CHAPERONIN 20 mediates iron superoxide dismutase (FeSOD) activity independent of its co-chaperonin role in Arabidopsis chloroplasts.

Iron superoxide dismutases (FeSODs; FSDs) are primary antioxidant enzymes in Arabidopsis thaliana chloroplasts. The stromal FSD1 conferred the only detectable FeSOD activity, whereas the thylakoid membrane- and nucleoid-co-localized FSD2 and FSD3 double mutant showed arrested chloroplast development. FeSOD requires cofactor Fe for its activity, but its mechanism of activation is unclear. We used reversed-phase high-performance liquid chromatography (HPLC), gel filtration chromatography, LC-MS/MS, protoplast transient expression and virus-induced gene silencing (VIGS) analyses to identify and characterize a factor involved in FeSOD activation. We identified the chloroplast-localized co-chaperonin CHAPERONIN 20 (CPN20) as a mediator of FeSOD activation by direct interaction. The relationship between CPN20 and FeSOD was confirmed by in vitro experiments showing that CPN20 alone could enhance FSD1, FSD2 and FSD3 activity. The in vivo results showed that CPN20-overexpressing mutants and mutants with defective co-chaperonin activity increased FSD1 activity, without changing the chaperonin CPN60 protein level, and VIGS-induced downregulation of CPN20 also led to decreased FeSOD activity. Our findings reveal that CPN20 can mediate FeSOD activation in chloroplasts, a role independent of its known function in the chaperonin system.

Effects of G6PD overexpression in NIH3T3 cells treated with tert-butyl hydroperoxide or paraquat.

The major physiological role of glucose-6-phosphate dehydrogenase (G6PD) is to provide NADPH, which is required for reductive biosynthesis and for detoxification of free radicals and peroxides in mature red blood cells. To study the function of G6PD in non-erythroid cells, we examined the sensitivity of NIH3T3 cells transfected with a plasmid containing human G6PD cDNA to tert-butyl hydroperoxide (TBH) and paraquat. Two transfected clones which had a sixteen-fold (H7 clone) and six-fold (H6 clone) increase in their intracellular G6PD activity were compared with control cells transfected with a vector alone. Cells with high-level expression of human G6PD were 2.3 (H6) to 3.7 (H7) times more resistant to TBH than control cells. The antioxidant (anti-TBH) abilities in H6 and H7 cells were revealed by (1) a significant increase in the intracellular level of NADPH and glutathione, (2) a reduction of fluorescent intensity of the oxidant-sensitive dye, 2',7'-dichlorofluorescin diacetate, and (3) a significant reduction in the production of oxidized adducts generated by lipid peroxidation. In contrast, cells overexpressing G6PD were very sensitive to paraquat, a superoxide-producing herbicide. The concentrations of paraquat required to produce a 50% decrease in cell viability of H7, H6 and control cells were 0.80 mM, 1.14 mM, and 2.19 mM, respectively. The cytotoxicity of paraquat correlated with the expression level of NADPH in the cells. In this study, overexpression of human G6PD in NIH3T3 cells had different effects on the toxicity of TBH vs. paraquat. Reduction of NADP+ to NADPH by G6PD protects cells from oxidative damage by TBH, but appears to enhance the toxicity of paraquat.

Analysis of lysophophatidylcholine-induced endothelial dysfunction.

Endothelial dysfunction caused by the early atherosclerotic process or by endothelial exposure to atherogenic lipids, including lysophosphatidylcholine (lysoPC), is characterized by a selective impairment of responses mediated by the pertussis toxin-sensitive Gi-2 protein. Experiments were performed to analyze the mechanisms underlying this effect. Bradykinin (BK: Gi-2 protein-independent), serotonin (5-HT: Gi-2 protein-dependent), or direct activation of the G(i-2)-protein by mastoparan increased the release of endothelium-derived nitric oxide (EDNO) from porcine arterial endothelial cells (EC). LysoPC decreased the release of EDNO caused by 5-HT, but did not affect the response to BK or mastoparan. LysoPC did not increase production of superoxide radicals detected by lucigenin-enhanced chemiluminescence. Western blot analysis showed no difference in the level of immunoreactive Gi alpha-2 between control and lysoPC-treated cells. Activation of the Gi-2 protein by serotonergic or alpha 2-adrenoceptor stimulation decreased the pertussis toxin-catalyzed ADP-ribosylation of Gi alpha-2 protein in membranes from control but not lysoPC-treated cells. However, direct activation of the Gi-2 protein by mastoparan inhibited the ADP-ribosylation in membranes from control and lysoPC-treated cells. The toxin-catalyzed reaction was reduced in lysoPC-treated cells or lysoPC-treated membranes. LysoPC reduced the ability of endothelin to increase GTP gamma S binding to the Gi-2 protein but did not affect the activity of mastoparan. These results suggest that lysoPC inhibits a pertussis toxin-sensitive signaling pathway in EC by an effect consistent with receptor:Gi-2-protein uncoupling.

Power spectrum analysis of heart rate variability in children with epilepsy.

OBJECT: Children with epilepsy have been found to be at increased risk of death during childhood. Sudden unexpected death (SUDEP) has accounted for at least 12% of deaths of children with epilepsy. The exact mechanisms of SUDEP are unknown; however, theories suggested have, to date, focused on autonomic instability. The purpose of this study was to investigate autonomic function in children with chronic epilepsy by means of power spectrum analysis of heart rate variability. METHODS: Thirty patients with epilepsy and 30 control subjects, all between the ages of 4 and 10 years, were enrolled in this study. Power spectrum analysis of heart rate variability (HRV) was performed under standardized conditions after the patients had rested for 15 min. Each patient was tested in a supine position first and then again in a head-up tilted position, with 15 min between the two tests. RESULTS: There was no significant difference between the low-frequency component (LF) and the high-frequency component (HF) of heart rate variability, or the LF/HF ratio, between the study and control groups, whether the test subjects were in the supine or the head-up tilt position. In the control group, however, the subjects showed a significantly greater LF component and a smaller HF component of heart rate variability, and a greater LF/HF ratio in the head-up position than in the supine position. This implies a normal sympathovagal balance. This phenomenon was not observed in the study group. This implies that the modulating effects on autonomic function deriving from the hemisphere were probably disturbed, owing to the brain lesions that each of the study group patients had already sustained. CONCLUSIONS: A disturbed balance of activity between the sympathetic and parasympathetic nervous system might result from the loss of hemispheric influence in patients with epilepsy. Nevertheless, further investigation is clearly necessary to ascertain the possible association of this disturbed balance with SUDEP. Further investigation is also needed to establish the exact location of the region in the brain that gives rise to this modulating influence.