Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019693 (HIV)
 170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A recombinant HIV-1 gp120 (rgp120) was expressed in a permanent Chinese Hamster Ovary (CHO) cell line (L761h) that constitutively secretes the product of clone p4 derived from the env gene of HIV-1 isolate GB8. The rgp120 was isolated from cell culture supernatants by a simple, rapid, non-denaturing and efficient purification procedure based on a novel combination of lectin affinity and FPLC ion-exchange chromatography. The purity of the isolated glycoprotein was rigorously confirmed by SDS-PAGE, capillary electrophoresis, laser desorption mass spectrometry, total amino acid analysis and N-terminal amino acid sequencing. The retention of biological activity by the purified rgp120 was assessed by determining the dissociation constant of rgp120 binding to sCD4. After formulation of this highly purified and biologically active rgp120 with "conventional" adjuvants, including types already used in clinical trials of candidate gp120-based HIV vaccines, antibody responses in immunised rabbits were analysed using panels of overlapping synthetic peptides. The consequences of using currently available adjuvants to deliver highly specialised and perhaps conformation-dependent molecules, like HIV gp120, are presented and discussed in the context of HIV vaccine development.
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PMID:Efficient purification and rigorous characterisation of a recombinant gp120 for HIV vaccine studies. 852 94

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

Matrix-associated laser desorption ionization/mass spectrometry (MALDI/MS) has been used to examine whole bacteria for the presence of a recombinant HIV p26 fusion protein. MALDI/MS, combined with affinity-purification techniques, is also shown to be very useful in monitoring the enzymatic cleavage of both affinity-bound fusion protein and fusion protein in solution. The combination of mass resolution, sensitivity, and speed of analysis makes MALDI/MS an attractive alternative to SDS-PAGE.
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PMID:Monitoring cleavage of fusion proteins by matrix-assisted laser desorption ionization/mass spectrometry: recombinant HIV-1IIIB p26. 866 Jun 21

The humoral immune response of patients infected with Cryptococcus neoformans var. neoformans and C. neoformans var. gattii to cytoplasmic (non-capsular) antigens from the two varieties of Cryptococcus has been investigated. Cytoplasmic antigens from C. neoformans (one clinical isolate and one acapsular mutant of var. neoformans and two clinical isolates from var. gattii) were subject to isoelectric focusing, SDS-PAGE and Western blotting; patients sera was then used in the immunoenzyme development of the Western blots. The humoral response from the 20 patients (all HIV+) infected with var. neoformans against the var. neoformans antigens was predominantly IgG based, with a large number of bands recognised: the most commonly recognised bands were at 26, 52, 74, 100, 115 and 144 kDa. The IgM response was less pronounced and the IgA response was practically non-existent. The humoral response of the sera from the 15 patients (all but one HIV-) infected with var. gattii against var. gattii antigens was also predominantly IgG based with bands at 37, 55, 65, 74, 94 and 115 kDa being most commonly recognised. Periodate treatment of cytoplasmic antigens reduced the intensity of antigen recognition, though it did not absolutely destroy reactivity to any individual antigen. Comparison of immunodevelopment of cytoplasmic antigens from both varieties grown at 25 degrees C and 37 degrees C revealed that culture temperature made no differences in the number of bands recognised although there were differences in the intensity of recognition. This is the first report on the pattern of serological recognition of the non-capsular antigens from the two varieties of Cryptococcus and it identifies a number of major antigenic components.
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PMID:Recognition of cytoplasmic yeast antigens of Cryptococcus neoformans var. neoformans and Cryptococcus neoformans var. gattii by immune human sera. 906 57

In the course of investigating effective biological active substances, we detected a substance in an extract of silkworm faeces that markedly suppresses viral production. The extract, prepared with hot phosphate-buffered saline and purified with ammonium sulfate precipitation, inhibited HVJ (Sendai virus), HSV (herpes simplex virus type-1), and HIV (human immunodeficiency virus type-1), but not poliovirus, suggesting that it is effective on enveloped virus production but not on non-enveloped ones. In the case of HVJ, indirect immunofluorescent staining using anti-HVJ antibody and Northern blotting analysis showed that, while viral adsorption and entry into the host cells were not affected, the synthesis of viral specific gene was inhibited by pretreatment of the virions with the extract. The extract affected more effectively aged virion, which losses membrane function as barrier and its envelope is leaky, than young virion that maintains barrier function. The active substance was partially purified by gel filtration after treatment of the extract with 1 N NaOH solution. From analysis with SDS-PAGE (SDS-polyacrylamide gel electrophoresis), protein bands were detected with molecular masses of about 25 kDa and near 14 kDa, while sugars were also detected with lectin blotting.
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PMID:Suppression of enveloped virus production with a substance from silkworm faeces. 907 8

The fusion domain of human immunodeficiency virus (HIV-1) envelope glycoprotein (gp120-gp41) is a conserved hydrophobic region located at the N terminus of the transmembrane glycoprotein (gp41). A V2E mutant has been shown to dominantly interfere with wild-type envelope-mediated syncytium formation and virus infectivity. To understand this phenomenon, a 33-residue peptide (wild type, WT) identical to the N-terminal segment of gp41 and its V2E mutant were synthesized, fluorescently labeled, and characterized. Both peptides inhibited HIV-1 envelope-mediated cell-cell fusion and had similar alpha-helical content in membrane mimetic environments. Studies with fluorescently labeled peptide analogues revealed that both peptides have high affinity for phospholipid membranes, are susceptible to digestion by proteinase-K in their membrane-bound state, and tend to self- and coassemble in the membranes. In SDS-polyacrylamide gel electrophoresis the WT peptide formed dimers as well as higher order oligomers, whereas the V2E mutant only formed dimers. The WT, but not the V2E mutant, induced liposome aggregation, destabilization, and fusion. Moreover, the V2E mutant inhibited vesicle fusion induced by the WT peptide, probably by forming inactive heteroaggregates. These data form the basis for an explanation of the mechanism by which the gp41 V2E mutant inhibits HIV-1 infectivity in cells when co-expressed with WT gp41.
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PMID:Fusion peptides derived from the HIV type 1 glycoprotein 41 associate within phospholipid membranes and inhibit cell-cell Fusion. Structure-function study. 915 94

Nef is a 27 kDa myristylated phosphoprotein expressed early in infection by HIV. The N terminus of Nef is thought to play a vital role in the functions of this protein through its interactions with membrane structures. The solution structure of a 25-residue polypeptide corresponding to the N terminus of Nef (Nef1-25) has been investigated by 1H NMR spectroscopy. In aqueous solution at pH 4.8 and 281 K, this peptide underwent conformational averaging, with Pro13 existing in cis and trans conformations in nearly equal proportions. In methanol solution, however, the peptide adopted a well-defined alpha-helical structure from residues 6 to 22, with the N- and C-terminal regions having a less ordered structure. On the basis of a comparison of chemical shifts and NOEs, it appeared that this helical structure was maintained in aqueous trifluoroethanol (50% v/v) and to a lesser extent in a solution of SDS micelles. When the N-acetyl group was replaced by either an N-myristyl or a free ammonium group, there was little effect on the three-dimensional structure of the peptide in methanol; deamidation of the C terminus also had no effect on the structure in methanol. In water, the myristylated peptide aggregated. The similarity between the sequences of Nef1-25 and melittin is reflected in the similar structures of the two molecules, although the N-terminal helix of melittin is more defined. This similarity in structure raises the possibility that Nef1-25 not only interacts with membranes but also may be capable of disrupting them and causing cell lysis. This type of interaction could contribute at least in part to the killing of bystander cells in lymphoid tissues during HIV infection.
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PMID:Solution structure of a polypeptide from the N terminus of the HIV protein Nef. 916 67

HIV-1 gp120 and truncated forms were expressed in HeLa T4 cells by vaccinia recombinant viruses. The truncated gp120 molecules consisted of N-terminal overlapping envelope proteins of 204, 287 and 393 amino acids respectively. Immunoprecipitation with specific monoclonal antibodies and SDS-PAGE analyses of HIV-1 gp120 revealed bands corresponding to low amounts of secreted and cell-bound stable dimers. In contrast, the truncated forms of gp120 expressed larger amounts of SDS-stable putative dimers and the amounts observed were inversely proportional to their size. The shortest gp120 mutant (204 aa) was found to be secreted almost exclusively as a dimer. The processing of gp120 and its truncated forms was further investigated in the presence of inhibitors of N-glycosylation. Monomers and dimers migrated on gels with the same relative changes, confirming that the protein with the higher molecular weight is a multimer of the smaller one. The putative dimeric form of the truncated gp120s could be stabilized by chemical cross-linking. Finally, the possible existence of an association domain in the N-terminal 204 amino acids (aa) of gp120 is discussed.
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PMID:Self-association of truncated forms of HIV-1 gp120. 921 91

Despite commonly applied clinical criteria, the early diagnosis of rheumatoid arthritis (RA) often remains difficult, thus delaying on suitable early treatment. In search for a test furthering the early and reliable diagnosis of RA, we have screened for novel disease specific autoantibodies. To this end proteins were isolated from synovial membranes and other tissues following a special protein purification protocol, and these were separated electrophoretically. Western blots were then used to screen sera of RA patients and of individuals suffering from other rheumatic diseases for antibodies to any of these proteins. The most prominent RA specific immunoreaction was with a 68k antigen, occurring in 110 of 167 RA patients (sensitivity is 66%). The antibody could also be identified in seronegative RA patients but not in healthy individuals (55 tested), in only 1 SLE patient of a group of 98 patients with other rheumatic diseases and in 1 out of 22 HIV patients, resulting in a specificity of 99%. Moreover, the anti-68k antibody could be correlated with a more severe course of RA. 13 out of 20 anti-68k positive RA patients (58%) had subcutaneous nodules, while only 2 out of 11 anti-68k negative (20%) did. The mean sedimentation rate of these antibody positive patients was 51 mm/h and 26 mm/h for the negative respectively. The 68k antigen was shown to be present in all human tissues investigated and is probably ubiquitously expressed. It is either located in the endoplasmatic reticulum or cytoplasm or both. Its isoelectric point is 5.1. It proved to be O-glycosylated and contains only one or a few sugar residues as the untreated and the deglycosylated antigen identical electrophoretical mobilities. The patient derived anti-68k antibodies were directed against the sugar residue: deglycosylation of the antigen completely abolished its immunoreactivity. N-acetylglucosamine competes with the antibody for binding the 68k antigen. The antigen physicochemical data of the 68k antigen argue against identity with one of the autoantigens in this molecular mass range already known to be associated with RA or other autoimmune diseases. It is neither identical to the 62k human antigen (EBNA-1) nor to RA33 (A2hnRNP), the 50k Sa antigen or the Hsp70 class of heats-hock proteins. It is argued that the particular method of protein purification applied in combination with separation via SDS-PAGE in the presence of urea, made it possible to detect a hitherto unidentified antigen. Considering the striking disease specificity of the anti-68k antibody it is now worthwhile to look for corresponding autoreactive T cells in order to analyse its role in the pathogenesis of RA.
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PMID:[RA-specific autoantibodies against a 68k antigen]. 923 11

Relative affinities of transcriptional regulatory elements for their respective factor have been essentially studied by bandshift analysis. Here we report a real-time study of factor/DNA interactions using a surface plasmon resonance approach and further characterization of recovered proteins involved in this interaction. For this purpose, human GATA-3, either recombinant or in nuclear extracts, and three natural GATA elements of the HIV-1 long terminal repeat (sites 1, 2, and 3) were chosen, in which only site 2 is a noncanonical GATA site. Direct analysis of sensorgrams, with recombinant huGATA-3, allowed the comparison of association and dissociation profiles of the three DNA regions and their ranking according to their relative affinities. This result, confirmed by competitions with each GATA site, demonstrated the higher relative affinity (at least sevenfold) of site 3. Interactions between the canonical and unique GATA site 3 and nuclear extracts were also studied in real time and provided information on its association and dissociation rates for native huGATA-3. Finally, recovered protein was identified as genuine huGATA-3 by SDS-PAGE, Western blotting, and bandshift assays.
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PMID:Analysis of interactions between huGATA-3 transcription factor and three GATA regulatory elements of HIV-1 long terminal repeat, by surface plasmon resonance. 935 44


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