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)

The free energies of dimer dissociation of the retroviral proteases (PRs) of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) were determined by measuring the effects of denaturants on the protein fluorescence upon the unfolding of the enzymes. HIV-1 PR was more stable to denaturation by chaotropes and extremes of pH and temperature than SIV PR, indicating that the former enzyme has greater conformational stability. The urea unfolding curves of both proteases were sigmoidal and single phase. The midpoints of the transition curves increased with increasing protein concentrations. These data were best described by and fitted to a two-state model in which folded dimers were in equilibrium with unfolded monomers. This denaturation model conforms to cases in which protein unfolding and dimer dissociation are concomitant processes in which folded monomers do not exist [Bowie, J. U., & Sauer, R. T. (1989) Biochemistry 28, 7140-7143]. Accordingly, the free energies of unfolding reflect the stabilities of the protease dimers, which for HIV-1 PR and SIV PR were, respectively, delta GuH2O = 14 +/- 1 kcal/mol (Ku = 39 pM) and 13 +/- 1 kcal/mol (Ku = 180 pM). The binding of a tight-binding, competitive inhibitor greatly stabilized HIV-1 PR toward urea-induced unfolding (delta GuH2O = 19.3 +/- 0.7 kcal/mol, Ku = 7.0 fM). There were also profound effects caused by adverse pH on the protein conformation for both HIV-1 PR and SIV PR, resulting in unfolding at pH values above and below the respective optimal ranges of 4.0-8.0 and 4.0-7.0
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PMID:Use of protein unfolding studies to determine the conformational and dimeric stabilities of HIV-1 and SIV proteases. 139 Jul 32

A method for the purification of a truncated, biologically active human immunodeficiency virus type 1 (HIV-1) trans-activator (rTAT) from recombinant Escherichia coli is reported here. The purification steps utilized include mild extraction (French press), concentration by ammonium sulfate precipitation, chromatography in 8 M urea on an S-Sepharose fast-protein liquid chromatography column, and finally, resolution by C-4 reverse-phase high-performance liquid chromatography. After the final step, the rTAT is dried and stored under salt-free conditions. Amino acid compositional analysis and N-terminal sequence analysis confirm that the purified protein is rTAT. Unlike other methods reported for purification of recombinant HIV-1 trans-activator, our protocol uses urea instead of guanidine HCl. The rTAT is fully soluble in buffered solutions at concentrations exceeding 10 mg/ml, migrates as a single 14 kDa species on both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and two-dimensional PAGE gels with a pI of 9.3 +/- 0.3. Additionally, the rTAT migrates as a monomer on size-exclusion chromatography columns under native conditions. Finally, purified rTAT exhibits trans-activator activity when introduced into appropriate reporter cells. Since rTAT is monomeric when tested by gel filtration, and yet exhibits biological activity, we conclude that the method of purification we have utilized is distinct from all other methods reported to date.
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PMID:Purification of an active monomeric recombinant HIV-1 trans-activator. 142 16

To understand whether disease caused by the human immunodeficiency virus (HIV) affects zidovudine disposition, we compared the drug's pharmacokinetics in six healthy volunteers; six persons with the acquired immunodeficiency syndrome (AIDS) and no evidence of gastrointestinal (nausea, vomiting, diarrhea), renal (elevated blood urea nitrogen, serum creatinine), or hepatic (elevated liver function tests) disease; and three patients with AIDS and hepatic disease. After a single oral dose of zidovudine, serial blood samples were analyzed for drug concentration by radioimmunoassay. A one-compartment oral absorption model was fit to the concentration-time data. The absorption rate constant (4.05 vs 2.11 hr-1) and time to maximum concentration (0.61 vs 1.03 hr) were significantly different in healthy volunteers versus patients with AIDS without hepatic disease. Differences in half-life, oral clearance, and area under the curve were not statistically significant. In the three patients with AIDS plus hepatic disease, clearance was reduced an average of 63%, and area under the curve was increased by a factor of 2.3. These comparative pharmacokinetic data do not support profound differences between zidovudine's disposition in healthy volunteers and individuals with AIDS; however, the differences and trends that were observed may represent an effect of HIV disease. Although the presence of hepatic disease clearly indicates a need to modify individual dosages, these pharmacokinetic data may have more generalized implications for zidovudine dosing as the relationships between drug concentration and therapeutic or toxic effects are clarified.
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PMID:Comparative pharmacokinetics of zidovudine in healthy volunteers and in patients with AIDS with and without hepatic disease. 149 5

Vpu as a human-immunodeficiency-virus-type-1-encoded 81-amino-acid integral-membrane protein was expressed in Escherichia coli using the inducible ptrc promoter of an ATG fusion vector. Recombinant Vpu is associated with membranes of E. coli and could be partially solubilized by detergents. Recombinant Vpu was phosphorylated in vitro with purified porcine casein kinase II (CKII) as well as with a CKII-related protein kinase found in cytoplasmic extracts of human and hamster cells. Recombinant Vpu associated with E. coli membranes has turned out to be the best substrate for in vitro phosphorylation with CKII. This reaction can be inhibited by heparin and the ATP analogue 5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (DRB), both known to be potent inhibitors of CKII. Radiolabelled gamma ATP and gamma GTP were used as phosphate donors in vitro phosphorylation of recombinant Vpu. In vivo phosphorylation of Vpu in HIV-1-infected H9 cells was also inhibited by DRB. We concluded therefrom that the Vpu protein is phosphorylated by the ubiquitous CKII in HIV-1-infected human host cells. Two seryl residues in the sequence of Vpu (position 52 and 56) correspond to the consensus S/TXXD/E for CKII. These potential phosphorylation sites are located within a well-conserved dodecapeptide of Vpu (residues 47-58), which is found in different HIV-1 strains as well as in a Vpu-like protein of SIVCPZ. Monoclonal and polyclonal antibodies directed against two different epitopes of Vpu were used for immunoprecipitation of Vpu from HIV-1-infected cells and for detection of Vpu in Western blot analyses. Vpu from HIV-1-infected cells as well as recombinant Vpu expressed in E. coli were determined by SDS/PAGE using 6 M urea to be 9 kDa, which corresponds to the calculated molecular mass of Vpu.
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PMID:Human-immunodeficiency-virus-type-1-encoded Vpu protein is phosphorylated by casein kinase II. 154 Dec 98

In this paper we report on the evaluation of several procedures that allow for the repeated use of an antigen-coated, enzyme-linked immunosorbent assay (ELISA) plate for enzyme immunoassay (EIA). We have shown that antigen-coated ELISA plates that were incubated once with an aqueous solution containing 8 M urea, 2% sodium dodecyl sulfate and 2% mercaptoethanol, after an EIA, can be reused again for EIA without loss of antigenic capacity. Thus, in this procedure, after an EIA, the ELISA plates were washed once with the above solution and then in a buffer containing 20 mM Tris-HCl, pH 7.5, 0.1% Tween 20 and 500 mM NaCl. This washing protocol was shown to remove the primary antibody, enzyme-conjugated secondary antibody and substrate without removing the antigen from the ELISA plate microwells. Thus, an antigen-coated ELISA plate previously used for an assay could be reused. We tested this repeat ELISA (R-ELISA) procedure on high antigen-binding ELISA plates coated with two different plant virus proteins, a synthetic peptide, the p25/24 gag and the gp120 proteins of the human immuno-deficiency virus, or the staphylococcus enterotoxin protein. In each case tested, the procedure allowed for the repeated use of the same antigen-coated plates for EIA of the respective antibodies. This procedure should prove to be particularly valuable for mass screening of samples tested for HIV and other disease-causing agents.
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PMID:R-ELISA: repeated use of antigen-coated plates for ELISA and its application for testing of antibodies to HIV and other pathogens. 157 Nov 53

The role of local immunity in relation to the frequent and heterogeneous pulmonary manifestations of HIV-1 infection in children is poorly understood. In order to examine lung immunity in pediatric AIDS patients, the cellular composition, immunoglobulin, and immune complex (IC) levels were evaluated in 23 samples of bronchoalveolar lavage (BAL) fluid and peripheral blood from 19 pediatric AIDS patients with acute pulmonary pathology. The patients were of two age groups: 4.0-21.5 months (N = 9) and 2.3-13.1 years (N = 10). In BAL, lymphocytes were elevated in 25-45% of samples, and neutrophils were elevated in 27-33%; BAL macrophages varied in percentage (28-99%) but had normal morphology. The blood differentials of pediatric AIDS patients undergoing BAL did not show significant differences when compared with a group of pediatric patients with tuberculosis, but leuko- and neutropenia was noted when compared with pediatric patients with pneumonia and no HIV disease. Of the immunoglobulins measured (IgG, IgM, IgA) only IgG was detectable in unconcentrated BAL fluid (1-37 mg/dl, equivalent to 12-630 mg/dl in the epithelial lining fluid after correction using urea as a marker of dilution). All patients were hypergammaglobulinemic and 83% had high levels of circulating IC (2-40 muEq/ml). Six BAL specimens (26%) also contained IC. The estimated level of IC in lung epithelial lining fluid (after correcting for dilution) was up to fivefold higher than IC concentration in corresponding sera. Specific antibodies to HIV-1 were demonstrated in 35% of the BAL samples by ELISA and in 65% by Western blotting.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of cells, immunoglobulins, and immune complexes present in the bronchoalveolar lavage of pediatric AIDS patients. 157 Dec 28

A case study is given of a 25-year old woman with rhabdomyolysis associated with HIV infection. The presenting symptoms were a 1-week history of backache, gross swelling of both hands and feet, and weakness and marked pain in most muscle groups; 3 days before admission the urine was black and she was unable to walk. Multiple, firm 1-2 cm lymph nodes were revealed during examination. White blood cell count (WBC) was 22,000/microliter with 12 pc lymphocytes, 7.3 pc monocytes, and 80.5 pc polymorphonuclear leukocytes. Hemoglobin concentration was 15.8 g/deciliter; platelet count was 124,000/microliter with a Westergren ESR of 109 mm/h. An antinuclear antibody test was negative. Serum concentration of urea was 3.8 mmol/liter, creatinine 42 microliter/liter, sodium 128 mmol/liter, and potassium 5.9 mmol/liter. Microscopic examination of urine revealed WBC 100/HPF, red blood cells 20/HBF, and granular casts. The dipstick test showed blood land protein in the urine. Electromyography showed inflammatory myopathy. Creatine Kinase (CK) concentration was 2359 IU/liter and lactate dehydrogenase concentration 1000 IU/liter. Hemolysis was present from clinical or laboratory signs. The patient tested HIV positive by ELISA (Abbott) and Western blot (Dupont). Treatment consisted of administration of 60 mg/day of prednisolone orally. Over 2 weeks, swelling of limbs was reduced and CK concentration was reduced to 931 IU/liter. The patient was discharged and did not keep a follow-up appointment. The patient did not have a history of other predisposing conditions, only HIV infection and persistent muscle weakness and inflammatory myopathy. There is evidence from other patient studies of myopathy associated with HIV infection and polymyositislike illness. In this case study, the patient may have had a acute form of polymyositis, or acute viral myositis such as occurs with echo, influenza, coxsackie, and other viral infections. A detailed viral investigation was not performed. HIV infection may have directly infected myocytes or immunosuppression predisposing to acute myositis by other pathogens. HIV-related muscle disease should include rhabdomyolysis.
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PMID:Rhabdomyolysis associated with human immunodeficiency virus (HIV) infection. 180 50

The high-level expression of HIV-1 Rev in Escherichia coli is described. Protein in crude bacterial extracts was dissociated from bound nucleic acid with urea. A simple purification and renaturation protocol, monitored by circular dichroism, is described which results in high yields of pure protein. The purified protein binds with high affinity to the Rev-responsive element mRNA and has nativelike spectroscopic properties. The protein exhibits concentration-dependent self-association as judged by analytical ultracentrifugation and gel filtration measurements. Purified Rev showed reversible heat-induced aggregation over the temperature range 0-30 degrees C. This hydrophobic-driven and nonspecific protein association was inhibited by low concentrations of sulfate ions. Rev solutions at greater than 80 micrograms/mL, incubated at 0-4 degrees C, slowly polymerized to form long hollow fibers of 20-nm diameter. Filament formation occurs at a lower protein concentration and more rapidly in the presence of Rev-responsive mRNA. The nucleic acid containing filaments are about 8 nm in diameter and up to 0.4 micron in length. On the basis of physical properties of the purified protein, we have suggested that in the nucleus of infected cells, Rev binding to the Rev-responsive region of env mRNA may be followed by helical polymerization of the protein which results in coating of the nucleic acid. Coated nucleic acid could be protected from splicing in the nucleus and exported to the cytoplasm.
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PMID:HIV-1 Rev expressed in recombinant Escherichia coli: purification, polymerization, and conformational properties. 185 52

In the first paper of this series a new structure with anti-HIV-1 activity was disclosed and analogues were synthesized to explore the structure-activity relationship of changes in the substituent (R) attached at the N-6 position of 9. This study describes the syntheses and anti-HIV-1 testing of analogues with variations of the five-membered urea ring of the 4,5,6,7-tetrahydro-5-methylimidazo[4,5,1-jk] [1,4]benzodiazepin-2(1H)-one (TIBO) structures. Although many different rings were synthesized to replace the cyclic urea of TIBO, most were found to be inactive in inhibiting the replication of the HIV-1 virus in MT-4 cells. The exceptions were replacement of the urea oxygen with sulfur or selenium to give the corresponding thio- or selenoureas. These were found to be more active than the oxygen counterparts. A small series of analogues was synthesized and tested which allowed direct comparison of urea and thiourea derivatives. Without exception, the latter were always more active than the former. The most active compound of this series (8d) was found to inhibit the HIV-1 virus with an IC50 of 0.012 microM which is comparable to that of AZT.
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PMID:Synthesis and anti-HIV-1 activity of 4,5,6,7-tetrahydro-5-methylimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-on e (TIBO) derivatives. 2. 195 37

Various constructs of the human immunodeficiency virus, type 1 (HIV-1) protease containing flanking Pol region sequences were expressed as fusion proteins with the maltose-binding protein of the malE gene of Escherichia coli. The full-length fusion proteins did not exhibit self-processing in E. coli, thereby allowing rapid purification by affinity chromatography on cross-linked amylose columns. Denaturation of the fusion protein in 5 M urea, followed by renaturation, resulted in efficient site-specific autoprocessing to release the 11-kDa protease. Rapid purification involving two column steps gave an HIV-1 protease preparations of greater than 95% purity (specific activity approximately 8500 pmol.min-1.micrograms protease-1) with an overall yield of about 1 mg/l culture. Incubation of an inactive mutant protease fusion protein with the purified wild-type protease resulted in specific trans cleavage and release of the mutant protease. Analysis of products of the HIV-1 fusion proteins containing mutations at either the N- or the C-terminal protease cleavage sites indicated that blocking one of the cleavage sites influences the cleavage at the non-mutated site. Such mutated full-length and truncated protease fusion proteins possess very low levels of proteolytic activity (approximately 5 pmol.min-1.micrograms protein-1).
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PMID:Autoprocessing of the HIV-1 protease using purified wild-type and mutated fusion proteins expressed at high levels in Escherichia coli. 207 Jul 93


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