Interaction of adriamycin with human erythrocyte membranes. Role of the negatively charged phospholipids.

The interaction of the antitumor compound adriamycin with human erythrocyte membranes, used as models of target cell membranes, has been studied using circular dichroism measurements. In order to elucidate the nature of the sites involved in the electrostatic interaction between adriamycin and erythrocyte membranes, its interaction with the following macromolecular systems was studied: phosphatidylserine-containing small unilamellar vesicles (SUV), prepared from total lipid extracts of erythrocytes, sialic acid-depleted erythrocyte ghosts and mucopolysaccharides. We have shown that the interaction between adriamycin and carboxylate groups is very weak and that negatively charged phosphate groups, in the case of membranes, or sulfate groups, in the case of mucopolysaccharides, are responsible for the prime interaction of adriamycin with these macromolecular systems.

The interaction of a glycosaminoglycan, heparin, with HIV-1 major envelope glycoprotein.

We demonstrate in vitro the occurrence of a specific but low-affinity interaction between soluble tetrameric rgp160 or soluble monomeric or tetrameric rgp120 and heparin-agarose (HA). This interaction is saturable, pH and temperature-dependent, and can be inhibited by soluble heparin, but not by soluble dextran. In buffer supplemented with 10 mM CaCl2, the C50 of soluble heparin, i.e., the concentration of soluble heparin which leads to 50% inhibition of the binding of [125I]rgp160 or of [125I]rgp120 to HA, is 1.1 x 10(-4) disaccharidic molar concentration for rgp160 and 3.2 x 10(-4) dissacharidic molar concentration for rgp120, which indicates low-affinity interactions. Upon chromatography on HA, [125I]rgp160 is repeatedly eluted as a retarded fraction when compared to the elution volume of [125I]rgp160-soluble heparin complex. Under the same experimental conditions, [125I]rgp120 is also eluted, but as a less retarded fraction than [125I]rgp160. Taken together, these results suggest that, at least part of the described anti HIV-1 activity of heparin might be mediated by interaction with HIV-1 major envelope glycoprotein.

Interactions of HIV-1 envelope glycoproteins with derivatized dextrans.

The present study demonstrates that derivatized dextrans, such as carboxylmethyl dextran benzylamide and carboxymethyl dextran benzylamide sulfonate, specifically interact with HIV-1 envelope glycoproteins (rgp160 and rgp41) with significantly higher affinities than those observed for dextran sulfate (MW 8 kDa). These results suggest the possible involvement in HIV infectivity of surface membrane molecules which may bind the virus at pre or post-CD4 binding steps. They also suggest the possible use of these compounds in anti-HIV therapy.

Anthracycline incorporation in human lymphocytes. Kinetics of uptake and nuclear concentration.

Fluorescence emission spectra from anthracycline-treated cells suspended in buffer have been studied. The kinetics of uptake and the nuclear concentration of anthracyclines in human lymphocytes have thus been determined using the fluorescence properties of these drugs. Four anthracyclines have been used: adriamycin (ADR), 4'-O-tetrahydropyranyl-adriamycin (THP-ADR), carminomycin (CAR) and aclacinomycin A (ACM). We have shown that no quenching of the drug fluorescence is obtained through interaction of the drugs with the various components of the cell except the nucleus. No quenching is observed with cells lacking nucleus such as platelets and erythrocytes. Quenching of drug fluorescence occurs when drugs intercalate between base pairs of DNA only. This allows rapid determination of the amount of drug intercalated between nuclear base pairs in the steady state. We have thus estimated that one molecule of drug can bind for every 10, 8.3, 10 and 6.7 DNA base pairs in the case of ADR, THP-ADR, ACM and CAR, respectively. The kinetics of drug incorporation into the nucleus, once the cells have been solubilized with triton X-100, is very fast for the four drugs. However, the kinetics of drug uptake by the intacts cells strongly depends on the nature of the drug. When 10(9) cells/l are incubated with 1 microM drug, 50% of the final nuclear concentration is reached within 97 min, 3.2 min, 3.0 min and 1.2 min in the case of ADR, THP-ADR, CAR and ACM, respectively. The kinetics directly correlates with the polarity of the molecule.

Glycans are involved in RANTES binding to CCR5 positive as well as to CCR5 negative cells.

We show that cell surface glycans, sialic acid and mannose-containing species, are involved beside glycosaminoglycans (GAGs), heparan sulfate and chondroitin sulfate in the binding of full length (1--68) RANTES not only to CCR5 positive human primary lymphocytes or macrophages but also to CCR5 negative monocytic U937 cells. Pretreating the cells with neuraminidase, heparitinase, chondroitinase or adding soluble glycans such as mannan or GAGs (heparin or chondroitin sulfate), significantly inhibited RANTES binding. Such effects were not observed with truncated (10--68) RANTES. Heat-denaturation of (1--68) RANTES strongly decreased its binding to the cells, demonstrating involvement of the three-dimensional structure. Accordingly, full length, but not truncated (10--68) RANTES, specifically bound to soluble mannan as well as to mannose-divinylsulfone-agarose affinity matrix and to soluble heparin or chondroitin sulfate as well as to heparin-agarose. Soluble heparin exerts, depending on its concentration, inhibitory or enhancing effects on RANTES binding to mannose-divinylsulfone-agarose, which indicates that RANTES interaction with glycans is modulated by GAGs. These data demonstrate that full length RANTES, but not its (10--68) truncated counterpart, interacts with glycans and GAGs, in soluble forms or presented either by affinity matrices or CCR5 positive as well as CCR5 negative cells.

Molecular interaction between HIV-1 major envelope glycoprotein and dextran sulfate.

We investigated at the molecular level the interaction between, HIV-1 recombinant gp160 (rgp160) and low-molecular-weight dextran sulfate. We demonstrate the occurrence of a specific interaction between rgp160 and sulfated dextran beads, which is saturable, pH-dependent and inhibitable by soluble dextran sulfate but not by soluble dextran. This specific interaction has a low affinity, with an estimated Kd in the 10(-4) M range. In addition, the binding of rgp160 to soluble recombinant CD4 (sT4) can only be inhibited by the preincubation of rgp160, but not of sT4, with dextran sulfate. Taken together, these results demonstrate the occurrence of a low affinity, but specific interaction between dextran sulfate and rgp160. This may account, at least in part, for the anti-HIV-1 activity of dextran sulfate.

A monoclonal antibody directed to sulfatide inhibits the binding of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein to macrophages but not their infection by the virus.

We show here that human immunodeficiency virus (HIV) envelope glycoproteins (gp160/gp120) bind to sulfatide and galactosyl ceramide. By immunofluorescence labeling with monoclonal antibody (mAb) A2B5, specific for ganglioside/sulfatide, we detect negatively charged glycolipids on CD4+ cells of the macrophage lineage and lymphocytes. Labeling of monocyte-derived macrophages (MDM) with mAb A2B5 was reproducibly found in 29 healthy donors, independently of the culture method and duration up to 11 days. The binding of the mAb to neuraminidase-treated MDM was unchanged relative to control cells, but mAb binding decreased after arylsulfatase treatment, which indicates that MDM membrane sulfatide is its major ligand. Preincubating MDM with the mAb partially (40-60%) but significantly inhibited the binding of HIV-1LAI radiolabeled recombinant gp160 to the cells. Similarly, the mAb entailed limited (32%) but significant inhibition of gp160 binding to cells of the monocytic U937 line but not to lymphoid CEM cells. However, mAb A2B5 did not inhibit the infection of CEM nor of U937 cells by HIV-1LAI strain, nor of MDM by monocytotropic HIV-1BaL. Thus, although sulfatide may be involved in the binding of HIV env glycoprotein to MDM or monocytic U937 cells, this does not play a significant role in HIV infection of these CD4+ cells.

Involvement of the HIV-1 external envelope glycoprotein 120 (gp120) C2 region in gp120 oligomerization.

A synthetic peptide resembling the C2 region of human immunodeficiency virus type 1 (HIV-1) gp120 (C2-Lai: amino acids (aa) 273-288), inhibited (C50 = 200 microM) gp120 calcium-dependent binding of N-acetyl-beta-D-glucosaminyl and mannosyl residues exposed on natural glycoprotein bovine fetuin whereas a peptide derived from an aa sequence downstream of C2-Lai (C2-SC19) had no such effect (C50 > 1000 microM). No calcium-carbohydrate-specific binding of C2-Lai to fetuin was detected. In addition, C2-Lai was also found to inhibit the calcium-dependent oligomerization of gp120: while recombinant gp120 (rgp120) was recovered mainly as oligomers (78%) in 10 mM CaCl2, in contrast to 100% monomers in 1mM CaCl2, mostly monomers (67%) were found in 10 mM CaCl2 in the presence of C2-Lai. Peptide C2-SC19 and carbohydrate structures such as fetuin, fucoidin, dextran or mannan did not significantly affect gp120 oligomerization. Electrophoresis and gel filtration analysis also showed that C2-Lai aggregated in the form of 20 kDa compounds, which is compatible with association of 10 molecules. Taken together, these results demonstrate that the C2 domain is involved in gp120 oligomerization and suggest that gp120 oligomers but not monomers have specific carbohydrate binding properties.

Antiviral activity of derivatized dextrans on HIV-1 infection of primary macrophages and blood lymphocytes.

The present study demonstrates at the molecular level that dextran derivatives carboxymethyl dextran benzylamine (CMDB) and carboxymethyl dextran benzylamine sulfonate (CMDBS), characterized by a statistical distribution of anionic carboxylic groups, hydrophobic benzylamide units, and/or sulfonate moieties, interact with HIV-1 LAI gp120 and V3 consensus clades B domain. Only limited interaction was observed with carboxy-methyl dextran (CMD) or dextran (D) under the same conditions. CMDBS and CMDB (1 microM) strongly inhibited HIV-1 infection of primary macrophages and primary CD4+ lymphocytes by macrophage-tropic and T lymphocyte-tropic strains, respectively, while D or CMD had more limited effects on M-tropic infection of primary macrophages and exert no inhibitory effect on M- or T-tropic infection of primary lymphocytes. CMDBS and CMDB (1 microM) had limited but significant effect on oligomerized soluble recombinant gp120 binding to primary macrophages while they clearly inhibit (> 50%) such binding to primary lymphocytes. In conclusion, the inhibitory effect of CMDB and the CMDBS, is observed for HIV M- and T-tropic strain infections of primary lymphocytes and macrophages which indicates that these compounds interfere with steps of HIV replicative cycle which neither depend on the virus nor on the cell.

A glucose-containing fraction extracted from the young erythrocyte membrane is capable of transferring glucose to hemoglobin in vitro.

Red blood cell (RBC) membranes are rich in a glycoconjugate that is extractable in chloroform/methanol solutions (2/1, v/v) and contains several hexoses, such as glucose. Old and young RBC are separated and their respective glycoconjugates are prepared. HbA0 is purified by column chromatography and incubated with solutions of this conjugate. After 24-h incubation, Hb is dialyzed and the amount of glycosylated Hb is measured by a method of column chromatography adapted from Trivelli. A very significant amount of HBAlc is formed when young RBC extracts are incubated: 3.6% of total Hb becomes HBAlc with the extracts, versus 3.2% with free glucose, and only 2.5% for controls. No increase in HbAlc is obtained when extracts of old RBC are incubated. Another difference between the action of the glycoconjugate and free glucose is that the former induces the increase of only the HBAlc fraction, whereas glucose induces the increase of all the minor Hb fractions. The evaluation of glucose contained in the conjugate before and after the glycosylation reaction demonstrates that it is due to an exchange of glucose units from the conjugate to Hb. The reaction is stereospecifically inhibited by p-nitrophenyl-beta-D-glucoside. The nature of the formed HbAlc is demonstrated by isoelectric focusing. A slight increase of HbAlc observed in the incubated controls may be due to an internal migration of some residues of glucose primitively bound to lysyl residues in an unstable form and also to some degree of denaturation during the incubation.

Inhibition by monoclonal anticomplement receptor type 1 on interactions between senescent human red blood cells and monocytic-macrophagic cells.

The effect of different murine monoclonal antibodies (Mab) specific for the glycoprotein complement receptor type 1 (CR1), type 2 (CR2), and type 3 (CR3) on the adhesion to and on the phagocytosis of human senescent red blood cells (S-RBC) by monocytes or by monocyte-derived macrophages (M phi) was investigated. Murine Mab anti-CR3 (anti-Leu 15 and OKM1) were found to inhibit, in the same order of magnitude, on one hand, the Fc receptors (FcR)-dependent rosetting and phagocytosis, and, on the other hand, the S-RBC rosetting and phagocytosis by adherent monocytes. Thus, the specific involvement of the CR3 epitopes recognized by Mab anti-Leu 15 or by OKM1 in the interactions between S-RBC and monocyte/macrophage could not be demonstrated. Murine Mab anti-CR1 was found to be a significant inhibitor of binding to and of phagocytosis of S-RBC (but not of young [Y] RBC) by monocytes or M phi, whereas Mab OKM5 carrying the same isotype as Mab anti-CR1, but a different specificity, was devoid of any significant inhibitory effect. Furthermore, Y-RBC or S-RBC opsonized with Mab anti-CR1 did not form FcR-dependent rosettes and were not internalized by monocytes; in addition, preincubation of phagocytes with Mab anti-CR1 did not inhibit FcR-dependent rosetting and phagocytosis. These results suggest that the effect of anti-CR1 is mediated through a specific binding to CR1 and not through an FcR blockade. As the role of specifically bound IgG on phagocytosis of human S-RBC by macrophages has previously been demonstrated by several authors, the present study suggests that monocyte-macrophage complement receptor type 1 may act in synergy with Fc receptors in the recognition of S-RBC by macrophages. It is shown in addition that the tripeptide Arg-Gly-Asp, identical to the region of iC3b recognized by CR3 and by several adhesion-promoting receptors that are structurally similar to CR3, such as fibronectin or vitronectin, is a significant inhibitor of the binding to and the phagocytosis of S-RBC by monocytic-macrophagic cells.

[Leucocytospermia, oxidative stress and male fertility: facts and hypotheses]. / Leucospermie, stress oxydatif et fertilité masculine: certitudes et hypothèses.

Leukocytospermia is frequent and significantly increased (over 10(6)/ml) in 20% of male factor infertility. It induces the production of highly toxic reactive oxygen species (ROS) which impair genital track accessory glands and sperm cell functions. The seminal medium contains extremely potent antioxidative defenses which usually balance the oxidative stress. In vivo, these defenses can be overwhelmed when ROS production is extremely important and/or when it lasts for a very long period of time. Infertility can then appear. In vitro, ROS have been univoqually demonstrated for being highly toxic since spermatozoa are no longer protected. Sperm cell defects are : decrease of acrosome reaction and fusiogenic ability and increase of DNA fragmentations. In case of male factor infertility, a leukocytospermia represents an essential or an additional risk factor that should be treated, specially when in vitro therapy is to be scheduled, in order to improve gamete quality.

Soluble L-selectin level is a marker for coronary artery disease in type 2 diabetic patients.

OBJECTIVE: To investigate whether the fall in soluble L-selectin (sL-selectin) level constitutes a marker for myocardial ischemia. RESEARCH DESIGN AND METHODS: The levels of soluble forms of adhesion molecules, i.e., intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVCAM-1), E-selectin (sE-selectin), P-selectin (sP-selectin), and L-selectin (sL-selectin), were compared in type 2 diabetic patients without inflammatory syndrome but with symptomatic coronary artery disease (CAD) (group 1, n = 11), with silent ischemic disorders and proven coronary stenoses (group 2, n = 11), with silent myocardial ischemia (SMI) and normal coronary angiography (group 3, n = 10), and without proven SMI (group 4, n = 13). These levels were compared with those of 22 control subjects. RESULTS: The sL-selectin level was significantly lower in groups 1, 2, or 3 with symptomatic CAD or with SMI as compared with the control group (P = 0.0004). Group 4 without myocardial ischemia did not significantly differ from the control subjects (P = 0.6). In type 2 diabetic patients, after controlling for HbA1c, a partial correlation between sL-selectin and the CAD status was significant (P = 0.001). sICAM-1 and sP- or sE-selectin did not differ significantly between type 2 diabetic patients and control subjects or among the different groups of patients. The sVCAM-1 level in type 2 diabetic patients was significantly higher than in the control subjects (P = 0.001), but there were no significant intergroup differences (P = 0.4). CONCLUSIONS: In type 2 diabetic patients, sVCAM-1 is increased with regard to glycemic control, whatever the CAD status. In type 2 diabetic patients with symptomatic CAD or SMI associated with coronary stenoses, sL-selectin is significantly decreased. A marked fall in sL-selectin might constitute a marker for silent CAD in type 2 diabetic patients.

Elevated concentrations of soluble E-selectin and vascular cell adhesion molecule-1 in NIDDM. Effect of intensive insulin treatment.

OBJECTIVE: To evaluate the effects of a 14-day intensive insulin therapy and short-term improvement of glycemic control on serum levels of soluble forms of adhesion molecules, i.e., intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVCAM-1), and E-selectin (sE-selectin) in NIDDM patients with poor glycemic control. RESEARCH DESIGN AND METHODS: A total of 16 NIDDM patients were compared with 23 healthy subjects (control group) and investigated before and after intensive insulin treatment. RESULTS: On day 0, sE-selectin and sVCAM-1 levels were significantly higher in NIDDM patients than in nondiabetic control subjects (median 87, range 63-115; median 544, range 408-797 vs. 58, 43-80; 443, 395-573 ng/ml, respectively) (P < 0.008 in both cases). On day 15, the fall in sE-selectin levels was significant (P < 0.0001) and at a lesser extent in sVCAM-1 levels (64, 48-85; 506, 417-678 ng/ml, respectively); these levels reached values that no longer differed from those of control subjects (P = 0.23 and 0.15, respectively). Moreover, the fall in sE-selectin was positively associated with the change in LDL cholesterol and the improvement of glycemia. CONCLUSIONS: In poorly controlled NIDDM patients, sE-selectin levels are increased and significantly fall to normal after short-term improvement of glycemic control. This suggests that assaying sE-selectin makes it possible to detect endothelium activation and to follow its reversal with euglycemia.

The role of T-agglutinin in the disappearance of erythrocytes artificially aged by desialylation.

The chance discovery of two mouse strains, one with and one without a high presence of serum T-agglutinin, permitted the investigation of the role of this antibody in the disappearance of desialylated erythrocytes, which may be regarded as a model for ageing. The proportional relationship between the quantity of sialic acid removed and the diminution of half-life is not affected by the presence or absence of T-agglutinin. Opsonization by T-agglutinin would therefore appear to be an improbable mechanism. Other possible theories are discussed.

Decrease of carbohydrate in membrane glycoproteins during human erythrocyte ageing in vivo.

Membrane glycoproteins from young human erythrocytes and erythrocytes aged in vivo were fractionated by gel filtration. Three major groups of glycoproteins were obtained. The neutral hexoses and sialic acid contents of each group of glycoproteins from the old cells were found to be significantly reduced by comparison to the values found in the glycoproteins of the young cells. Thus, the previously observed decrease in neutral hexoses and in sialic acid contents of the full erythrocyte membrane during in vivo ageing does not affect only one particular group of glycoproteins but each of the groups of glycoproteins tested, including the major glycoproteins of the erythrocyte membrane, that is to say glycophorins. It is shown in addition, that the previously observed decrease per cell of the surface galactose and N-acetylgalactosamine residues of the ageing erythrocyte affect several groups of membrane glycoproteins including band 3, PAS-1, PAS-4 and PAS-3. The physiological significance of these experimental data is discussed.

Does red blood cell size correlate with red blood cell age in mouse?

Mouse red blood cells (RBC) can be fractionated according to their size by counterflow centrifugation. The mean corpuscular hemoglobin content and the enzyme activities (ASAT, LDH, PK and acetylcholinesterase) increase when the mean corpuscular volume (MCV) rises. However, the in-vivo survival of size-separated RBC is similar whatever their MCV is; thus, counterflow centrifugation is not a suitable procedure to achieve an age fractionation of mouse RBC. Moreover, RBC subpopulations collected by counterflow centrifugation are different from those obtained when RBC are fractionated according to their density.

Enhancement of phagocytic activity of human monocytic-macrophagic cells by immunostimulating peptides from human casein.

Two immunostimulating peptides, Val-Glu-Pro-Ile-Pro-Tyr and Gly-Leu-Phe, obtained from human caseins, were demonstrated to significantly stimulate binding of human senescent red blood cells to human monocytic-macrophagic cells and their phagocytosis by these cells.

Modifications of sialidase activity during the monocyte-macrophage differentiation in vitro.

Human mononuclear cells were isolated from peripheral blood by centrifugation over Ficoll Hypaque, followed by adherence to plastic dishes. Monocyte-derived macrophages were obtained after culture for 3 or 5 days of the adherent cells in RPMI medium containing 20% heat-inactivated foetal calf serum. The sialidase activities were assayed in the whole homogenate using sodium 4-methyl-umbelliferyl-alpha-D-neuraminate as substrate, at various pHs, ranging from 3.6 to 6. The in vitro differentiation of monocytes into macrophages from day 0 up to day 5 was accompanied by a significant (P less than or equal to 0.01) increase in the sialidase activity on both a per-cell (+360%) and a per-mg protein in the homogenate (+125%) basis.

Lectin-carbohydrate interactions and infectivity of human immunodeficiency virus type 1 (HIV-1).

The aim of this study was to determine whether mannosyl-specific lectins, especially Concanavalin A (ConA), may bridge HIV-1 env glycoproteins to cell membranes to increase virus binding to its targets, and to what extent this lectin-carbohydrate interaction can modify HIV-1 infectivity for monocytic compared with lymphoid cells. Monocytic U937 and lymphoid CEM cells, which both express surface mannose, were utilized. Whether first incubated with env glycoprotein or with the cells, lectins bound both to the cells and to radiolabeled recombinant gp160 (rgp160). Thus, they enhanced rgp160 adsorption to the cells in a methyl-alpha-mannose inhibitable manner. ConA did not appear to bind to the V1 domain of CD4 at the U937 cell surface since Leu3a binding was not blocked in the presence of ConA, nor was recombinant CD4 retained on a ConA-agarose affinity matrix. Moreover, enhanced rgp160 binding to the cells was CD4 independent, since it was not modified by preincubating the cells with Leu3a. Finally, ConA did not inhibit the binding of CD4-IgG3 chimeric molecules to virions immobilized on nitrocellulose membrane, which argues against the possibility that it interferes with the interaction of gp120 and CD4. However, both when incubated with the virus or with the cells and despite mediating enhanced binding of env glycoprotein, ConA neutralized HIV-1 infectivity for monocytic U937 as well as for lymphoid CEM cells. In this respect, ConA behaves like neutralizing antibodies which do not interfere with CD4 binding of gp120 but rather with some later event that leads to virus entry. These findings obtained with plant lectins may be of relevance in vivo, inasmuch as endogenous mannosyl-binding proteins, which are known to function as opsonins, have been reported to inhibit in vitro infection by HIV-1.