Separation and characterization of nonphosphorylated and serine-phosphorylated urokinase. Catalytic properties and sensitivity to plasminogen activator inhibitor type 1.

Urokinase synthesized by human A431 epidermoid carcinoma cells is phosphorylated on serine (Mastronicola, M. R., Stoppelli, M. P., Migliaccio, A., Auricchio, F., and Blasi, F. (1990) FEBS Lett. 266, 109-114). To test the possibility that phosphorylation may have specific effects on urokinase function, the phosphorylated and nonphosphorylated forms of urokinase were separated by Fe(3+)-Sepharose chromatography. Both forms exhibit indistinguishable Km and kcat for plasminogen activation. On the other hand, their sensitivity toward the specific plasminogen activator inhibitor type 1 is different as assessed by measuring both the stability of the covalent complex and the residual enzymatic activity. Phosphorylated urokinase was 50% inhibited at a concentration of plasminogen activator inhibitor type 1 4-fold higher than nonphosphorylated urokinase (0.7 versus 0.15 nM). Furthermore about 10% of phosphorylated urokinase was resistant to plasminogen activator inhibitor type 1 at a concentration as high as 20 nM. Thus, phosphorylation affects urokinase sensitivity to plasminogen activator inhibitor type 1, therefore resulting in a net, although indirect, increase of urokinase activity. These results suggest the existence of a novel cellular regulatory mechanism of extracellular proteolysis

New Evidence for Cross Talk between Melatonin and Mitochondria Mediated by a Circadian-Compatible Interaction with Nitric Oxide

Extending our previous observations, we have shown on HaCat cells that melatonin, at ~10−9 M concentration, transiently raises not only the expression of the neuronal nitric oxide synthase (nNOS) mRNA, but also the nNOS protein synthesis and the nitric oxide oxidation products, nitrite and nitrate. Interestingly, from the cell bioenergetic point of view, the activated NO-related chemistry induces a mild decrease of the oxidative phosphorylation (OXPHOS) efficiency, paralleled by a depression of the mitochondrial membrane potential. The OXPHOS depression is apparently balanced by glycolysis. The mitochondrial effects described have been detected only at nanomolar concentration of melatonin and within a time window of a few hours’ incubation; both findings compatible with the melatonin circadian cycle

Local anesthetics alter mitochondrial energization in human sh-sy5y neuroblastoma cells-the roles of cytosolic Ca++ and endogenous nitric oxide.

Dallas - Texas US

OMIP-090: A 20-parameter flow cytometry panel for rapid analysis of cell diversity and homing capacity in human conventional and regulatory T cells

Supporting Information is available online at: https://onlinelibrary.wiley.com/doi/10.1002/cyto.a.24720#support-information-section .The panel was developed and optimized for monitoring changes in homing capacity and functional diversity of human CD4+ conventional and regulatory T cell subsets. The analysis was based on expression of only surface markers in freshly isolated peripheral blood mononuclear cells (PBMCs) to reduce at minimum any alteration due to permeabilization or freezing/thawing procedures. We included markers to assess the distribution of naïve and memory populations based on the expression of CD45RA, CCR7, CD25, CD28 and CD95 in both conventional and regulatory T cells. The identification of major functional subsets was performed using CCR4, CCR6, CCR10, CXCR3 and CXCR5. Homing capacity of these subsets to skin, airway tract, gut and inflammatory lesions could finally be assessed with the markers CLA, CCR3, CCR5 and integrin β7. The panel was tested on freshly isolated PBMCs from healthy donors and patients with allergic rhinitis or autoimmune disorders.British Heart Foundation, Grant/AwardNumber: FS/16/57/32733; King's HealthPartners; LUPUS UK; National Institute forHealth Research; Biomedical Research Centrebased at Guy's and St Thomas' NHSFoundation Trust and King's College Londonand the NIHR Clinical Research Facility;Wellcome Trust, Grant/Award Number:108874/B/15/Z; BD Biosciences ResearchProgram Award

Nitric oxide and cytochrome oxidase: mechanisms of inhibition and NO degradation

NO inhibits mitochondrial respiration by reacting with either the reduced or the oxidized binuclear site of cytochrome c oxidase, leading respectively to accumulation of cytochrome a(3)(2+)-NO or cytochrome a(3)(3+)-NO2- species. Exploiting the unique light sensitivity of the cytochrome a(3)(2+)-NO, we show that under turnover conditions, depending on the cytochrome c(2+) concentration, either the cytochrome a(3)(2+)-NO or the nitrite-bound enzyme is formed. The predominance of one of the two inhibitory pathways depends on the occupancy of the turnover intermediates. In the dark, the respiration recovers at the rate of NO dissociation (k' = 0.01 s(-1) at 37 degrees C). Illumination of the sample speeds up recovery rate only at higher reductant concentrations, indicating that the inhibited species is cytochrome a(3)(2+)-NO. When the reaction occurs with the oxidized binuclear site, light has no effect and NO is oxidized to harmless nitrite eventually released in the bulk, accounting for catalytic NO degradation

Nitric oxide reversibly inhibits cytochrome oxidase, which in turn degrades NO to nitrite: the mechanism of a two-faced interaction.

Kimball Union Academy, New Hampshire - US