Peroxynitrite inhibits T lymphocyte activation and proliferation by promoting impairment of tyrosine phosphorylation and peroxynitrite-driven apoptotic death

Peroxynitrite (ONOO-) is a potent oxidizing and nitrating agent produced by the reaction of nitric oxide with superoxide. It readily nitrates phenolic compounds such as tyrosine residues in proteins, and it has been demonstrated that nitration of tyrosine residues in proteins inhibits their phosphorylation. During immune responses, tyrosine phosphorylation of key substrates by protein tyrosine kinases is the earliest of the intracellular signaling pathways following activation through the TCR complex. This work was aimed to evaluate the effects of ONOO- on lymphocyte tyrosine phosphorylation, proliferation, and survival. Additionally, we studied the generation of nitrating species in vivo and in vitro during immune activation. Our results demonstrate that ONOO-, through nitration of tyrosine residues, is able to inhibit activation-induced protein tyrosine phosphorylation in purified lymphocytes and prime them to undergo apoptotic cell death after PHA- or CD3-mediated activation but not upon phorbol ester-mediated stimulation. We also provide evidence indicating that peroxynitrite is produced during in vitro immune activation, mainly by cells of the monocyte/macrophage lineage. Furthermore, immunohistochemical studies demonstrate the in vivo generation of nitrating species in human lymph nodes undergoing mild to strong immune activation. Our results point to a physiological role for ONOO- as a down-modulator of immune responses and also as key mediator in cellular and tissue injury associated with chronic activation of the immune system.     

Peroxynitrite-mediated alpha-tocopherol oxidation in low-density lipoprotein: a mechanistic approach

Previous reports proposed that peroxynitrite (ONOO-) oxidizes alpha-tocopherol (alpha-TOH) through a two-electron concerted mechanism. In contrast, ONOO- oxidizes phenols via free radicals arising from peroxo bond homolysis. To understand the kinetics and mechanism of alpha-TOH and gamma-tocopherol (gamma-TOH) oxidation in low-density lipoprotein (LDL) (direct vs. radical), we exposed LDL to ONOO- added as a bolus or an infusion. Nitric oxide (.NO), ascorbate and CO2 were used as key biologically relevant modulators of ONOO- reactivity. Although approximately 80% alpha-TOH and gamma-TOH depletion occurred within 5 min of incubation of 0.8 microM LDL with a 60 microM bolus of ONOO-, an equimolar infusion of ONOO- over 60 min caused total consumption of both antioxidants. gamma-Tocopherol was preserved relative to alpha-TOH, probably due to gamma-tocopheroxyl radical recycling by alpha-TOH. alpha-TOH oxidation in LDL was first order in ONOO- with approximately 12% of ONOO- maximally available. Physiological concentrations of.NO and ascorbate spared both alpha-TOH and gamma-TOH through independent and additive mechanisms. High concentrations of.NO and ascorbate abolished alpha-TOH and gamma-TOH oxidation. Nitric oxide protection was more efficient for alpha-TOH in LDL than for ascorbate in solution, evidencing the kinetically highly favored reaction of lipid peroxyl radicals with.NO than with alpha-TOH as assessed by computer-assisted simulations. In addition, CO2 (1.2 mM) inhibited both alpha-TOH and lipid oxidation. These results demonstrate that ONOO- induces alpha-TOH oxidation in LDL through a one-electron free radical mechanism; thus the inhibitory actions of.NO and ascorbate may determine low alpha-tocopheryl quinone accumulation in tissues despite increased ONOO- generation.     

Association of β<Subscript>2</Subscript>-microglobulin with the α<Subscript>3</Subscript> domain of H-2D<Superscript>b</Superscript> heavy chain

MHC class I molecules are heterotrimeric complexes composed of heavy chain, ₂-microglobulin (₂m) and short peptide. This trimeric complex is generated in the endoplasmic reticulum (ER), where a peptide loading complex (PLC) facilitates transport from the cytosol and binding of the peptide to the preassembled ER resident heavy chain/₂m dimers. Association of mouse MHC class I heavy chain with ₂m is characterized by allelic differences in the number and/or positions of amino acid interactions. It is unclear, however, whether all alleles follow common binding patterns with minimal contributions by allele-specific contacts, or whether essential contacts with ₂m are different for each allele. While searching for the PLC binding site in the ₃ domain of the mouse MHC class I molecule H-2D^b, we unexpectedly discovered a site critical for binding mouse, but not human, ₂m. Interestingly, amino acids in the corresponding region of another MHC class I heavy chain allele do not make contacts with the mouse ₂m. Thus, there are allelic differences in the modes of binding of ₂m to the heavy chain of MHC class I.     

Protein flexibility and intrinsic disorder

Comparisons were made among four categories of protein flexibility: (1) low-B-factor ordered regions, (2) high-B-factor ordered regions, (3) short disordered regions, and (4) long disordered regions. Amino acid compositions of the four categories were found to be significantly different from each other, with high-B-factor ordered and short disordered regions being the most similar pair. The high-B-factor (flexible) ordered regions are characterized by a higher average flexibility index, higher average hydrophilicity, higher average absolute net charge, and higher total charge than disordered regions. The low-B-factor regions are significantly enriched in hydrophobic residues and depleted in the total number of charged residues compared to the other three categories. We examined the predictability of the high-B-factor regions and developed a predictor that discriminates between regions of low and high B-factors. This predictor achieved an accuracy of 70% and a correlation of 0.43 with experimental data, outperforming the 64% accuracy and 0.32 correlation of predictors based solely on flexibility indices. To further clarify the differences between short disordered regions and ordered regions, a predictor of short disordered regions was developed. Its relatively high accuracy of 81% indicates considerable differences between ordered and disordered regions. The distinctive amino acid biases of high-B-factor ordered regions, short disordered regions, and long disordered regions indicate that the sequence determinants for these flexibility categories differ from one another, whereas the significantly-greater-than-chance predictability of these categories from sequence suggest that flexible ordered regions, short disorder, and long disorder are, to a significant degree, encoded at the primary structure level.     

Holistic approach to functional anatomy of the injured ankle joint

Talocrural joint injuries are among the most common injuries of the joints and therefore there is a need for a holistic approach to analysis of morphology, biomechanics and visualization of the talocrural joint ligamentary apparatus in different positions. The research was carried out on 20 fresh and conserved anatomical specimens of the lower leg on which X-ray, computed tomography, ultrasonography and stress analysis were performed before and after the lesion of ligaments. Also the gait of 130 adults without (100) and with ligament and joint capsule lesion (30) was analyzed by infrared telemetry. After complete discission of the lateral ligaments, arthrography and CT could register the lesion, while X-ray and ultrasonography could not detect it. Gait analysis of healthy and injured leg showed that the injured leg was significantly less loaded.     

Reaction of human hemoglobin with peroxynitrite. Isomerization to nitrate and secondary formation of protein radicals

Peroxynitrite, a strong oxidant formed intravascularly in vivo, can diffuse onto erythrocytes and be largely consumed via a fast reaction (2 x 10(4) m(-1) s(-1)) with oxyhemoglobin. The reaction mechanism of peroxynitrite with oxyhemoglobin that results in the formation of methemoglobin remains to be elucidated. In this work, we studied the reaction under biologically relevant conditions using millimolar oxyhemoglobin concentrations and a stoichiometric excess of oxyhemoglobin over peroxynitrite. The results support a reaction mechanism that involves the net one-electron oxidation of the ferrous heme, isomerization of peroxynitrite to nitrate, and production of superoxide radical and hydrogen peroxide. Homolytic cleavage of peroxynitrite within the heme iron allows the formation of ferrylhemoglobin in approximately 10% yields, which can decay to methemoglobin at the expense of reducing equivalents of the globin moiety. Indeed, spin-trapping studies using 2-methyl-2-nitroso propane and 5,5 dimethyl-1-pyrroline-N-oxide (DMPO) demonstrated the formation of tyrosyl- and cysteinyl-derived radicals. DMPO also inhibited covalently linked dimerization products and led to the formation of DMPO-hemoglobin adducts. Hemoglobin nitration was not observed unless an excess of peroxynitrite over oxyhemoglobin was used, in agreement with a marginal formation of nitrogen dioxide. The results obtained support a role of oxyhemoglobin as a relevant intravascular sink of peroxynitrite.     

Peroxynitrite decay in the presence of hydrogen peroxide, mannitol and ethanol: a reappraisal

We have reported previously that the apparent rate of peroxynitrite (ONOO(-) ) decay, as followed from its absorbance at 302 nm, decreases in the presence of hydrogen peroxide, mannitol and ethanol (Alvarez et al., 1995, Chem. Res. Toxicol. 8:859-864; Alvarez et al., 1998, Free Radic. Biol. Med. 24:1331-1337). Recently, two papers confirmed the observation and proposed that this slowing effect was due to the formation of absorbing peroxynitrate (O(2) NOO(-) ) as intermediate (Goldstein and Czapski, 1998, J. Am. Chem. Soc. 120:3458-3463; Hodges and Ingold, 1999, J. Am. Chem. Soc. 121:10695-10701). Peroxynitrate would be formed from the reaction of peroxynitrite-derived nitrogen dioxide with superoxide. Superoxide, in turn, would arise from the one-electron oxidation of hydrogen peroxide, or from the reaction of reductive radicals derived from mannitol and ethanol with dioxygen. In agreement with this concept, we show herein that under the conditions of our previous work, the slowing effect is prevented by superoxide dismutase and, in the case of mannitol and ethanol, by reducing the dioxygen concentration of the reaction solutions. Thus, superoxide formation is necessary for the decrease in the rate of absorbance decay. In addition, by simulations using known rate constants and absorption coefficients, we show that the slowing effect can be quantitatively accounted for by the formation of peroxynitrate.     

Slow rate during AF improves ventricular performance by reducing sensitivity to cycle length irregularity

Atrial fibrillation (AF) is characterized by short and irregular ventricular cycle lengths (VCL). While the beneficial effects of heart rate slowing (i.e., the prolongation of VCL) in AF are well recognized, little is known about the impact of irregularity. In 10 anesthetized dogs, R-R intervals, left ventricular (LV) pressure, and aortic flow were collected for >500 beats during fast AF and when the average VCL was prolonged to 75%, 100%, and 125% of the intrinsic sinus cycle length by selective atrioventricular (AV) nodal vagal stimulation. We used the ratio of the preceding and prepreceding R-R intervals (RR(p)/RR(pp)) as an index of cycle length irregularity and assessed its effects on the maximum LV power, the minimum of the first derivative of LV pressure, and the time constant of relaxation by using nonlinear fitting with monoexponential functions. During prolongation of VCL, there was a pronounced decrease in curvature with the formation of a plateau, indicating a lesser dependence on RR(p)/RR(pp). We conclude that prolongation of the VCL during AF reduces the sensitivity of the LV performance parameters to irregularity.