Electron spin resonance--spin stabilization in enzymatic systems: detection of semiquinones produced during peroxidatic oxidation of catechols and catecholamines

The electron spin resonance-spin stabilization technique has been applied in an enzymatic system. This technique, which generates radicals in high steady-state concentration under static conditions, involves the use of limited quantities of enzyme and substrate while allowing facile spectral interpretation. In this work $\text{o}$-semiquinone intermediates produced during peroxidase-catalyzed oxidation of catechols and catecholamines have been detected as their metal complexes with Zn²⁺. No significant effect on the peroxidase activity was found for the concentrations of Zn²⁺ ions employed.     

Accumulation of Inositol Phosphates Induced by Chlorpromazine in C6 Glioma Cells

Abstract: Chlorpromazine, a cationic amphiphilic drug known to affect phospholipid metabolism, greatly increases the generation of inositol phosphates in C6 glioma cells. When a pulse-chase protocol with myo-[2-³H]inositol as the radioactive precursor was used, the peak increase in radioactivity of inositol phosphates was observed at 20 min. The drug decreased inositol tetrakisphosphate labeling as a percentage of inositol trisphosphate in a dose-dependent manner. It also increased the labeling of the inositol-containing phospholipids, the precursors of the inositol phosphates. The increase in radioactivity of both phospholipids and inositol phosphates was dose-dependent, but appeared also to be a function of the time of exposure of the cultures to the drug, suggesting that the concentration of chlorpromazine in the cell, and not that in the medium, is the critical factor. The optimum concentration for maximum phospholipid labeling was lower than that eliciting maximum generation of inositol phosphates. The data suggest that the mechanism probably does not involve cell-surface receptors, but rather may consist of a direct effect of chlorpromazine on phosphoinositidase C and possibly other enzymatic reactions concerned with the metabolism of inositol phosphates.     

HIV‐1 infection of CD4 T cells impairs antigen‐specific B cell function

Failures to produce neutralizing antibodies upon HIV‐1 infection result in part from B‐cell dysfunction due to unspecific B‐cell activation. How HIV‐1 affects antigen‐specific B‐cell functions remains elusive. Using an adoptive transfer mouse model and ex vivo HIV infection of human tonsil tissue, we found that expression of the HIV‐1 pathogenesis factor NEF in CD4 T cells undermines their helper function and impairs cognate B‐cell functions including mounting of efficient specific IgG responses. NEF interfered with T cell help via a specific protein interaction motif that prevents polarized cytokine secretion at the T‐cell–B‐cell immune synapse. This interference reduced B‐cell activation and proliferation and thus disrupted germinal center formation and affinity maturation. These results identify NEF as a key component for HIV‐mediated dysfunction of antigen‐specific B cells. Therapeutic targeting of the identified molecular surface in NEF will facilitate host control of HIV infection.     

Tyrosinase-catalyzed oxidation of dopa and related catechol(amine)s: a kinetic electron spin resonance investigation using spin-stabilization and spin label oximetry

The oxidation of four catechol(amine)s by tyrosinase has been studied by electron spin resonance and optical methods. Rates of oxygen consumption and of dopaquinone and dopachrome formation during the oxidation of dopa have been measured, and compared with rates of dopasemiquinone production measured using spin-stabilization procedures. In the presence of spin-stabilizing metal ions, production of semiquinone is approximately quantitative. Time-dependent ESR spectra obtained from dopa and dopamine show a slow regeneration of semiquinone, suggesting that a semiquinone precursor is slowly reformed. In contrast, time-dependent spectra for 4-methylcatechol and N-acetyldopamine show decay of the primary semiquinone together with buildup of a secondary semiquinone apparently derived from the corresponding 6-hydroxy-catechol(amine). Thus, catecholamines that give rise to a cyclizable quinone show a pattern of behavior that differs from those that produce a non-cyclizable quinone. These results are discussed in terms of their possible significance to melanogenesis and the toxicity of catechol(amine)s, which has been attributed to production of semiquinones and/or other oxygen radicals.