Biomedical subjects

P S Pine

Publications and source records attributed to P S Pine.

8 recordsLinked to original sources

PubMed1992

CD4 changes conformation upon ligand binding.

Aurintricarboxylic acid (ATA) has been shown to block the binding site for both HIV gp120 and mAb anti-Leu 3a on CD4. We have unexpectedly found that brief treatment with > or = 1 micrograms/ml ATA rapidly disengages another mAb, OKT4E, after it has been bound to CD4 on human PBL. OKT4E is specific for a discontinuous epitope overlapping the MHC class II-binding region in the N-terminal CD4 domain. Interestingly, among 10 other mAb tested, only anti-Leu 8, specific for a leukocyte homing receptor is also quickly released from the cells by ATA treatment. Disengagement of the OKT4E mAb is also seen on a CD4-positive cell line (HPB-ALL) and with recombinant soluble CD4 (sCD4) bound to immobilized OKT4E. In all of these cases, disengagement is prevented if OKT4E is cross-linked, or the Leu 3a site is blocked by the mAb, but not by gp120. Photobleaching fluorescence resonance energy transfer (pFRET) measurements suggest that OKT4E is released as an indirect consequence of ATA-evoked conformational changes of CD4. Similar changes were detected as a result of gp120 binding to PBL. These data raise the possibility of a novel type of immunomodulation: induced disengagement of a bound ligand from its Ag.

Antibodies, Monoclonal↗

PubMed1992

Prevention of binding of rgp120 by anti-HIV active tannins.

Several tannins with anti-HIV activity have been described previously (Nonaka et al., J Nat Prod 53: 587-595, 1990). We have shown that the tannins chebulinic acid and punicalin were able to block the binding of HIV rgp120 to CD4. These compounds were not toxic to stimulated human peripheral blood lymphocytes at concentrations ten times above their maximal effective concentration.

Antiviral Agents↗

PubMed1992

Epitope mapping by photobleaching fluorescence resonance energy transfer measurements using a laser scanning microscope system.

The donor photobleaching method (T. M. Jovin and D. J. Arndt-Jovin. 1989. Annu. Rev. Biophys. Biophys. Chem. 18:271-308.) has been adapted to an ACAS 570 (laser scanning microscope) system to measure fluorescence resonance energy transfer (FRET) on individual human peripheral blood T cells. Photobleaching was completed in approximately 100 ms in our case and it followed double-exponential kinetics. The energy transfer efficiency (E) was approximately 20% between the CD4 epitopes OKT4-FITC and Leu-3a-PE as well as between OKT4E-FITC and OKT4-PE. E was approximately 8% between OKT4-FITC and Leu-4-PE (alpha CD3) and barely detectable (approximately 4%) from OKT4-FITC to Leu-5b-PE (alpha CD2). The E values obtained by the photobleaching method were highly reproducible both in repeated measurement of identical samples and in experiments with different batches of cells and were in agreement with the flow cytometric donor quenching measurements. As expected, E measured between primary and secondary layers of antibodies increased (from approximately 14% to approximately 28%) when F(ab')2 fragments were substituted for whole antibody molecules as the donor. On a T cell line we mapped the distance between the idiotypic determinant of the T cell receptor (TcR) and the Leu-4 epitope of CD3 as proximal as E = 28%, as compared to E = 4% between a framework TcR epitope and Leu-4. In the latter case, however, approximately 40% less Leu-4 was bound suggesting that the antigen binding site of TcR is in close proximity with one of the two CD3 epsilon chains, which hence are not equivalent.

Antibodies, Monoclonal↗

PubMed1991

Laser scanning and confocal microscopy of daunorubicin, doxorubicin, and rhodamine 123 in multidrug-resistant cells.

The multidrug-resistant gene (MDR1) encodes an energy-dependent drug efflux pump (P-glycoprotein) for many anti-cancer drugs. We have studied the intracellular distribution of rhodamine 123 (R123), daunorubicin (DN), and doxorubicin (DOX) in cells expressing a human MDR1 gene. The distribution of these fluorescent drugs was measured by laser scanning microscopy and confocal microscopy. We devised a new method for analysis of fluorescence line scan data to determine the intracellular distribution of fluorescent probes. This method and confocal microscopy showed that R123, DN, and DOX are localized to both plasma membrane and intracellular compartments in multidrug-resistant cells. When the cells are treated with verapamil, an inhibitor of the multidrug transporter, the amount of DOX, DN, and R123 associated with the cell rises. After inhibition, the relative distribution of DOX and DN between the cell surface and intracellular structures does not change dramatically. However, R123 tends to relocalize to intracellular sites from predominantly plasma membrane sites, indicating that this dye behaves differently than the anti-cancer drugs. These results show the subcellular distributions of R123, DN, and DOX in plasma membrane, cytoplasm, and intracellular membrane systems, but do not allow definitive distinctions among existing models of how P-glycoprotein affects the distribution of drugs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1991

Comparison of the in vitro and biophysical effects of cyclosporine A, FK-506, and mycophenolic acid on human peripheral blood lymphocytes.

The immunosuppressive drugs FK-506 and mycophenolic acid (MPA) have recently been described, but their mode(s) of action are not well understood. We have compared them to cyclosporine A (CsA) in several assays. We have shown that CsA (1 microgram/ml), MPA (0.1 microgram/ml), and FK-506 (0.5 microgram/ml) all induce a state of unresponsiveness to anti-CD3 stimulation as measured by [3H]-thymidine uptake. This suggests that the target of these drugs may be present only after mitogenic stimulation. These drugs also cause a hyperpolarization of the plasma membrane of lymphocytes. This effect is blocked by quinine or verapamil. All three immunosuppressors only slightly modulate the increase in intracellular Ca++ caused by Con-A or by anti-CD3 stimulation but do not affect Ca++ levels alone. They also decrease expression of IL-2 receptors on alpha CD3-stimulated lymphocytes. Similarities in their modes of action, as measured by these biophysical and cell biological tests, indicate the possibility that these three drugs will show similarities in their clinical performance.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1990

Aurin tricarboxylic acid, the anti-AIDS compound, prevents the binding of interferon-alpha to its receptor.

Binding of HIV to its receptor, the CD4 molecule of lymphocytes, can be prevented by chemical agents. These agents could be considered as potential anti-AIDS drugs. We have shown that aurin tricarboxylic acid (ATA, 3 microM) specifically blocks the binding of gp120, the HIV coat protein, to the CD4 molecule. We have also found that ATA prevents the binding of interferon-alpha to its receptor in a dose-dependent manner (12-50 microM range). Membrane potential shift, associated with binding of interferon-alpha to its receptor, was also blocked by ATA in a dose-dependent fashion. Our results indicate that potential anti-AIDS drugs should be screened for such undesired side effects.

Antiviral Agents↗

PubMed1990

Polyionic compounds selectively alter availability of CD4 receptors for HIV coat protein rgp120.

We studied the ability of several polyionic compounds, previously shown to have activity in vitro against human immunodeficiency virus (anti-HIV) to block binding of anti-CD4 and recombinant HIV gp120 to the CD4 receptor on human lymphocytes. We found that Evans blue and aurin tricarboxylic acid could completely inhibit binding of anti-CD4 (Leu3a) and rgp120 and have selectivity for the CD4 receptor. A number of other compounds, including dextran sulfate and heparin had no effect on binding of rgp120 and were shown to be nonspecific for inhibition of binding of monoclonal antibodies to different T-cell receptors. Studies using a number of membrane-active drugs showed that changes in membrane potential or ion fluxes were not involved in the inhibition of binding of rgp120 by Evans blue or aurin tricarboxylic acid.

Antibodies, Monoclonal↗

PubMed1988

Dynamic analyses of lymphoblast membranes exposed to alpha interferon using flow cytometry and fluorescence recovery after photobleaching.

Interferons represent a major group of the biologic response modifiers which exert multipotent effects upon cell growth, cyto-differentiation and immune functions. Previous experimental studies with alpha interferon (IFN-alpha) have suggested that modulation of transmembrane signaling could be a critical determinant in the bioregulatory diversity. To determine whether any initial changes at the plasma membrane would directly correlate with one or more actions of IFN-alpha, we investigated cultures of Daudi lymphoblasts which are uniquely susceptible to growth inhibition. Complementary biophysical techniques were applied. In one approach, changes in plasma membrane ion flux were measured by flow cytometry, using a fluorescent dye indicator of membrane potential: Cells briefly exposed (5-10 min) to a DNA-recombinant IFN-alpha 2 (100 to 800 U/ml) manifested a consistent plasma membrane hyperpolarization (-60 to -90 mV) which could be blocked by ouabain. In a second approach, changes in diffusion coefficients of plasma membrane-associated macromolecules were determined by measuring the fluorescence redistribution after pulse photobleaching (FRAP): Individual plasma membrane proteins (sIgM, Leu 12 or Leu 16) were labelled with FITC conjugated goat antibodies [F(ab')2 or Fab'] or with phycoerythrin-B conjugated monoclonal mouse antibodies. Statistical comparisons of cells exposed to IFN-alpha 2 for 10 to 30 min showed immediate 27 to 88% increases in mean lateral diffusion rates. Mutant Daudi cells, cloned for resistance to growth inhibition showed no plasma membrane hyperpolarization with IFN-alpha 2 (up to 1000 U/ml), and baseline lateral diffusion coefficients matched those of IFN-alpha 2-treated, non-resistant cells. We conclude that biophysical status and responses of the plasma membrane must be closely linked to the molecular mechanisms of anti-proliferative signal transduction.

Cell Membrane↗