Superoxide anion formation from lucigenin: an electron spin resonance spin-trapping study.

Lucigenin (LC2+) is frequently used as a superoxide probe. To detect superoxide, lucigenin must be reduced to the lucigenin cation radical (LC.+).

Apoptosis occurs in endothelial cells during hypertension-induced microvascular rarefaction.

Disappearance of microvessels (microvascular rarefaction) during hypertension is a process that exacerbates the hypertensive condition. The cellular process by which the vessels disappear is not known. In the present study, we investigate the pathogenic role of cell death, specifically apoptosis, in hypertension-induced microvascular rarefaction.

Molecular identification of a novel fibrinogen binding site on the first domain of ICAM-1 regulating leukocyte-endothelium bridging.

Binding of fibrinogen to intercellular adhesion molecule 1 (ICAM-1) enhances leukocyte adhesion to endothelium by acting as a bridging molecule between the two cell types. Here, a panel of four monoclonal antibodies (mAbs) to ICAM-1 was used to dissect the structure-function requirements of this recognition. All four mAbs bound to ICAM-1 transfectants and immunoprecipitated and immunoblotted ICAM-1 from detergent-solubilized JY lymphocyte extracts.

Functional relevance during lymphocyte migration and cellular localization of activated beta1 integrins.

The state of integrin activation can be assessed by monoclonal antibodies (mAb) that selectively recognize integrins in their active form. We demonstrate herein that the expression of the epitope recognized by mAb HUTS-21 is induced on T lymphoblasts upon binding of soluble vascular cell adhesion molecule (VCAM)-1 and an 80-kDa tryptic fragment of fibronectin (FN80) to the beta1 integrins very late activation antigen (VLA)-4 and VLA-5, and that this effect is dependent on ligand concentration and is specific for beta1 integrins. On T lymphoblasts adhering to immobilized fibronectin, the HUTS-21 epitope localized exclusively to sites of integrin binding to fibronectin.

Where the outside meets the inside: integrins as activators and targets of signal transduction cascades.

In fibroblasts, signaling through the adhesion receptors known as integrins synergizes with other cellular stimulators such as the growth factors. There is currently great interest in the details of the ensuing 'outside in' signal transduction mechanisms, and the focal adhesion kinase in particular, has been a focus of attention. Less is understood of signalling through integrins on leukocytes which also perform a costimulator role.

Mechanism of nitric oxide release from S-nitrosothiols.

S-Nitrosothiols have many biological activities and have been suggested to be intermediates in signal transduction. The mechanism and products of S-nitrosothiol decomposition are of great significance to the understanding of nitric oxide (.NO) biochemistry.

The reaction between nitric oxide and alpha-tocopherol: a reappraisal.

Recently Gorbunov et al. reported that nitric oxide (.NO) can directly oxidize alpha-tocopherol to alpha-tocopheroxyl radical (Gorbunov et al.

Regulation of leukocyte integrin function: affinity vs. avidity.

Leukocytes circulate freely in the bloodstream until receiving signals which activate adhesive mechanisms essential for immune responsiveness. Key mediators of these adhesion events are heterodimeric cell surface receptors called integrins. It is now apparent that several components may contribute to successful integrin-mediated adhesion: alterations in individual receptors lead to enhanced affinity for ligand; integrin clustering causes an increase in avidity; by spreading, the adhering cell is less susceptible to shear force.

Effect of superoxide dismutase mimics on radical adduct formation during the reaction between peroxynitrite and thiols--an ESR-spin trapping study.

We have reexamined the formation and reactions of radicals formed from peroxynitrite (ONOO-)-mediated oxidation of glutathione (GSH), L-cysteine (Cys), N-acetyl-D,L-penicillamine (NAP), and sodium bisulfite (NaHSO3). Sulfur-centered and superoxide union radicals were trapped using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and the radical adducts were analyzed by electron spin resonance (ESR) spectroscopy. The following sulfur-centered radicals were detected: glutathionyl radical (GS') from GSH, L-cysteinyl radical ('Scys) from L-cysteine, N-acetyl-D,L-penicillamine thiyl radical ('SNAP) from NAP, and sulfite anion radical (SO3-.

The role of glutathione in the transport and catabolism of nitric oxide.

Nitric oxide acts as a neuronal and vascular messenger implying diffusion through intracellular environments containing 5-10 mM glutathione. Nitric oxide reacts with glutathione under aerobic conditions generating S-nitrosoglutathione (GSNO). GSNO reacts with glutathione (k= 8.

Characterization of sulfur-centered radical intermediates formed during the oxidation of thiols and sulfite by peroxynitrite. ESR-spin trapping and oxygen uptake studies.

Using a novel phosphorylated spin trap, 5-diethoxy-phosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO), an analog of the commonly used trap 5,5'-dimethyl-1-pyrroline N-oxide (DMPO), we have investigated the reactions of sulfur-centered radicals produced from the oxidation of thiols and sulfite by peroxynitrite. The predominant species trapped in all cases are the corresponding sulfur-centered radicals, i.e.

T cell adhesion to intercellular adhesion molecule-1 (ICAM-1) is controlled by cell spreading and the activation of integrin LFA-1.

Many leukocyte integrins require activation before they can adhere to their ligands. For example, stimulation of T cells enables the integrin LFA-1 to bind to ligand. This study compares two well known protocols for inducing T cell LFA-1 mediated adhesion to intercellular adhesion molecule-1 (ICAM)-1.

The beta 2 integrin Mac-1 but not p150,95 associates with Fc gamma RIIA.

In this study we have compared the ligand binding activity of the two closely related beta 2 integrins, Mac-1 and p150,95, which are expressed separately as receptors permanently transfected into K562 cells. Mac-1 has previously been shown to associate with Fc gamma R, particularly Fc gamma RIII, but K562 cells express only endogenous Fc gamma RIIA. We have, therefore, taken advantage of this situation to examine a possible relationship between Fc gamma RIIA with Mac-1 and p150,95 in the absence of other Fc gamma R.

Interaction of nitric oxide with photoexcited rose bengal: evidence for one-electron reduction of nitric oxide to nitroxyl anion.

The interaction of nitric oxide (.NO) with Rose Bengal (RB) in the presence of electron donors was investigated. Upon illumination of a mixture of RB and .

Leukocyte integrins.

Lymphocytes, monocytes and granulocytes, which are collectively known as 'leukocytes', circulate primarily within the vascular system and lymphoid tissue but are found in essentially all tissues of the body. This mobile lifestyle necessitates the constant making and breaking of adhesive contacts with targets in their immediate environment. The adhesion receptors termed integrins, which are expressed in abundance by leukocytes, are well suited to carry out the transient interactions in which these cells engage.

The S100 family protein MRP-14 (S100A9) has homology with the contact domain of high molecular weight kininogen.

The heterodimeric molecule MRP-8/MRP-14 (S100A8/S100A9) is abundantly expressed in circulating monocytes and neutrophils. We report here an homology between the C-terminal 'tail' region of MRP-14 (S100A9) and sequences within the plasma protein, high molecular weight kininogen (HMWK) which are involved in binding to negatively charged surfaces such as kaolin. MRP-14 also binds to kaolin and is competitively inhibited by HMWK and by peptides corresponding to MRP-14 tail and the HMWK 'contact' regions.

Inhibition of macrophage-dependent low density lipoprotein oxidation by nitric-oxide donors.

We have previously shown that nitric oxide donors inhibit the oxidation of low density lipoprotein (LDL) initiated by copper ions or by azo-bis-amidinopropane (Hogg et al., 1993. FEBS Lett.

The antioxidant effect of spermine NONOate in human low-density lipoprotein.

The nitric oxide (*NO) donor N-[4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl]-1,3- propanediamine, also referred to as Spermine NONOate (SNN), inhibited the copper(II) sulfate-initiated oxidative modification of human low-density lipoprotein (LDL) as measured by the formation of thiobarbituric acid reactive substances, conjugated diene formation, and changes in electrophoretic mobility. The presence of the nitronyl nitroxide 1-oxy-2-[p-(trimethylammoniumyl)phenyl]-4,4,5,5- tetramethylimadazoline 3-oxide, a scavenger of *NO, antagonized the inhibitory activity of SNN. The inhibition of copper-dependent LDL oxidation had a nonlinear dependence on SNN concentration.

Spin-labeling study of the oxidative damage to low-density lipoprotein.

In this study, we have spin-labeled the lysine and cysteine residues of low-density lipoprotein (LDL) using N-4-(2,2,6,6-tetramethylpiperidinyl-1-oxyl-4-yl) maleimide (MAL-6) and succinimidyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylate (SSL), respectively. The electron spin resonance (ESR) spectrum of SSL bound to LDL indicated that the nitroxide moiety was relatively mobile. In contrast, the ESR spectrum of MAL-6 bound to LDL showed that the nitroxide moiety was rotationally restricted.

Role of apolipoprotein B-derived radical and alpha-tocopheroxyl radical in peroxidase-dependent oxidation of low density lipoprotein.

The peroxidation of low density lipoprotein (LDL) may play an important role in the modification of the lipoprotein to an atherogenic form. The oxidation of LDL by peroxidases has recently been suggested as a model for in vivo transition metal ion-independent oxidation of LDL (Wieland, E., S.

Trapping of nitric oxide formed during photolysis of sodium nitroprusside in aqueous and lipid phases: an electron spin resonance study.

Photolytic decomposition of sodium nitroprusside (SNP), a widely used nitrovasodilator, produced nitric oxide (.NO), which was continuously monitored by electron spin resonance (ESR) spectroscopy. The .

Nitric oxide donor compounds inhibit the toxicity of oxidized low-density lipoprotein to endothelial cells.

Photo-oxidized low-density lipoprotein is cytotoxic to bovine aortic endothelial cells in a concentration-dependent manner. Total cell killing occurs at a concentration of 600 mumol/l lipid hydroperoxide (LOOH). Selenium deficiency enhances the toxicity of LOOH such that 300 mumol/l LOOH is cytotoxic.

Antibodies that activate beta 2 integrins can generate different ligand binding states.

A human erythroleukemic cell line (K562) that does not normally express beta 2 integrins has been transfected with the genes encoding these integrins. The resulting cell lines show minimal background adhesion but can be stimulated to bind to appropriate substrates when activated with either of two different antibodies to CD18. The two antibodies appear to generate different ligand binding states in LFA-1 such that different members of the ICAM family are recognized.