Membranal cholesterol is not required for L-selectin adhesiveness in primary lymphocytes but controls a chemokine-induced destabilization of L-selectin rolling adhesions.

Cholesterol-enriched lipid microdomains regulate L-selectin signaling, but the role of membrane cholesterol in L-selectin adhesion is unclear. Arrest chemokines are a subset of endothelial chemokines that rapidly activate leukocyte integrin adhesiveness under shear flow. In the absence of integrin ligands, these chemokines destabilize L-selectin-mediated leukocyte rolling.

Talin 1 and paxillin facilitate distinct steps in rapid VLA-4-mediated adhesion strengthening to vascular cell adhesion molecule 1.

VLA-4 (alpha4beta1) is a key integrin in lymphocytes, interacting with endothelial vascular cell adhesion molecule 1 (VCAM-1) on blood vessels and stroma. To dissect the contribution of the two cytoskeletal VLA-4 adaptor partners paxillin and talin to VLA-4 adhesiveness, we transiently knocked them down in Jurkat T cells and primary resting human T cells by small interfering RNA silencing. Paxillin was required for VLA-4 adhesiveness to low density VCAM-1 under shear stress conditions and was found to control mechanical stability of bonds mediated by the alpha4 subunit but did not affect the integrin affinity or avidity to VCAM-1 in shear-free conditions.

Alpha4beta1-dependent adhesion strengthening under mechanical strain is regulated by paxillin association with the alpha4-cytoplasmic domain.

The capacity of integrins to mediate adhesiveness is modulated by their cytoplasmic associations. In this study, we describe a novel mechanism by which alpha4-integrin adhesiveness is regulated by the cytoskeletal adaptor paxillin. A mutation of the alpha4 tail that disrupts paxillin binding, alpha4(Y991A), reduced talin association to the alpha4beta1 heterodimer, impaired integrin anchorage to the cytoskeleton, and suppressed alpha4beta1-dependent capture and adhesion strengthening of Jurkat T cells to VCAM-1 under shear stress.

Lymphocyte arrest requires instantaneous induction of an extended LFA-1 conformation mediated by endothelium-bound chemokines.

It is widely believed that rolling lymphocytes require successive chemokine-induced signaling for lymphocyte function-associated antigen 1 (LFA-1) to achieve a threshold avidity that will mediate lymphocyte arrest. Using an in vivo model of lymphocyte arrest, we show here that LFA-1-mediated arrest of lymphocytes rolling on high endothelial venules bearing LFA-1 ligands and chemokines was abrupt. In vitro flow chamber models showed that endothelium-presented but not soluble chemokines triggered instantaneous extension of bent LFA-1 in the absence of LFA-1 ligand engagement.

[Artificially functionalized polyenoic fatty acids--a new lipid bioregulators].

Dopamine, histamine, serotonin, and serotonin analogs were acylated with arachidonic and eicosapentaenoic acids, and the reaction products were named as artificially functionalized fatty acids (AFFA). The amides of arachidonic acid with serotonin, dopamine, and histamine were found to inhibit human platelet aggregation induced by ADP, arachidonic acid and adrenaline. Amides of arachidonic and eicosapentaeonic acids with serotonin and dopamine protect sea urchin early embryos against cytotoxic action of serotonin and histamine antagonists.

The interaction of prostaglandins with serum low-density lipoproteins.

The interaction of human serum low-density lipoproteins (LDL) with various types of prostaglandins (PG) was studied using equilibrium dialysis, steady-state fluorescence polarization spectroscopy and photolabeling methods. Low concentrations (10(-13)-10(-9) M) of PGE1 and PGF2 alpha were shown to induce specific rearrangements of the lipids on the LDL surface, whereas the closely related PGE2 and PGF1 alpha had no effect. With fluorescent labeled LDL, the PGE1-induced changes of the steady-state fluorescence polarization (P) were shown to be time- and concentration-dependent, saturable and reversible.

A new approach to studies of cell-antibody interactions using fluorescent lipid probes.

The interaction of poly- and monoclonal antibodies against the L-chain of human Ig with Burkitt lymphoma EB-3 cells was studied using a fluorescent lipid probe, anthrylvinyl-labelled sphingomyelin, incorporated into the cell plasma membrane. Binding of the antibodies to Ig receptors on the surface was shown to induce changes in the fluorescence polarization of the probe. The high sensitivity of the method allows one to detect less than 100 antibody molecules per cell.

Binding of specific ligands to muscarinic receptors alters the fluidity of membrane fragments from rat brain. A fluorescence polarization study with lipid-specific probes.

The previously suggested method of following ligand-receptor interactions by measuring ligand-induced changes in membrane fluidity [(1986) FEBS Lett. 194, 313-316] was employed to study the binding of specific ligands of the muscarinic receptor to rat brain membrane fragments containing a fluorescent analogue of phosphatidylcholine (APC) as a membrane probe. Upon addition of carbachol and atropine in low concentrations the fluorescence polarization of the APC-labeled membranes decreased significantly demonstrating that binding of these ligands to the muscarinic receptor increases the fluidity of its lipid environment.

[Interaction of prostaglandins with low-density lipoproteins in human blood].

Interaction of prostaglandins (PG) with human plasma low density lipoproteins (LDL) was studied, using fluorescent spectroscopy and photoreactive labeling. It was demonstrated that PGE1 at low concentrations (less than 10(-9) M) induces specific lipid rearrangements on the surface of LDL globules. It was assumed that these rearrangements are brought about by the interaction of PG with apolipoprotein B to form short-living complexes.

The interaction of prostaglandins with high-density lipoproteins: a non-equilibrium model of ligand-receptor interaction.

Using high-density lipoproteins (HDL) labeled with a fluorescent phospholipid probe (an anthrylvinyl-labeled analogue of sphingomyelin) it was found that low amounts (10(-12) M) of the prostaglandins E1 and F2 alpha induced different structural changes of the HDL surface, whereas prostaglandin E2 had no effect. The effects of prostaglandin E1 on HDL were largely paralleled by those of this prostaglandin on synthetic recombinants prepared from apolipoprotein A1, phospholipids and cholesterol. The prostaglandin E1-HDL interaction resembled that of a ligand with a receptor site because it was specific, reversible, concentration- and temperature-dependent and saturable.

[Experimental research on the action of isoptin and alkylenediamine derivatives on the aggregation capacity and the membrane-bound calcium level of thrombocytes].

It was found in experiments on rabbit platelets that isoptin and derivatives of N,N'-di(beta-phenylisopropyl)polymethylene diamines produce a dose-dependent decrease of the platelet aggregation activity. The effect correlates with a lowering of membrane-bound calcium level in platelets as shown by the fluorescent technique with the use of chlortetracycline probe. The results obtained demonstrate that the derivatives of alkylenediamines significantly suppress platelet aggregation and block membrane-bound calcium at the lower concentrations than those of isoptin.

[DNA-phospholipid interactions. A study using lipid-specific fluorescent and photoreactive probes].

The interaction of phospholipids with phage T7 DNA was investigated using anthryl-vinyl-labeled and photoactivable phosphatidylcholine and sphingomyelin. Fluorescence polarization studies demonstrated that, in the presence of DNA, the fluorophore mobility is diminished as its distance from the polar head-group is increased. Immobilization of lipid chains is enhanced by Ca2+ ions, the effect being more pronounced for sphingomyelin than for phosphatidylcholine derivatives.

The binding of the B-chain of ricin to Burkitt lymphoma cells. A new approach to ligand-receptor interaction studies.

It is shown that conformational changes of receptor proteins brought about by binding of a ligand induce changes in the lipid environment of the receptor that can be monitored by fluorescent lipid probes. On this basis a new approach to studies of ligand-receptor binding is proposed. Using the interaction of the ricin B-chain with Burkitt lymphoma cells as an example and fluorescent labelled sphingomyelin as a probe, the ligand-induced changes of fluorescence anisotropy were shown to be concentration-dependent and to permit determination of the binding constant and the number of receptor-binding sites.

Influence of cholesterol and prostaglandin E1 on the molecular organization of phospholipids in the erythrocyte membrane. A fluorescent polarization study with lipid-specific probes.

Anthryl-labeled fluorescent probes closely mimicking phosphatidylcholine and sphingomyelin were applied to study the state of these phospholipids in the rabbit erythrocyte membrane. At normal cholesterol levels both probes exhibited higher fluorescence polarization values in the membranes than in phospholipid vesicles of similar lipid composition, indicating a decreased fluidity of the probe environment in erythrocyte ghosts. In ghosts prepared from normal erythrocytes no evidence of lateral separation of phosphatidylcholine and sphingomyelin was found.

Interaction of prostaglandin E1 with human high density lipoproteins.

Prostaglandin E1 has been shown to interact with serum high density lipoproteins (HDL) in a manner resembling the interaction of a ligand with a high affinity binding site. The presence of 10(-12)-10(-10) M prostaglandin E1 induces a rearrangement of the HDL surface lipids and probably influences the biological functions of the lipoproteins.

[Membrane structure of remantadine-sensitive and remantadine-resistant influenza virus studied with fluorescent phospholipid probes].

Significant differences in the molecular organization of lipid bilayer in remantadin-resistant and remantadin-sensitive strains of influenza virus were demonstrated by means of fluorescent phospholipid probes, analogues of phosphatidylcholine and sphingomyelin. The data on fluorescence polarization and transfer of excitation energy from protein tryptophanes on probe fluorophores showed phosphatidylcholine and sphingomyelin to be segregated in influenza virion membrane. Gradients of mobility of lipid chains in virion membrane and in phospholipid vesicles have opposite directions.