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)

We previously reported that the pleckstrin homology (PH) domain of Bruton's tyrosine kinase (Btk) binds Ins(1,3,4,5)P4 and that missense mutations in this domain which cause either human X-linked agammaglobulinemia (XLA) or murine X-linked immunodeficiency (Xid) also dramatically reduce the Ins(1,3,4,5)P4 binding activity. In this paper, we describe the inositol phosphate binding specificity of the Btk PH domain and different inositol polyphosphate binding properties among the PH domains of Tec family kinases. Our results suggest that certain inositol phosphates and/or phosphoinositides are physiological ligands of some Tec family kinases and that Tec family members are differently regulated by inositol molecules.
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PMID:Characterization of the pleckstrin homology domain of Btk as an inositol polyphosphate and phosphoinositide binding domain. 924 Apr 35

Lamivudine (2'-deoxy-3'-thiacytidine; 3TC) is a dideoxynucleoside analogue that inhibits the replication of human immunodeficiency virus (HIV). We are currently investigating the intracellular metabolism of 3TC to its active triphosphate (3TCTP) in peripheral blood mononuclear cells (PBMC) and a monocytic cell line (U937). Optimal phosphorylation of 3TC was achieved after incubation for 24 hr, with 3TC diphosphate (3TCDP) the predominant metabolite formed, in both cell types investigated. Further studies in PBMCs followed preincubation with the mitogen phytohaemagglutinin (PHA) for 72 hr. This enabled greater detection of phosphates, compared to resting cells. A 3TC concentration of 1 microM was chosen for future interaction studies, allowing good detection of 3TC and phosphates on radiochromatograms whilst being similar to the plasma level found in clinical studies (i.e. 3 microM). With a shift in treatment to combination therapy, it is essential that potential interactions between nucleoside analogues are investigated at the phosphorylation level, as this could affect antiviral activity. Both deoxycytidine (dC) and 2',3'-dideoxycytidine (ddC) significantly inhibited 3TC phosphorylation (e.g. at dC 100 microM, no 3TCTP was detected in PBMCs; P < 0.001, whereas 66% of control 3TCTP production was observed in U937 cells; P < 0.01). Zidovudine (ZDV) caused a small but significant reduction of 3TC phosphate production in both PBMCs and U937 cells. However, this may be due to toxicity or an effect on endogenous dCTP pools. Neither 2',3'-dideoxyinosine (ddI) or 2',3'-didehydro-2',3'-dideoxythymidine (d4T) significantly inhibited 3TC phosphorylation. These results suggest it would be better to coadminister two nucleoside analogues with different activation pathways.
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PMID:Lamivudine (3TC) phosphorylation and drug interactions in vitro. 933 75

Phosphorylation is important in the regulation of many cellular processes, yet the precise role of protein phosphorylation for many RNA-binding protein substrates remains obscure. In this report, we demonstrate that phosphorylation of a recombinant human immunodeficiency virus type-1 Rev protein promotes rapid formation of an efficient RNA-binding state. The apparent dissociation constant for ligand binding is enhanced 7-fold for the protein following phosphorylation; however, phosphate addition leads to a 1. 6-fold decrease in RNA ligand-protein complex stability. RNA ligand binding stimulates slow formation of an equally competent binding state for the unphosphorylated protein, indicating that the addition of phosphate or ligand binding promotes a similar conformational change in Rev. Phosphorylation directly alters the conformation of Rev, as revealed by modification experiments that monitor the solvent accessibility of cysteines in the protein. These biochemical properties are attributed to the addition of phosphate at one of two serine residues (Ser-54 or Ser-56) that lie within the multimerization domain adjacent to the RNA-binding helix. Glutaraldehyde-mediated cross-linking experiments revealed that phosphorylation of Rev does not affect Rev multimerization activity. The Rev protein from the less pathogenic HIV-2 isolate lacks this phosphorylation site in the amino acid sequence; thus, the described biochemical properties of the phosphorylated protein may contribute to Rev activity and possibly to HIV-1 virulence during natural infection.
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PMID:Site-specific phosphorylation of the human immunodeficiency virus type-1 Rev protein accelerates formation of an efficient RNA-binding conformation. 934 Dec 15

Members of the chemokine receptor family CCR5 and CXCR4 have recently been shown to be involved in the entry of human immunodeficiency virus (HIV) into target cells. Here, we investigated the regulation of CXCR4 in rat basophilic leukemia cells (RBL-2H3) stably transfected with wild type (Wt CXCR4) or a cytoplasmic tail deletion mutant (DeltaCyto CXCR4) of CXCR4. The ligand, stromal cell derived factor-1 (SDF-1) stimulated higher G-protein activation, inositol phosphate generation, and a more sustained calcium elevation in cells expressing DeltaCyto CXCR4 relative to Wt CXCR4. SDF-1 and phorbol 12-myristate 13-acetate (PMA), but not a membrane permeable cAMP analog induced rapid phosphorylation as well as desensitization of Wt CXCR4. Phosphorylation of DeltaCyto CXCR4 was not detected under any of these conditions. Despite lack of receptor phosphorylation, calcium mobilization by SDF-1 in DeltaCyto CXCR4 cells was partially desensitized by prior treatment with SDF-1. Of interest, the rapid release of calcium was inhibited without affecting the sustained calcium elevation, indicating independent regulatory pathways for these processes. PMA completely inhibited phosphoinositide hydrolysis and calcium mobilization in Wt CXCR4 but only partially inhibited these responses in DeltaCyto CXCR4. cAMP also partially inhibited these responses in both Wt CXCR4 and DeltaCyto CXCR4. SDF-1, PMA, and cAMP caused phosphorylation of phospholipase Cbeta3 in Wt and DeltaCyto CXCR4 cells. Both SDF-1 as well as PMA induced rapid internalization of Wt CXCR4. SDF-1 but not PMA induced internalization of DeltaCyto CXCR4 albeit at reduced levels relative to Wt CXCR4. These results indicate that signaling and internalization of CXCR4 are regulated by receptor phosphorylation dependent and independent mechanisms. Desensitization of CXCR4 signaling, independent of receptor phosphorylation, appears to be a consequence of the phosphorylation of phospholipase Cbeta3.
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PMID:Regulation of human chemokine receptors CXCR4. Role of phosphorylation in desensitization and internalization. 935 42

The conformation of the DNA and the interactions of the nucleic acid with the protein in a complex of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and 19-mer/18-mer double-stranded DNA template-primer (dsDNA) are described. The structure of this HIV-1 RT complex with dsDNA serves as a useful paradigm for studying aspects of nucleotide polymerases such as catalysis, fidelity, drug inhibition, and drug resistance. The bound dsDNA has a bend of approximately 41 degrees at the junction of an A-form region (first five base pairs near the polymerase active site) and a B-form region (the last nine base pairs toward the RNase H active site). The 41 degrees bend occurs smoothly over the four base pairs between the A-form portion and the B-form portion in the vicinity of helices alpha H and alpha I of the p66 thumb subdomain. The interactions between the dsDNA and protein primarily involve the sugar-phosphate backbone of the nucleic acid and structural elements of the palm, thumb, and RNase H of p66, and are not sequence specific. Amino acid residues from the polymerase active site region, including amino acid residues of the conserved Tyr-Met-Asp-Asp (YMDD) motif and the "primer grip," interact with 3'-terminal nucleotides of the primer strand and are involved in positioning the primer terminal nucleotide and its 3'-OH group at the polymerase active site. Amino acid residues of the "template grip" have close contacts with the template strand and aid in positioning the template strand near the polymerase active site. Helix alpha H of the p66 thumb is partly inserted into the minor groove of the dsDNA and helix alpha I is directly adjacent to the backbone of the template strand. Amino acid residues of beta 1', alpha A', alpha B', and the loop containing His539 of the RNase H domain interact with the primer strand of the dsDNA.
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PMID:Protein-nucleic acid interactions and DNA conformation in a complex of human immunodeficiency virus type 1 reverse transcriptase with a double-stranded DNA template-primer. 935 57

AZT-P-ddI is an antiviral heterodimer composed of one molecule of 3'-azido-3'-deoxythymidine (AZT) and one molecule of 2',3'-dideoxyinosine (ddI) linked through their 5' positions by a phosphate bond. The metabolic fate of the dimer was studied with isolated rat, monkey, and human hepatocytes and was compared with that of its component monomers AZT and ddI. Upon incubation of double-labeled [14C]AZT-P-[3H]ddI in freshly isolated rat hepatocytes in suspension at a final concentration of 10 microM, the dimer was taken up intact by cells and then rapidly cleaved to AZT, AZT monophosphate, ddI, and ddI monophosphate. AZT and ddI so formed were then subject to their respective catabolisms. High-performance liquid chromatography analyses of the extracellular medium and cell extracts revealed the presence of unchanged dimer, AZT, 3'-azido-3'-deoxy-5'-beta-D-glucopyranosylthymidine (GAZT), 3'-amino-3'-deoxythymidine (AMT), ddI, and a previously unrecognized derivative of the dideoxyribose moiety of ddI, designated ddI-M. Trace extracellular but substantial intracellular levels of the glucuronide derivative of AMT (3'-amino-3'-deoxy-5'-beta-D-glucopyranosylthymidine [GAMT]) were also detected. Moreover, the extent of the formation of AMT, GAZT, and ddI-M from the dimer was markedly lower than that with AZT and ddI alone by the hepatocytes. With hepatocytes in primary culture obtained from rat, monkey, and human, large interspecies variations in the metabolism of AZT-P-ddI were observed. While GAZT and ddI-M, metabolites of AZT and ddI, respectively, as well as AZT 5'-monophosphate (MP) and ddI-MP were detected in the extracellular media of all species, AMT and GAMT were produced only by rat and monkey hepatocytes. No such metabolites were formed by human hepatocytes. The metabolic fate of the dimer by human hepatocytes was consistent with in vivo data recently obtained from human immunodeficiency virus-infected patients.
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PMID:Comparative metabolism of the antiviral dimer 3'-azido-3'-deoxythymidine-P-2',3'-dideoxyinosine and the monomers zidovudine and didanosine by rat, monkey, and human hepatocytes. 937 57

A novel fluorogenic substrate for continuous feline immunodeficiency virus (FIV) protease (PR) assay was developed in which 2-aminobenzoic acid (Abz) and p-nitrophenylalanine (F(NO2)) were used as the fluorescent donor and acceptor, respectively. The 14-amino-acid fluorogenic substrate of sequence RALTK(Abz) VQ approximately F(NO2)VQSKGR (approximately indicates cleavage site) was modeled after a naturally occurring FIV PR capsid/nucleocapsid cleavage site in the gag polyprotein. The 2-aminobenzoyl group was attached to the epsilon amino group of a lysine (K(Abz)) in position P3 and the F(NO2) is in position P1' in order to promote efficient intramolecular quenching prior to cleavage by FIV PR. We measured a K(m) of 33 +/- 6 microM and a kcat of 0.29 +/- 0.02 s-1 for the enzymatic hydrolysis of this fluorogenic substrate by FIV PR under the conditions of our assay (0.05 M sodium citrate/0.1 M sodium phosphate buffer, pH 5.25, 0.2 M NaCl, 0.1 mM EDTA, and 1 mM dithiothreitol). This assay affords a rapid and convenient means for quantitating FIV PR activities and promises to be useful for judging the relative strength of inhibitors.
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PMID:A continuous fluorometric assay for the feline immunodeficiency virus protease. 941 81

Cleavage and DNA joining reactions, carried out by human immunodeficiency virus type 1 (HIV-1) integrase, are necessary to effect the covalent insertion of HIV-1 DNA into the host genome. For the integration of HIV-1 DNA into the cellular genome to be completed, short gaps flanking the integrated proviral DNA must be repaired. It has been widely assumed that host cell DNA repair enzymes are involved. Here we report that HIV-1 integrase multimers possess an intrinsic DNA-dependent DNA polymerase activity. The activity was characterized by its dependence on Mg2+, resistance to N-ethylmaleimide, and inhibition by 3'-azido-2',3'-dideoxythymidine-5'-triphosphate, coumermycin A1, and pyridoxal 5'-phosphate. The enzyme efficiently utilized poly(dA)-oligo(dT) or self-annealing oligonucleotides as a template primer but displayed relatively low activity with gapped calf thymus DNA and no activity with poly(dA) or poly(rA)-oligo(dT). A monoclonal antibody binding specifically to an epitope comprised of amino acids 264 to 273 near the C terminus of HIV-1 integrase severely inhibited the DNA polymerase activity. A deletion of 50 amino acids at the C terminus of integrase drastically altered the gel filtration properties of the DNA polymerase, although the level of activity was unaffected by this mutation. The DNA polymerase efficiently extended a hairpin DNA primer up to 19 nucleotides on a T20 DNA template, although addition of the last nucleotide occurred infrequently or not at all. The ability of integrase to repair gaps in DNA was also investigated. We designed a series of gapped molecules containing a single-stranded region flanked by a duplex U5 viral arm on one side and by a duplex nonviral arm on the other side. Molecules varied structurally depending on the size of the gap (one, two, five, or seven nucleotides), their content of T's or C's in the single-stranded region, whether the CA dinucleotide in the viral arm had been replaced with a nonviral sequence, or whether they contained 5' AC dinucleotides as unpaired tails. The results indicated that the integrase DNA polymerase is specifically designed to repair gaps efficiently and completely, regardless of gap size, base composition, or structural features such as the internal CA dinucleotide or unpaired 5'-terminal AC dinucleotides. When the U5 arm of the gapped DNA substrate was removed, leaving a nongapped DNA template-primer, the integrase DNA polymerase failed to repair the last nucleotide in the DNA template effectively. A post-gap repair reaction did depend on the CA dinucleotide. This secondary reaction was highly regulated. Only two nucleotides beyond the gap were synthesized, and these were complementary to and dependent for their synthesis on the CA dinucleotide. We were also able to identify a specific requirement for the C terminus of integrase in the post-gap repair reaction. The results are consistent with a direct role for a heretofore unsuspected DNA polymerase function of HIV-1 integrase in the repair of short gaps flanking proviral DNA integration intermediates that arise during virus infection.
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PMID:Efficient gap repair catalyzed in vitro by an intrinsic DNA polymerase activity of human immunodeficiency virus type 1 integrase. 949 61

Dysregulated apoptosis may underlie the etiology of T cell depletion by human immunodeficiency virus type 1 (HIV-1). We show that HIV-induced apoptosis is preceded by an exponential increase in reactive oxygen intermediates (ROIs) produced in mitochondria. This leads to caspase-3 activation, phosphatidylserine (PS) externalization, and GSH depletion. Since mitochondrial ROI levels are regulated by the supply of NADPH from the pentose phosphate pathway (PPP), the effect of transaldolase (TAL), a key enzyme of PPP, was investigated. Jurkat and H9 human CD4+ T cells were transfected with TAL expression vectors oriented in the sense or antisense direction. TAL overexpression down-regulated glucose-6-phosphate dehydrogenase activities and GSH levels. Alternatively, decreased TAL expression up-regulated glucose-6-phosphate dehydrogenase activities and GSH levels. HIV-induced 1) mitochondrial ROI production, 2) caspase-3 activation, 3) proteolysis of poly(ADP-ribose) polymerase, and 4) PS externalization were accelerated in cells overexpressing TAL. In contrast, suppression of TAL abrogated these four activities. Thus, susceptibility to HIV-induced apoptosis can be regulated by TAL through controlling the balance between mitochondrial ROI production and the metabolic supply of reducing equivalents by the PPP. The dominant effect of TAL expression on oxidative stress, caspase activation, PS externalization, and cell death suggests that this balance plays a pivotal role in HIV-induced apoptosis.
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PMID:Molecular ordering in HIV-induced apoptosis. Oxidative stress, activation of caspases, and cell survival are regulated by transaldolase. 956 23

Detergent-based vaginal microbicides, in addition to their high contraceptive failure rates, cause mucosal erosion and local inflammation that might increase the risk of heterosexual human immunodeficiency virus (HIV) transmission. In a systematic effort to identify a microbicide contraceptive potentially capable of preventing the sexual transmission of HIV as well as providing fertility control, a series of novel aryl phosphate derivatives of 5-bromo-6-methoxy-3'-azido-3'-deoxythymidine (AZT; zidovudine) were synthesized and examined for dual anti-HIV and sperm-immobilizing activity (SIA). Whereas AZT displayed potent anti-HIV activity (IC50 = 0.006 microM) but lacked SIA (EC50 > 300 microM), two 5-bromo-6-methoxy-aryl phosphate derivatives of AZT, compounds WHI-05 and WHI-07, exhibited potent anti-HIV activity as well as SIA. The IC50 (HIV) and EC50 (SIA) values for WHI-07 were 439-fold and 13.5-fold lower, respectively, than those for the detergent-based virucidal spermicide, nonoxynol-9 (N-9). Sperm motion kinematics using computer-assisted sperm motion analysis combined with confocal laser scanning microscopy, high-resolution low-voltage scanning, and transmission electron microscopy demonstrated that both WHI-05 and WHI-07 cause a complete and irreversible loss of sperm motility in a concentration- and time-dependent fashion without concomitantly affecting the sperm acrosomal membrane integrity. In experiments designed to assess the fertilizing capacity of treated sperm, preincubation of sperm with either compound resulted in a concentration-dependent loss of the ability to adhere to and penetrate zona-free hamster eggs as well as inhibition of binding to human zona. WHI-07 applied intravaginally prior to artificial insemination of epididymal sperm drastically reduced fertility in hormonally primed CD-1 mice. Unlike the intravaginal application of N-9, repetitive intravaginal application of WHI-07 did not damage the vaginal epithelium or cause local inflammation. Structure-function relationship analyses showed that the addition of bromo-methoxy functional groups to AZT was essential for, and the aryl phosphate derivatization contributory to, the SIA of both compounds. Compounds WHI-05 and WHI-07 may be useful as dual-function vaginal contraceptives for women who are at high risk for acquiring HIV/acquired immunodeficiency virus syndrome by heterosexual vaginal transmission.
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PMID:Aryl phosphate derivatives of bromo-methoxy-azidothymidine are dual-function spermicides with potent anti-human immunodeficiency virus. 971 47


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