5-Lipoxygenase compartmentalization in granulocytic cells is modulated by an internal bipartite nuclear localizing sequence and nuclear factor kappa B complex formation.

A region of basic amino acids spanning residues 639-656 in the human 5-lipoxygenase sequence resembles a consensus bipartite nuclear localizing sequence. A synthetic peptide consisting of the Kaposi fibroblast growth factor signal sequence fused to the 5-lipoxygenase639-656 bipartite nuclear localizing sequence has a prominent inhibitory effect on 5-lipoxygenase catalysis in granulocytic HL-60 cells activated by calcium ionophor A23187. Recombinant 5-lipoxygenase was not affected by the peptide.

5-Lipoxygenase and 5-lipoxygenase activating protein (FLAP) immunoreactivity in lungs from patients with primary pulmonary hypertension.

Inflammatory infiltrates and endothelial cell proliferation have been appreciated in plexiform and concentric lesions, which characterize the vascular remodeling in primary pulmonary hypertension (PPH). Leukotriene production by perivascular and alveolar macrophages relies on activation of 5-lipoxygenase (5-LO), with translocation of the enzyme to the nuclear membrane, and association with the 5-LO activating protein (FLAP). Using immunohistochemical staining, we localized and semi-quantitatively estimated the abundance of 5-LO and FLAP in lungs obtained from patients with PPH, patients with interstitial lung disease (ILD), and normal control subjects.

Inhibition of mitogen-activated protein kinase kinase blocks activation and redistribution of 5-lipoxygenase in HL-60 cells.

In Ca2+ ionophore-activated HL-60 granulocytes the mitogen-activated protein kinase kinase-1 inhibitor, PD098059, blocked translocation of 5-lipoxygenase from the cytosol to the nuclear membrane and the corresponding enzyme activation. PD098059 inhibited 5-HETE formation with an IC50 = 9.4 microM in cells stimulated with A23187 alone, and with an IC50 = 12 microM in cells stimulated with A23187 plus 20 microM arachidonic acid.

Inhibition of 5-lipoxygenase-activating protein (FLAP) reduces pulmonary vascular reactivity and pulmonary hypertension in hypoxic rats.

Chronically elevated shear stress and inflammation are important in hypertensive lung vessel remodeling. We postulate that 5-lipoxygenase (5-LO) is a molecular determinant of these processes. Immunohistology localized the 5-LO to macrophages of normal and chronically hypoxic rat lungs and also to vascular endothelial cells in chronically hypoxic lungs only.

Tyrosine kinase activity modulates catalysis and translocation of cellular 5-lipoxygenase.

Tyrosine kinase activity, a determinant of Src homology domain interactions, has a prominent effect on cellular localization and catalysis by 5-lipoxygenase. Six separate inhibitors of tyrosine kinase each inhibited 5(S)-hydroxyeicosatetraenoic acid formation by HL-60 cells stimulated with calcium ionophore, in the presence or absence of exogenous arachidonic acid substrate, indicating that they modulated cellular 5-lipoxygenase activity. The tyrosine kinase inhibitors also blocked the translocation of 5-lipoxygenase from cytosol to membranes during cellular activation, consistent with their effects on its catalytic activity.

5-Lipoxygenase contains a functional Src homology 3-binding motif that interacts with the Src homology 3 domain of Grb2 and cytoskeletal proteins.

A short, proline-rich region spanning residues 566-577 in human 5-lipoxygenase is a binding site for the Src homology 3 (SH3) domain of growth factor receptor-bound protein 2 (Grb2), an "adaptor" protein for tyrosine kinase-mediated cell signaling. Purified 5-lipoxygenase bound to glutathione S-transferase fusion products of Grb2 and a truncated version of Grb2 containing its SH3 domain. A peptide corresponding to the proline-rich, SH3-binding motif inhibited formation of the 5-lipoxygenase.

Irreversible inactivation of 5-lipoxygenase by leukotriene A4. Characterization of product inactivation with purified enzyme and intact leukocytes.

We report that leukotriene A4, the electrophilic product of 5-lipoxygenase catalysis, irreversibly inactivates the enzyme. Leukotriene A4 inhibits 5-hydroxyeicosatetraenoic acid formation by human neutrophils and differentiated granulocytic HL-60 cells in a concentration-dependent manner with IC50 values = 22.4 +/- 2.

Absence of persistence and transfer of genetic material by recombinant Escherichia coli in conventional, antibiotic-treated mice.

Strain BST-1 is a derivative of Escherichia coli K-12 that carries a plasmid designated pURA-4 and is the expression system used by The Upjohn Company in the production of recombinant bovine somatotropin (rbSt). This plasmid also encodes an ampicillin resistance gene. The plasmidless carrier strain, BST-1C, contains a gene for tetracycline resistance which is provided by the chromosomal insertion of the transposon Tn10.

Purification of a high-molecular-weight inhibitor of the calcium-activated proteinase.

An inhibitor of the muscle calcium-activated proteinases has been purified from porcine skeletal muscle by using DEAE-cellulose column chromatography, thermal treatment, Sephacryl S-400 column chromatography in 6 M urea and Sephacryl S-300 column chromatography in 6 M urea. Sodium dodecyl sulfate polyacrylamide slab gel electrophoresis shows that the purified inhibitor is homogeneous and has a subunit molecular weight of 172 000. The inhibitor inactivates both the low- and high-calcium-requiring forms of the calcium-activated proteinase but does not inhibit other proteinases against which it has been tried.

A calcium-activated protease possibly involved in myofibrillar protein turnover. Isolation of a low-calcium-requiring form of the protease.

Two forms of calcium-activated neutral protease were isolated and purified from porcine skeletal muscle. The two forms of the protease differ markedly in their requirement for calcium with the low-calcium-requiring form showing one-half maximal activation at 45 micro M calcium while the high-calcium-requiring form shows one-half maximal activation at 0.74 micro M calcium.