization of cyclophilin A found in the virions of human immunodeficiency virus type 1 MN strain. 855 96

We have previously demonstrated that the membrane of the Staphylococcus aureus L form induced tumor necrosis factor alpha (TNF-alpha) from murine macrophages. In this study, we purified two proteins which induce TNF-alpha production from a human monocytic cell line, THP-1, and murine macrophages. These molecules were purified from delipidated membranes by deoxycholic acid extraction, two-step anion-exchange chromatography, and preparative electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified proteins showed for each a single band with a molecular mass of 30, and 36 kDa. These proteins were heat stable. Polymyxin B did not affect the production of TNF-alpha induced by these proteins. Furthermore, these proteins induced comparable levels of TNF-alpha in both lipopolysaccharide-responsive and -nonresponsive mouse macrophages. Pretreatment of murine macrophages with gamma interferon enhanced 30- and 36-kDa protein-mediated TNF-alpha production. The 30-kDa protein showed lethal toxicity to D-galactosamine-treated mice. The 30- and 36-kDa proteins stimulated the human immunodeficiency virus type 1 long terminal repeat in a monocytic cell line but not a T-cell line. This effect appeared to be mediated through the induction of nuclear factor kappaB. These results indicate that the 30- and 36-kDa proteins, membrane constituents of the S. aureus L form, may play a role in S. aureus infection and/or in human immunodeficiency virus type 1-infected individuals.
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PMID:Proteins of 30 and 36 kilodaltons, membrane constituents of the Staphylococcus aureus L form, induce production of tumor necrosis factor alpha and activate the human immunodeficiency virus type 1 long terminal repeat. 875 63


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