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1.
Can J Physiol Pharmacol ; 94(3): 272-7, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26762617

ABSTRACT

The role of hyperhomocysteinemia in coronary artery disease (CAD) patients remains unclear. The present study evaluated the relationship between homocysteine (HCys), adenosine plasma concentration (APC), plasma uric acid, and CAD severity evaluated using the SYNTAX score. We also evaluated in vitro the influence of adenosine on HCys production by hepatoma cultured cells (HuH7). Seventy-eight patients (mean age ± SD: 66.3 ± 11.3; mean SYNTAX score: 19.9 ± 12.3) and 30 healthy subjects (mean age: 61 ± 13) were included. We incubated HuH7 cells with increasing concentrations of adenosine and addressed the effect on HCys level in cell culture supernatant. Patients vs. controls had higher APC (0.82 ± 0.5 µmol/L vs 0.53 ± 0.14 µmol/L; p < 0.01), HCys (15 ± 7.6 µmol/L vs 6.8 ± 3 µmol/L, p < 0.0001), and uric acid (242.6 ± 97 vs 202 ± 59, p < 0.05) levels. APC was correlated with HCys and uric acid concentrations in patients (Pearson's R = 0.65 and 0.52; p < 0.0001, respectively). The SYNTAX score was correlated with HCys concentration. Adenosine induced a time- and dose-dependent increase in HCys in cell culture. Our data suggest that high APC is associated with HCys and uric acid concentrations in CAD patients. Whether the increased APC participates in atherosclerosis or, conversely, is part of a protective regulation process needs further investigations.


Subject(s)
Adenosine/blood , Coronary Artery Disease/blood , Homocysteine/blood , Uric Acid/blood , Aged , Case-Control Studies , Female , Humans , Hyperhomocysteinemia/blood , Male , Middle Aged , Tumor Cells, Cultured
3.
Europace ; 15(9): 1328-32, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23594931

ABSTRACT

AIMS: Adenosine is a possible mediator in vasovagal syncope (VVS) via the activation of its receptors. High expression of adenosine A2A receptors (A2AR) has been reported in VVS. The function of these over-expressed receptors in this population has never been evaluated. METHODS AND RESULTS: We used Adonis, a specific-made antibody with A2AR agonist properties, to evaluate binding parameters (i.e. dissociation constant KD) and cAMP production (i.e. EC50) by peripheral blood mononuclear cells of 16 VVS patients. Eight healthy volunteers served as controls. A2AR expression was higher in patients than controls; mean: 11.5 ± 1.2 vs. 7.7 ± 0.8 AU, P = 0.04. Also, KD values were higher in patients than controls: 2.1 ± 0.02 × 10(-7) vs. 5 ± 1 × 10(-8) M, P < 0.01 In controls, KD values were lower than EC50 (5 ± 1.7 × 10(-8) vs. 2.8 ± 0.4 10(-7) M, P < 0.01), but in patients, KD values did not differ from EC50: 2. ± 0.2 × 10(-7) vs. 2.5 ± 0.4 × 10(-7) M, P > 0.05. However, four patients had lower EC50 (3.5 ± 0.3 × 10(-8) M) than KD (2.9 ± 1.2 × 10(-7) M; KD/EC50 = 9.6), suggesting the presence of spare receptors. CONCLUSION: The function of A2AR of patients with VVS was preserved since their stimulation by Adonis led to cAMP production with an EC50 comparable with those in controls. However, their affinity was lower than those of controls. Our results suggest that A2AR are implicated in the physiopathology of VVS.


Subject(s)
Receptor, Adenosine A2A/blood , Syncope, Vasovagal/blood , Syncope, Vasovagal/diagnosis , Adult , Aged , Biomarkers/metabolism , Humans , Male , Middle Aged , Reproducibility of Results , Sensitivity and Specificity , Young Adult
4.
AIDS Res Hum Retroviruses ; 18(3): 201-6, 2002 Feb 10.
Article in English | MEDLINE | ID: mdl-11839154

ABSTRACT

A multiple antigen peptide [CLIV; (PTKAKRR1VVQREKR2)4-K2-K-betaA] from the C terminus of the gp120 subunit of HIV Env inhibits Env-mediated cell-to-cell fusion through direct interference with the process (Virology 2000;273:169). We have examined various CLIV analogs using a cell-to-cell fusion assay, receptor binding assays, and molecular modeling to further address the characteristics of the peptide responsible for its anti-HIV activity. We show that (1) CLIV does not interfere with Env binding to CD4 and does not interact with the binding site of Env on CXCR4; (2) CLIV does not inhibit protease activities already reported to play a role in fusion; and (3) the pharmacophore is composed of cleavage site1 with amino acid residues at its C terminal end. Based on our data and on the literature, we propose that CLIV interferes with processing of the gp120 C terminus at site1 by the lymphocyte surface after CD4 binding. Our hypothesis implies that the cleavage region of Env is submitted to a stepwise processing including the known intracellular cleavage of gp160 at site2 in order to set the activation of the fusion peptide and a yet unexplored cleavage at site1 by the target cell surface that triggers fusion.


Subject(s)
Antiviral Agents/metabolism , HIV Envelope Protein gp120/metabolism , Membrane Fusion , Peptides/metabolism , Amino Acid Sequence , CD4 Antigens/metabolism , Dipeptidyl Peptidase 4/metabolism , Humans , Molecular Sequence Data , Receptors, CXCR4/metabolism
5.
J Investig Med ; 49(1): 56-67, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11217148

ABSTRACT

BACKGROUND: Infections and hypotension are serious complications that develop during hemodialysis (HD) treatment. Adenosine (ADO), a strong hypotensive and immunosuppressive agent, may participate in these two HD complications, because high concentrations of ADO metabolites are found in dialyzed human plasma. ADO, which is released by endothelial cells, is quickly transformed into inosine (INO) by plasmatic ADO deaminase (ADA) and mononuclear cell ADO deaminase (MCADA). In plasma, the degradation of ADO into INO and its uptake by red blood cells (RBC) are both very rapid, resulting in the short half-life of ADO in blood. METHODS: Using liquid chromatography, we evaluated ADO and INO plasma concentrations before and after HD session. RESULTS: Before the HD session, ADO and INO plasma concentrations were higher in hemodialyzed patients than in controls and in peritoneally dialyzed patients. At the end of the HD session, ADO plasma concentration was increased. ADO plasma concentration for the undialyzed patients was in the same range as that of the controls. Before HD, ADA activity was higher in hemodialyzed patients (559 +/- 349 IU) than in controls (219 +/- 48 IU), and the activity rose during the session (665 +/- 135 IU). ADA activity in the undialyzed patients (222 +/- 80 IU) was in the same range as that of the controls (219 +/- 48 IU). Before the HD session, the MCADA activity (247 +/- 144 IU) was lower than in controls (624 +/- 99 IU). HD did not modify ADO RBC uptake. ADO inhibited mononuclear cell proliferation and interferon-gamma production in humans. Finally, as much as 50 microM INO does not inhibit ADO uptake by RBC and does not modify ADA and MCADA activities. CONCLUSIONS: These data indicate that chronic HD inhibited MCADA activity and increased ADO plasma concentration. Both high ADO plasma concentration and low MCADA activity may be involved in dialysis-induced immune system failure and thereby favor infectious diseases.


Subject(s)
Adenosine/blood , Renal Dialysis/adverse effects , Adenosine Deaminase/blood , Adult , Aged , Aged, 80 and over , Case-Control Studies , Female , Humans , Hypotension/etiology , Infections/etiology , Inosine/blood , Male , Middle Aged
6.
J Infect Dis ; 183(5): 744-52, 2001 Mar 01.
Article in English | MEDLINE | ID: mdl-11181151

ABSTRACT

Protein disulfide isomerase (PDI) is a multifunctional protein with thiol-disulfide redox-isomerase activities. It catalyzes thiol-disulfide interchange reactions on the cell surface that may cause structural modifications of exofacial proteins. PDI inhibitors alter human immunodeficiency virus (HIV) spread, and it has been suggested that PDI may be necessary to trigger HIV entry. This study examined this hypothesis by using cell-to-cell fusion assays, in which the HIV envelope (Env) expressed on the cell surface interacts with CD4(+) lymphocytes. PDI is clustered at the lymphocyte surface in the vicinity of CD4-enriched regions, but both antigens essentially do not colocalize. Anti-PDI antibodies and 2 inhibitors of its catalytic function altered Env-mediated membrane fusion at a post-CD4 cell binding step. The fact that the PDI catalytic activity present on lymphocytes is required for fusion supports the hypothesis that catalysts assist post-CD4 cell binding conformational changes within Env.


Subject(s)
CD4-Positive T-Lymphocytes/enzymology , HIV Infections/etiology , HIV/physiology , Membrane Fusion/physiology , Protein Disulfide-Isomerases/metabolism , Viral Envelope Proteins/physiology , Blotting, Western , CD4 Lymphocyte Count , CD4-Positive T-Lymphocytes/ultrastructure , CD4-Positive T-Lymphocytes/virology , Cell Aggregation , Cells, Cultured , Dithionitrobenzoic Acid/pharmacology , HIV/ultrastructure , Membrane Fusion/immunology , Microscopy, Confocal , Oxidation-Reduction , Protein Disulfide-Isomerases/antagonists & inhibitors , Sulfhydryl Reagents/pharmacology , Viral Envelope Proteins/immunology
7.
Virology ; 273(1): 169-77, 2000 Jul 20.
Article in English | MEDLINE | ID: mdl-10891419

ABSTRACT

CLIV is a multiple antigen peptide ([PTKAKRRVVQREKR](4)-K(2)-K-betaA) that encompasses the cleavage region of the human immunodeficiency virus type 1 (HIV-1) envelope precursor. It displays an antiviral activity against HIV-1 and HIV-2 and inhibits HIV-1 Env-mediated cell-to-cell fusion. This effect has previously been attributed to interference with Env processing, resulting in the expression of a nonfusogenic envelope [Virology (1998) 247, 137]. However, we show here that CLIV does not alter the status of Env cleavage at steady state. Using various aggregation/syncytium assays that allow us to discriminate between gp120/CD4 binding and binding followed by gp41-mediated fusion, we demonstrate that CLIV inhibits a step of the cell-to-cell fusion process after CD4 binding. We demonstrate also that CLIV binds at 37 degrees C to a single class of protein present at the CD4(+) cell surface (Scatchard analysis: K(d) = 8 nM; B(max) = 10(4) sites/cell) and that the fusion inhibition activity seems to correlate with binding to this proteic component. In contrast, CLIV interacts with neither membrane-inserted nor CD4-associated Env. We therefore propose that CLIV interferes after Env/CD4 binding with a step of the membrane fusion process that may involve the C-terminal domain of gp120.


Subject(s)
Anti-HIV Agents/pharmacology , CD4 Antigens/metabolism , Gene Products, env/chemistry , HIV Envelope Protein gp120/pharmacology , HIV-1/drug effects , Membrane Fusion/drug effects , Peptide Fragments/pharmacology , Amino Acid Sequence , Anti-HIV Agents/chemistry , Anti-HIV Agents/metabolism , Cell Line , Cell Membrane/drug effects , Cell Membrane/metabolism , Gene Products, env/metabolism , Gene Products, env/pharmacology , Genetic Vectors/genetics , Giant Cells/cytology , Giant Cells/drug effects , Giant Cells/metabolism , Giant Cells/virology , HIV Antigens/chemistry , HIV Antigens/metabolism , HIV Antigens/pharmacology , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/metabolism , HIV-1/metabolism , HIV-2/drug effects , HIV-2/metabolism , Humans , Lymphocytes/cytology , Lymphocytes/drug effects , Lymphocytes/metabolism , Lymphocytes/virology , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Binding/drug effects , Protein Processing, Post-Translational/drug effects , Vaccinia virus/genetics
8.
FEMS Microbiol Lett ; 183(2): 235-40, 2000 Feb 15.
Article in English | MEDLINE | ID: mdl-10675590

ABSTRACT

SPC(3) is a multiple antigen peptide derived from the V(3) loop of human immunodeficiency virus (HIV) envelope (Env). It exerts a potent anti-HIV activity whereas it alters neither Env expression nor binding to CD(4). Here, SPC(3) binding characteristics, its subsequent intracellular fate and the fact that it inhibited SDF(1)alpha binding to the lymphocyte surface provided strong arguments to conclude that it exerts its anti-HIV activity through interference with the CXCR(4) coreceptor. In contrast, it interferes with none of the other major surface proteins and mechanisms involving V(3) and implicated in infection, as shown here. This work identifies the target mechanism of SPC(3).


Subject(s)
Anti-HIV Agents/pharmacology , HIV Envelope Protein gp120/pharmacology , Lymphocytes/drug effects , Receptors, CXCR4/physiology , Animals , Cell Line , Cell Membrane/metabolism , Cricetinae , Dipeptidyl Peptidase 4/metabolism , HIV Envelope Protein gp120/metabolism , Humans , Phosphorylation , Receptors, CXCR4/drug effects , Recombinant Proteins , Thrombin/pharmacology
9.
Eur J Biochem ; 260(2): 482-9, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10095785

ABSTRACT

Analysis of the fate of HIV-1 envelope protein gp160 (Env) has shown that newly synthesized proteins may be degraded within the biosynthetic pathway and that this degradation may take place in compartments other than the lysosomes. The fate of newly synthesized Env was studied in living BHK-21 cells with the recombinant vaccinia virus expression system. We found that gp160 not only undergoes physiological endoproteolytic cleavage, producing gp120, but is also degraded, producing proteolytic fragments of 120 kDa to 26 kDa in size, as determined by SDS/PAGE in non reducing conditions. Analysis of the 120-kDa proteolytic fragment, and comparison with gp120, showed that it is composed of peptides linked by disulfides bonds and lacks the V3-loop epitope and the C-terminal domain of gp120 (amino acids 506-516). A permeabilized cell system, with impaired transport of labeled Env from the endoplasmic reticulum (ER) to Golgi compartments, was developed to determine the site of degradation and to define some biochemical characteristics of the intracellular degradation process. In the semipermeable BHK-21 cells, there was: (a) no gp120 production (b), a progressive decrease in the amount of newly synthesized gp160 and a concomitant increase in the amount of a 120-kDa proteolytic fragment. This fragment had the same biochemical characteristics as the 120-kDa proteolytic fragment found in living nonpermeabilized cells, and (c) susceptibility of the V3 loop. This degradation process occurred in the ER, as shown by both biochemical and indirect immunofluorescence analysis. Furthermore, there was evidence that changes in redox state are involved in the ER-dependent envelope degradation pathway because adding reducing agents to permeabilized cells caused dose-dependent degradation of the 120-kDa proteolytic fragment and of the remaining gp160 glycoprotein. Thus our results provide direct evidence that regulated degradation of the HIV-1 envelope glycoprotein may take place in the ER of infected cells.


Subject(s)
Endoplasmic Reticulum/metabolism , HIV Envelope Protein gp160/metabolism , Animals , Cell Line , Cricetinae , HIV-1 , Humans , Microscopy, Fluorescence , Oxidation-Reduction , Sheep
10.
Toxicon ; 36(12): 1939-47, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9839678

ABSTRACT

We have purified from Vipera lebetina venom a family of inhibitors of platelet aggregation, named Lebetins. They are composed of two peptide groups of short (Lebetin 1: L1alpha: GDNKPPKKGPPNG; L1beta: DNKPPKKGPPNG) and long (Lebetin 2: L2alpha: GDNKPPKKGPPNGCFGHKIDRIGSHSGLGCNKVDDNKG; L2beta: DNKPPKKGPPNGCFGHKIDRIGSHSGLGCNKVDDNKG) size. The sequence presenting anti-platelet activity is mainly present within the Lebetin 1 sequence [Barbouche, R. Marrakchi, N., Mansuelle, P., Krifi, M., Fenouillet, E., Rochat, H. and El Ayeb, M. (1996) Novel anti-platelet aggregation polypeptides from Vipera lebetina venom: isolation and characterization. FEBS Lett. 392, 6-10]. Here, the peptides that compose the Lebetin 1 family were synthesized. Their respective activity was determined. Synthetic L1alpha and L1beta inhibited collagen-induced platelet aggregation in the nanomolar range. A peptide corresponding to L1beta deleted by D at its N terminus (L1gamma) also inhibited platelet aggregation potently; further truncation of L1gamma impaired its activity. Because L1 peptides efficiently inhibited fibrinogen-induced alpha-chymotrypsin treated-platelet aggregation, we tested whether they act mainly through the inhibition of platelet binding to fibrinogen and showed that they failed to inhibit platelet binding to fibrinogen-coated wells. The activity of L1 peptides was also tested in vivo: their intravenous administration strongly inhibited collagen-induced thrombocytopenia in rats.


Subject(s)
Fibrinolytic Agents/pharmacology , Peptides/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Viper Venoms/pharmacology , Amino Acid Sequence , Animals , Collagen/adverse effects , Fibrinolytic Agents/toxicity , In Vitro Techniques , Mice , Molecular Sequence Data , Peptides/toxicity , Platelet Aggregation Inhibitors/chemical synthesis , Platelet Aggregation Inhibitors/toxicity , Rats , Rats, Wistar , Thrombocytopenia/chemically induced
11.
J Pept Res ; 52(4): 283-8, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9832306

ABSTRACT

A multibranched peptide construct (SPC3) derived from the conserved sequence of the third variable domain (V3) of the human immunodeficiency virus (HIV) envelope (Env) inhibits HIV infectivity. It is being tested in phase II clinical trials (FDA protocol 257A). Because some Env-derived peptides inhibit HIV infectivity through alteration of Env biosynthetic pathway, we studied whether SPC3 displays its activity through interference with Env biosynthesis or with its functions at the membrane. Syncytium formation was impaired when human CD4+ cells expressed recombinant HIV Env in the presence of SPC3. This inhibition was not due to an effect of SPC3 on the amount of Env expressed at the cell membrane. As assessed using antibodies, the conformation of the receptor binding site and of V3 presented on membrane Env was not affected by the presence of SPC3 during biosynthesis. Finally, despite the ability of SPC3 to bind to CD4+ cell membrane, SPC3 did not interfere with Env binding to CD4. These data suggest that SPC3 interferes with the infection process at a post-CD4 binding step, and not with the folding of Env.


Subject(s)
AIDS Vaccines/chemistry , HIV Envelope Protein gp120/chemistry , HIV Infections/prevention & control , HIV-1/isolation & purification , Peptide Fragments/chemistry , AIDS Vaccines/therapeutic use , Binding Sites , Cells, Cultured , Clinical Trials, Phase II as Topic , HIV Envelope Protein gp120/pharmacology , HIV Envelope Protein gp120/therapeutic use , HIV-1/chemistry , Humans , Peptide Fragments/pharmacology , Peptide Fragments/therapeutic use , Protein Conformation , Receptors, HIV/antagonists & inhibitors
12.
Virology ; 247(2): 137-43, 1998 Aug 01.
Article in English | MEDLINE | ID: mdl-9705906

ABSTRACT

A 22-amino-acid-long multibranched peptide construct (CLV) derived from the cleavage region (KIEPLGVAPTKAKRR*VVQREKR*) of the human immunodeficiency virus (HIV) type-1 envelope precursor inhibits HIV infection (Virology, 1996, 223, 406-408). We attempted to characterize its activity for Env expressed via a recombinant vaccinia virus (rVV): gp 160 cleavage was delayed, but not impaired, in the presence of CLV (10 microM), whereas neither Env production nor Env membrane expression was significantly altered. Through the synthesis of analogs, we concluded that the presence of a cleavage sequence was required for inhibition of syncytium formation by CLV in rVV-infected CD(4+) cell cultures: indeed, a single amino acid residue substitution (R* > S) in the cleavage sites presented by CLV abolished its activity. Other analogs allowed us to further determine the region of CLV which mediates its activity. The ability of a radiolabeled CLV analog to enter cells was also shown. Although, these data strongly suggest that CLV acts on Env fusogenicity at least partially through interference with gp160 processing.


Subject(s)
Anti-HIV Agents/pharmacology , Gene Products, env/metabolism , HIV Envelope Protein gp160/metabolism , Peptides/pharmacology , Amino Acid Sequence , Animals , Anti-HIV Agents/chemical synthesis , Anti-HIV Agents/metabolism , Cell Line , Cell Membrane/virology , Cells, Cultured , Cricetinae , Gene Products, env/drug effects , Giant Cells/virology , HIV Envelope Protein gp160/drug effects , Humans , Molecular Sequence Data , Peptides/chemical synthesis , Peptides/metabolism , Protein Precursors/chemistry , Protein Precursors/pharmacology , Protein Processing, Post-Translational/drug effects
13.
Virology ; 242(2): 338-45, 1998 Mar 15.
Article in English | MEDLINE | ID: mdl-9514971

ABSTRACT

The requirement for glycosylation in the transmembrane protein, gp41, of human immunodeficiency virus type 1 envelope protein for fusion activity has been studied. By using a mutant gene in which three conserved sites have been removed and which shows no fusion ability, genes were constructed which replace one, two, or three sites in all possible combinations. Following expression of the resultant proteins using the vaccinia T7 system, each Env variant was assessed by visual and quantitative syncytium assays. Our data indicate that two sites are sufficient for high levels of fusion and that the single site at position 621 is the most critical of all positions. We interpret our data in the light of previous contradictory reports on the role of gp41 glycosylation in bioactivity and the emerging structure of gp41.


Subject(s)
HIV Envelope Protein gp41/physiology , Animals , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/virology , Chlorocebus aethiops , Giant Cells/enzymology , Giant Cells/virology , Glycosylation , HIV Envelope Protein gp160/metabolism , HIV Envelope Protein gp41/genetics , HeLa Cells , Humans , Mutagenesis , Mutation , Vero Cells , beta-Galactosidase/metabolism
14.
Virology ; 241(1): 163-7, 1998 Feb 01.
Article in English | MEDLINE | ID: mdl-9454727

ABSTRACT

The influence of HIV Env glycosylation on the conformation of the third variable domain (V3) of Env was studied by both deglycosylation of mature Env and the use of Env produced by recombinant systems in which alpha-glucosidase activity was inhibited by either deoxynojirimycin (DNM) or mutation. Selective deglycosylation affected anti-V3 antibody binding. The immunoreactivity and sensitivity to thrombin cleavage of V3 presented on Env produced in baby hamster kidney cells were changed by DNM treatment. In contrast, Env expressed in alpha-glucosidase I-deficient Chinese hamster ovary cells or in their parental cells treated by DNM fully retained these V3 properties. These results are discussed in relation to the inconsistent data obtained on V3 property changes resulting from Env glycosylation changes.


Subject(s)
HIV Envelope Protein gp120/metabolism , HIV-1/metabolism , Peptide Fragments/metabolism , Thrombin/metabolism , 1-Deoxynojirimycin/pharmacology , Animals , Cell Line , Cricetinae , Enzyme Inhibitors/pharmacology , Glycoside Hydrolases/metabolism , Glycosylation , HIV Antibodies/immunology , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp120/immunology , HIV-1/drug effects , HIV-1/genetics , HIV-1/immunology , Humans , Peptide Fragments/genetics , Peptide Fragments/immunology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism
15.
J Pept Sci ; 4(8): 479-85, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9927254

ABSTRACT

SPC3 is a peptide construct (eight branches of the GPGRAF motif) derived from the consensus sequence present at the apex of the third variable domain of the human immunodeficiency virus (HIV) envelope (Env). It presents a potent anti-HIV activity and is currently tested in phase II clinical trials (FDA protocol 257A). Its mode of action remains unclear. It was thought that SPC3 exerts its effect both during HIV interaction with CD4+ cells but also through interference either with a post-binding event or with Env processing. Accordingly, SPC3 was supposed to be able to bind and to enter CD4+ cells. In this work, we addressed these points. SPC3 was found to interact with CD4+ cell membrane with a K0.5 value in the range of 500 nM. The binding of SPC3 to CD4+ cells involves its interaction with a cell membrane associated protein which is pronase sensitive and different from CD4. This interaction was similar from 2 to 37 degrees C. The maximum binding occurred at acidic pH whereas the interaction was inhibited in alkaline conditions. We observed also that SPC3 was internalized rapidly into the cells - the maximal intracell amount was reached within 30 min - where it remained stable for at least 24 h. Altogether, these data suggest that SPC3 can exert its antiviral activity via interference with events occurring at the cell surface but also into the target cell.


Subject(s)
Antiviral Agents/metabolism , CD4-Positive T-Lymphocytes/metabolism , HIV Envelope Protein gp120/metabolism , HIV , Antiviral Agents/chemical synthesis , CD4-Positive T-Lymphocytes/virology , Cell Line , Gene Products, env/chemistry , HIV Envelope Protein gp120/chemistry , Humans , Recombinant Proteins , Surface Properties
16.
FEBS Lett ; 406(1-2): 191-5, 1997 Apr 07.
Article in English | MEDLINE | ID: mdl-9109416

ABSTRACT

Using a CD4-binding assay to assess the conformation of the human immunodeficiency virus envelope glycoprotein (CHO+ Env), we studied the effect of treatment with various glycosidases on the stability of Env in denaturing environments and in biological media: cleavage from Env of either high-mannose-type glycans (HMT- Env) by endoglycosidase H or sialic acid residues (Sial- Env) by sialidase did not alter Env stability whereas its complete deglycosylation (CHO- Env) by N-glycanase had a large effect. The influence of glycan removal on Env sensitivity to proteases was also studied. Thrombin cleavage within V3 was affected by N-glycanase treatment; both HMT- Env and CHO- Env displayed an increased sensitivity to other endoproteases. Thus, partial deglycosylation increases Env sensitivity to proteases but only its total deglycosylation alters its stability.


Subject(s)
Gene Products, env/metabolism , Glycoside Hydrolases/metabolism , Animals , Binding Sites , Blood , CD4 Antigens/metabolism , CHO Cells , Cricetinae , Glycosylation , Hydrolysis , Thrombin/metabolism
17.
Virology ; 231(1): 89-95, 1997 Apr 28.
Article in English | MEDLINE | ID: mdl-9143306

ABSTRACT

alpha-Glucosidase inhibitors-e.g., 1-deoxynojirimycin (DNM)-interfere with HIV infectivity in CD4+ cell cultures but have proven unsuccessful in clinical trials. In vitro, several HIV Env properties, including the cleavage of the Env precursor gp 160, the immunoreactivity of the third variable domain (V3) of Env, the binding to the CD4 receptor, and the induction of the membrane fusion between the virus and the host cell, have been reported to be altered by such inhibitors. We have studied these properties for Env expressed via a recombinant vaccinia virus in two Chinese hamster ovary cell lines, an alpha-glucosidase I-deficient cell line and its parental cell line, treated with DNM under conditions that have been reported to alter Env properties. The glycosylation of Env, but not the quantity produced, varied in accordance with the experimental conditions. However, irrespective of these conditions, Env cleavage, V3 immunoreactivity, CD4 binding, membrane expression, and ability to induce syncytium formation were similar. Thus, neither the alpha-glucosidase I deficiency nor DNM treatment had a significant effect on the properties of Env produced here. Cellular mechanisms that may allow the normal expression of Env are discussed and may offer an explanation for the many discrepant results obtained to date on the effects of DNM on HIV Env.


Subject(s)
1-Deoxynojirimycin/pharmacology , Anti-HIV Agents/pharmacology , Enzyme Inhibitors/pharmacology , Gene Products, env/metabolism , HIV-1/metabolism , alpha-Glucosidases/metabolism , Animals , CD4-Positive T-Lymphocytes/metabolism , CHO Cells , Cell Fusion , Cell Line , Cricetinae , Gene Deletion , Gene Products, env/genetics , Glycosylation , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp120/metabolism , HIV Envelope Protein gp41/genetics , HIV Envelope Protein gp41/metabolism , Humans , Peptide Fragments/genetics , Peptide Fragments/metabolism , Recombinant Proteins , alpha-Glucosidases/genetics
18.
Virology ; 223(2): 406-8, 1996 Sep 15.
Article in English | MEDLINE | ID: mdl-8806580

ABSTRACT

Multibranched peptides (SPCs) derived either from the fusion protein (gp41) sequence or from the cleavage sequence of the human immunodeficiency virus type 1 envelope were chemically synthesized and tested for their ability to inhibit both syncytium formation and HIV production in CD4+ cells. The gp41-derived SPCs had no effect. In contrast, an SPC encompassing the envelope cleavage sites strongly inhibited both HIV Env-induced syncytium formation and viral production.


Subject(s)
HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp120/pharmacology , HIV Envelope Protein gp41/genetics , HIV Envelope Protein gp41/pharmacology , HIV-1/genetics , Amino Acid Sequence , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/virology , Cells, Cultured , Giant Cells/drug effects , Giant Cells/virology , HIV Infections/virology , Molecular Sequence Data , Recombinant Proteins/chemical synthesis , Recombinant Proteins/pharmacology
19.
FEBS Lett ; 392(1): 6-10, 1996 Aug 19.
Article in English | MEDLINE | ID: mdl-8769304

ABSTRACT

Lebetins 1 and Lebetins 2, two polypeptide groups that inhibit platelet aggregation, were isolated from Vipera lebetina venom by gel filtration and reverse phase chromatography. Amino acid sequencing indicated that the first group contains two major polypeptides of 13 and 12 residues; their molecular weight was determined by electrospray mass spectrometry. The second was composed of two peptides of 38 and 37 residues, each with one disulfide bond. Sequence analysis revealed neither RGD sequence nor homology with other proteins including known snake or tick polypeptides. Lebetins 1 were Pro and Lys rich peptides and their sequences were identical to the N-terminus of Lebetins 2. Lebetins inhibited platelet aggregation induced by thrombin, collagen and PAF-acether. The 50% concentration that inhibited human and rabbit platelet aggregation induced by thrombin was 590 nM and 125 nM for Lebetins 1 and 100 nM and 8 nM for Lebetins 2, respectively. Lebetins 1 and Lebetins 2 also inhibited fibrinogen-induced aggregation of alpha-chymotrypsin-treated platelets as well as in vivo collagen-induced thrombocytopenia in rats with half effective doses of 2 nmol/kg and 4.2 nmol/kg, respectively. Lebetins were not toxic after intravenous injection into mice and rats. These polypeptides form novel platelet inhibitors that could be used to delineate further the mechanisms of platelet aggregation and serve as a model for developing antithrombotic agents.


Subject(s)
Platelet Aggregation Inhibitors/isolation & purification , Viper Venoms/chemistry , Viper Venoms/isolation & purification , Amino Acid Sequence , Animals , Collagen , Humans , In Vitro Techniques , Mice , Molecular Sequence Data , Peptides/chemistry , Peptides/isolation & purification , Peptides/pharmacology , Platelet Aggregation Inhibitors/chemistry , Platelet Aggregation Inhibitors/pharmacology , Rabbits , Rats , Rats, Wistar , Thrombocytopenia/chemically induced , Thrombocytopenia/prevention & control , Viper Venoms/pharmacology
20.
Virology ; 218(1): 224-31, 1996 Apr 01.
Article in English | MEDLINE | ID: mdl-8615025

ABSTRACT

N-glycosylation of the human immunodeficiency virus type-1 envelope (Env) glycoprotein precursor (gp160) occurs by transfer of Glc3Man9GlcNAc2 onto the nascent protein. Maturation then occurs via cleavage of the three Glc residues, which starts during translation. These events are considered necessary to create Env functional conformation: treatment with "alpha"-glucosidase inhibitors, but not alpha-mannosidase inhibitors (i) impairs gp160 cleavage into gp120 and gp41, (ii) diminishes the accessibility of gp120 V3 region, (iii) prevents gp120 binding to its CD4 receptor, and (iv) prevents gp41-mediated membrane fusion. These inhibitors are of therapeutic interest. Here, using a collection of parent and mutant CHO cells that possess mutations in different steps of glycosylation, we reassessed the role of glycans in both the processing and the properties of recombinant gp160 expressed from a vaccinia virus vector. Mutant cells were as follows: Lec23 (which lacks alpha-glucosidase I activity) produces a collection of triglucosylated structures (Glc3Man7-9GlcNAc2); LEC10 (which has increased GlcNAc transferase III activity) produces complex glycans with a bisected GlcNAc residue; Lec1 (which lacks GlcNAc transferase I) and Lec3.2.8.1 (which lacks GlcNAc transferase I and has decreased activity of CMP-NeuNAc and UDP-Gal translocases) produce Man5GlcNAc2 glycans at complex or hybrid sites. As expected, glycosylation of Env produced from mutants was affected but, irrespective of the glycosylation phenotype, (i) similar quantities of Env were synthesized, (ii) the immunoreactivity of V3 was similar, (iii) gp160 was efficiently cleaved into gp120 and gp41, (vi) Env was exposed at the cell membrane, (v) secreted gp120 bound CD4, and (vi) membrane gp41 was able to induce membrane fusion with CD4+ cells. Thus, the glycosylation alterations examined are dispensable for Env processing and biological activity in CHO cells. In particular, removal of the three outer Glc residues was not required per se for Env folding in this system because functional Env is obtained from Lec23 cells: it appears therefore that lack of modification is not equivalent to drug inhibition of modification. These data are discussed in the light of previous reports describing the use of glycosidase inhibitors to alter glycosylation.


Subject(s)
Gene Products, env/metabolism , HIV-1/metabolism , Protein Precursors/metabolism , Animals , CD4 Antigens/metabolism , CHO Cells , Cell Line , Cricetinae , Gene Products, env/chemistry , Gene Products, env/genetics , Glycosylation , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp120/immunology , HIV Envelope Protein gp160 , HIV-1/chemistry , HIV-1/genetics , Lymphocytes/metabolism , Membrane Fusion , Molecular Weight , Peptide Fragments/genetics , Peptide Fragments/immunology , Protein Precursors/chemistry , Protein Precursors/genetics , Recombinant Proteins , Vaccinia virus
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