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 Nef protein encoded by human immunodeficiency virus type 1 (HIV-1) has been shown to be an important factor of progression of viral growth and pathogenesis in both in vitro and in vivo. The lack of a simple procedure to purify Nef in its native conformation has limited molecular studies on Nef function. A two-step procedure that includes heparin and immobilized metal ion affinity chromatographies (IMACs) was developed to purify His-tagged Nef (His(6)-Nef) expressed in bacteria in native condition. During the elaboration of this purification procedure, we identified two closely SDS-PAGE-migrating contaminating bacterial proteins, SlyD and GCHI, that co-eluted with His(6)-Nef in IMAC in denaturing condition and developed purification steps to eliminate these contaminants in native condition. Overall, this study describes a protocol that allows rapid purification of His(6)-Nef protein expressed in bacteria in native condition and that removes metal affinity resin-binding bacterial proteins that can contaminate recombinant His-tagged protein preparation.
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PMID:Two-step purification of His-tagged Nef protein in native condition using heparin and immobilized metal ion affinity chromatographies. 1282 Nov 99

A novel antifungal protein, designated castamollin, was isolated from Chinese chestnut (Castanea mollisima) seeds with a procedure involving ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-Sepharose and FPLC-gel filtration on Superdex 75. Castamollin possessed a novel N-terminal sequence demonstrating little similarity to N-terminal sequences of Castanea sativa chitinase. Castamollin exhibited a molecular mass of 37kDa in gel filtration and SDS-PAGE. It inhibited the activity of human immunodeficiency virus-1 reverse transcriptase with an IC(50) of 7microM and translation in a cell-free rabbit reticulocyte lysate system with an IC(50) of 2.7microM. Castamollin displayed antifungal activity against Botrytis cinerea, Mycosphaerella arachidicola, Physalospora piricola, and Coprinus comatus but was devoid of lectin activity.
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PMID:Purification of castamollin, a novel antifungal protein from Chinese chestnuts. 1468 Sep 38

Sulfated polymannuroguluronate (SPMG), a marine sulfated polysaccharide, has entered the Phase II clinical trial in China as the first anti-acquired immune deficiency syndrome (AIDS) drug candidate obtained from marine organisms. To determine the binding site(s) (receptors) of SPMG in lymphocytes mediating its anti-AIDS activities, fluorescein-5-isothiocyanate (FITC)-labeled SPMG was used to investigate SPMG binding to lymphocytes. Flow cytometry (FCM) and fluorescence microscopy analysis showed that the SPMG binds to lymphocytes in a rapid, specific, reversible, and saturable fashion. Several SPMG binding proteins were purified by affinity chromatography from lymphocyte membrane preparations. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis revealed that a 55kDa lymphocyte membrane protein is CD4. To characterize the SPMG and CD4 interaction, inhibition assay and surface plasmon resonance (SPR) assay were carried out. SPMG bound to CD4 in a multivalent fashion with specificity. The binding of SPMG to human lymphocyte CD4 was competitively inhibited by human soluble CD4 (hsCD4). Likewise, the binding between hsCD4 and immobilized SPMG was blocked by excess free SPMG. These results indicate that CD4 is one of the specific SPMG binding sites (receptors) in lymphocytes. The interaction between SPMG and CD4 may provide a mechanistic explanation of the immunopotentiating and anti-AIDS activities of SPMG in human immunodeficiency virus (HIV) infected individuals.
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PMID:Sulfated polymannuroguluronate, a novel anti-acquired immune deficiency syndrome (AIDS) drug candidate, targeting CD4 in lymphocytes. 1527 71

A contaminant protein complex found in pharmaceutical urinary human chorionic gonadotrophin preparations is reported to have anti-human immunodeficiency virus-associated Kaposi's sarcoma activity. The aim of this study was to isolate and characterize this protein complex by proteomic approaches. Size exclusion chromatography was used in the isolation of these human chorionic gonadotrophin-associated fragments. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of a protein complex that dissociated into two protein bands under reducing conditions. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of this complex showed three polypeptides at approximately 6.2, 11.4, and 15.8 kDa. Peptide mass mapping and N-terminal amino acid sequencing identified two polypeptides as metabolites of placental transforming growth factor-beta (11.4 kDa) and bikunin (15.8 kDa). Subsequent matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of the anti-human immunodeficiency virus-associated Kaposi's sarcoma active preparations CG-10 (Sigma), Pregnyl (Organon), and Profasi (Serono) revealed the presence of metabolites of placental transforming growth factor-beta in all three; no other non-human chorionic gonadotrophin-related protein species were observed in these preparations. Our findings present evidence that urinary human chorionic gonadotrophin preparations are contaminated with metabolites of placental transforming growth factor-beta, which may have transforming growth factor-beta agonist actions, and metabolites of bikunin, which is a protease inhibitor. In combination these molecules may be responsible for the anti-human immunodeficiency virus-associated Kaposi's sarcoma activity demonstrated for these urinary human chorionic gonadotrophin preparations.
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PMID:Identification of placental transforming growth factor-beta and bikunin metabolites as contaminants of pharmaceutical human chorionic gonadotrophin preparations by proteomic techniques. 1585 73

Two-dimensional gel electrophoresis (2-DE) is widely used for initial protein separation in proteomics. Commercial products using neutral pH sodium dodecyl sulfate-polyacrylamide gel electrophoresis ((SDS-PAGE)/(Bis (2-hydroxyethyl) imino-tris (hydroxymethyl) methane-HCl, or Bis-Tris)) have greatly improved this technique, but cost and limited sizes restrict their applications. An "in-house" system is presented, resulting in better resolution, separation, and new spot visualization and improved resolution when compared to Tris-HCl gels. Their utility is demonstrated using albumin-depleted serum samples, rabbit heart left ventricle, and human immunodeficiency virus type 1 (HIV-1).
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PMID:Improvements in two-dimensional gel electrophoresis by utilizing a low cost "in-house" neutral pH sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. 1588 85

The structure of the Vpu protein of human immunodeficiency virus type 1 (HIV-1) is composed of a short Nterminal domain (NTD), a transmembrane domain (TM), and a cytoplasmic domain (CD). Previous studies have shown that the Vpu protein from subtype B HIV-1 is transported predominantly to the rough endoplasmic reticulum (RER)/Golgi complex compartments of the cell and is not incorporated into virions. Using a previously described VpuEGFP reporter system in which the Vpu protein was fused to the gene for enhanced green fluorescent protein (EGFP), we showed that the subtype B Vpu fusion protein was localized to the RER/Golgi region of the cell, similar to the native protein. In the present study, we show that fusion of the subtype C Vpu to EGFP results in a fusion protein that is transported to the cell surface. Using this reporter system, chimeric Vpu proteins in which the CD of the subtype B and C proteins were exchanged showed that the CD was sufficient for targeting the subtype B protein to the Golgi complex of the cell. Following identification of the cytoplasmic domain as being responsible for intracellular targeting, we then generated a series of mutants in which 13, 23, 31, 38, 51, and 56 amino acids were deleted from the cytoplasmic domain of subtype B Vpu. These deletion mutants were analyzed by SDS-PAGE for size, for membrane localization, and intracellular localization by confocal fluorescence microscopy. Our results indicate that the mutant with the carboxyl-terminal 13 amino acids deleted was still localized to the Golgi complex but mutants with 23, 31, 38, 51, and 56 amino acids from the carboxyl-terminus of the subtype B Vpu were transported to the cell surface. These results suggest that a signal for the retention of the subtype B Vpu within the Golgi complex resides in the second alpha-helical domain.
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PMID:Identification of a region within the cytoplasmic domain of the subtype B Vpu protein of human immunodeficiency virus type 1 (HIV-1) that is responsible for retention in the golgi complex and its absence in the Vpu protein from a subtype C HIV-1. 1592

Retrocyclins are circular mini-defensins with significant potential as agents against human immunodeficiency virus, influenza A, and herpes simplex virus. Retrocyclins bind carbohydrate-containing surface molecules such as gp120 and CD4 with high affinity (Kd, 10-100 nM), promoting their localization on cell membranes. The structural features important for activity have yet to be fully elucidated, but here, we have determined the first three-dimensional structure of a retrocyclin, namely, one of the most potent forms, retrocyclin-2. In the presence of SDS micelles, a well-defined beta-hairpin braced by three disulfide bonds that defines the cystine ladder motif is present. By contrast, a well-defined structure could not be determined in aqueous solution, suggesting that the presence of SDS micelles stabilizes the extended conformation of retrocyclin-2. Translational diffusion measurements indicate that retrocyclin-2 interacts with the SDS micelles, and such a membrane-like interaction may be an important feature in the mechanism of action of these antimicrobial peptides. Analytical ultracentrifugation and the NMR data indicated that retrocyclin-2 self-associates to form a trimer in a concentration-dependent manner. The ability to self-associate may contribute to the high-affinity binding of retrocyclins for glycoproteins by increasing the valency and enhancing the ability of retrocyclins to cross-link cell surface glycoproteins.
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PMID:Retrocyclin-2: structural analysis of a potent anti-HIV theta-defensin. 1768 59

The transmembrane glycoprotein gp41 of human immunodeficiency virus has been proposed to form trimer-of-hairpin during virus-cell membrane fusion. To investigate its oligomerization propensity under soluble and membrane-mimic conditions, sodium salt of perfluorooctanoate (PFO) was applied. A recombinant gp41 ectodomain devoid of disulfide linkage was overexpressed in Escherichia coli and characterized by MS and circular dichroism spectropolarimetry in PFO solution in comparison to SDS. The helical content of this ectodomain in PFO is higher than that in SDS. Notably, PFO employed in PAGE clearly conduced to the formation of trimer under the optimized condition as visualized in the gel. In addition, the proteins expressed from the two mutants in the heptad repeat (HR) domains of gp41, I62P, and N126K, were also examined by the PFO-PAGE analysis for functional ramification of molecular organization. Remarkably, the I62P mutation completely abolished the gp41 trimer formation, whereas the N126K mutation resulted in a more stable trimer. The data suggested that PFO-PAGE analysis is appropriate for evaluating the effect of mutations on the trimerization of gp41 and other fusion proteins which may be implicated in the alteration of their fusogenicity.
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PMID:The application of perfluorooctanoate to investigate trimerization of the human immunodeficiency virus-1 gp41 ectodomain by electrophoresis. 1863 39

The signature for human immunodeficiency virus type 1 (HIV-1) neurovirulence remains a subject of intense debate. Macrophage viral tropism is one prerequisite but others, including virus-induced alterations in innate and adaptive immunity, remain under investigation. HIV-1-infected mononuclear phagocytes (MPs; perivascular macrophages and microglia) secrete toxins that affect neurons. The authors hypothesize that neurovirulent HIV-1 variants affect the MP proteome by inducing a signature of neurotoxic proteins and thus affect cognitive function. To test this hypothesis, HIV-1 isolates obtained from peripheral blood of women with normal cognition (NC) were compared to isolates obtained from women with cognitive impairment (CI) and to the laboratory adapted SF162, a spinal fluid R5 isolate from a patient with HIV-1-associated dementia. HIV-1 isolates were used to infect monocyte-derived macrophages (MDMs) and infection monitored by secreted HIV-1 p24 by enzyme-linked immunosorbent assay (ELISA). Cell lysates of uninfected and HIV-1-infected MDMs at 14 days post infection were fractionated by cationic exchange chromatography and analyzed by surface enhanced laser desorption ionization time of flight (SELDI-TOF) using generalized estimating equations statistics. Proteins were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D SDS-PAGE) and identified by tandem mass spectrometry. Levels of viral replication were similar amongst the HIV-1 isolates, although higher levels were obtained from one viral strain obtained from a patient with CI. Significant differences were found in protein profiles between virus-infected MDMs with NC, CI, and SF162 isolates (adjusted P value after multiple testing corrections, or q value <.10). The authors identified 6 unique proteins in NC, 7 in SF162, and 20 in CI. Three proteins were common to SF162 and CI strains. The MDM proteins linked to infection with CI strains were related to apoptosis, chemotaxis, inflammation, and redox metabolism. These findings support the hypothesis that the macrophage proteome differ when infected with viral isolates of women with and without CI.
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PMID:Proteomic analyses of monocyte-derived macrophages infected with human immunodeficiency virus type 1 primary isolates from Hispanic women with and without cognitive impairment. 1911 25

Thirteen different antiretrovirals are commonly used in hospital protocols for suppressing the activity of the human immunodeficiency virus (HIV) and associated opportunistic diseases in patients with acquired immunodeficiency syndrome (AIDS). In this work, three micellar mobile phases are recommended for screening these substances, using UV detection, and the process can be performed in less than 18 min. The first mobile phase (sodium dodecyl sulphate or SDS 50mM) is used for the group consisting of acyclovir, didanosine, ganciclovir, stavudine and zidovudine. The second mobile phase (SDS 120 mM/4.5% propanol) is used for the group containing abacavir, lamivudine, nevirapine, valaciclovir and zalcitabine, whereas the third mobile phase (SDS 150 mM/5% pentanol) is used for efavirenz, indinavir and ritonavir. The use of micellar liquid chromatography (MLC) as an analytical tool allows serum samples to be injected directly. The method was validated over the range of 0-10 microg mL(-1). The limits of detection (signal-to-noise ratio of 3), which ranged from 6 to 30 ng mL(-1), were adequate for monitoring these substances. Intra- and inter-day relative standard deviations of the assay were below 3% for all compounds. The recoveries in spiked serum samples were in the 89.5-104.4% range. The method can be applied to the screening, monitoring and control of patients' treatment with antiretrovirals and antivirals.
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PMID:Screening and monitoring antiretrovirals and antivirals in the serum of acquired immunodeficiency syndrome patients by micellar liquid chromatography. 1922 34


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