Temporal Association Between Pulmonary Inflammation and Antioxidant Induction Following Hyperoxic Exposure of the Preterm Guinea Pig

The time course and nature of the pulmonary inflammatory and antioxidant responses, both during and after hyperoxic-induced acute lung injury were studied in the preterm guinea pig. Three-day preterm (65 days gestation) guinea pigs were randomly exposed to either 21% O₂ (control) or 95% O₂ (hyperoxia) for 72 hours. All pups were then maintained in ambient conditions for up to a further 11 days, during which time lung damage was monitored. In animals exposed to hyperoxia, evidence of acute lung injury and inflammation was characterized by a marked increase in microvascular permeability and elevated numbers of neutrophils in bronchoalveolar lavage fluid. Protein concentration, elastase-like activity and elastase-inhibitory capacity in lavage fluid were at a maximum at the end of the 72 hours hyperoxic exposure. Four days later, all values had returned to control levels. In contrast, increased numbers of neutrophils, macrophages and lymphocytes were recovered in the lavage fluid during this early recovery period. Coinciding with the influx of inflammatory cells, there was a significant increase in glutathione peroxidase, manganese superoxide dismutase and catalase activities in immature lung. Lung copper/zinc superoxide dismutase activity remained unchanged during both experimental periods. The strong temporal relationship between the influx of inflammatory cells to the lung and the induction of pulmonary antioxidant enzyme defences suggests that a common mechanism underlies both responses. These findings have led us to regard inflammation in the hyperoxic-injured immature lung as a beneficial event and not, as previously suggested, as part of the injurious process.     

超氧化物歧化酶切割超螺旋DNA的活性

在体外系统中,发现超氧化物歧化酶(SOD)具有切割超螺旋DNA的活性. 猪血和牛血Cu/Zn-SOD以及烟草Mn-SOD都能将超螺旋DNA转变为非超螺旋结构的缺刻环状DNA,进一步产生线状DNA. 它们只作用于超螺旋DNA而不作用于线状DNA. 这个事实排除了SOD样品中污染核酸酶的可能性. 用H₂O₂、胍基抑制或蛋白酶降解的实验结果表明,这两种酶的活性中心处于酶蛋白的不同部位.     

Isolation of Mn-SOD and Low Active Fe-SOD from Methylomonas J; Consisting of Identical Proteins

Cultures of Methylomonas J. an aerobic methylotrophic bacterium, were grown both in Mn-rich and Fe-rich media. Crude extracts of the cultures from the Mn-rich and Fe-rich medium showed a specific activity of 12.2 and 0.6 units/mg by a cytochrome c-xanthine oxidase method and 19.4 and 1.3 units/mg by an ESR method, respectively. We isolated Mn-SOD and Fe-SOD from the bacteria grown in the Mn-rich and Fe-rich mediums, respectively. Specific activity and metal contents of the Mn-enzyme were 2,250 units/mg/g-atom Mn and Mn = 0.98 and Fe = 0.12 (g-atoms/mol dirner), while those of the Fe-enzyme were 61 units/mg/g-atom Fe and Mn = 0.02 and Fe = 1.08. No difference of physicochemical properties of the Fe-and Mn-enzymes were detected. Furthermore, enzyme activity was restored by dialysis of an apoprotein obtained from the Fe-enzyme with either manganese sulfate or ferrous ammonium sulfate.     

根癌农杆菌介导麝香百合遗传转化体系的建立

以麝香百合"white elegance"叶片为外植体,通过根癌农杆菌介导法,研究了影响其转化的因素,建立了稳定而高效的麝香百合遗传转化体系。结果表明,以叶片为受体材料,外植体预培养有利于农杆菌的侵染;3 d的共培养,根癌农杆菌OD600值为0.6左右、侵染10 min,能获得较高的转化率;50 mg.L-1的卡那霉素对叶片有很好的筛选效果。抗性植株经PCR检测,初步证明Mn-SOD基因已转入到麝香百合植株中。     

The RING Domain of TRAF2 Plays an Essential Role in the Inhibition of TNFα-Induced Cell Death but Not in the Activation of NF-κB

Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) and receptor-interacting protein 1 (RIP1) play critical roles in activating c-Jun N-terminal kinase (JNK) and inhibitor of κB kinase (IKK), as well as in inhibiting apoptosis induced by TNFα. The TRAF2 RING domain-mediated polyubiquitination of RIP1 is believed to be essential for TNFα-induced IKK activation, and the RING-domain-deleted TRAF2 (TRAF2-ΔR) has been widely used as a dominant negative in transient overexpression systems to block TNFα-induced JNK and IKK activation. Here, we report that stable expression of TRAF2-ΔR at a physiological level in TRAF2 and TRAF5 double knockout (TRAF2/5 DKO) cells almost completely restores normal TNFα-induced IKK activation, but not RIP1 polyubiquitination. In addition, stable expression of TRAF2-ΔR in TRAF2/5 DKO cells efficiently inhibited the TNFα-induced later phase of prolonged JNK activation, yet failed to inhibit TNFα-induced cell death. Although the basal and inducible expression of anti-apoptotic proteins in TRAF2-ΔR-expressing TRAF2/5 DKO cells was normal, the cells remained sensitive to TNFα-induced cell death because anti-apoptotic proteins were not recruited to the TNFR1 complex efficiently. Moreover, stable expression of TRAF2-ΔR in TRAF2/5 DKO cells failed to suppress constitutive p100 processing in these cells. These data suggest that (i) the TRAF2 RING domain plays a critical role in inhibiting cell death induced by TNFα and is essential for suppressing the noncanonical nuclear factor κB pathway in unstimulated cells; (ii) RIP1 polyubiquitination is not essential for TNFα-induced IKK activation; and (iii) prolonged JNK activation has no obligate role in TNFα-induced cell death.     

Molecular cloning and characterization of Mn-superoxide dismutase from disk abalone (Haliotis discus discus)

The mitochondrial enzyme manganese superoxide dismutase (mitMn-SOD) is one of the antioxidant enzymes involved in cellular defense against oxidative stress and catalyzes the conversion of O₂⁻ into the stabler H₂O₂. In this study, a putative gene encoding Mn-SOD from disk abalone (Haliotis discus discus, aMn-SOD) was cloned, sequenced, expressed in Escherichia coli K12(TB1) and the protein was purified using pMAL protein purification system. Sequencing resulted ORF of 681 bp, which corresponded to 226 amino acids. The protein was expressed in soluble form with molecular weight of 68 kDa including maltose binding protein and pI value of 6.5. The fusion protein had 2781 U/mg activity. The optimum temperature of the enzyme was 37 °C and it was active in a range of acidic pH (from 3.5 to 6.5). The enzyme activity was reduced to 50% at 50 °C and completely heat inactivated at 80 °C. The alignment of aMn-SOD amino acid sequence with Mn-SODs available in NCBI revealed that the enzyme is conserved among animals with higher than 30% identity. In comparison with human mitMn-SOD, all manganese-binding sites are also conserved in aMn-SOD (H28, H100, D185 and H189). aMn-SOD amino acid sequence was closer to that of Biomphalaria glabrata in phylogenetic analysis.     

Human brain cortex: mitochondrial oxidative damage and adaptive response in Parkinson disease and in dementia with Lewy bodies

Frontal cortex samples from frozen human brains were used to assess tissue respiration; content of mitochondria; mitochondrial oxygen uptake; activity of respiratory complexes and of mitochondrial nitric oxide synthase (mtNOS); content of cytochromes a, b, and c; oxidative damage (protein carbonyls and TBARS); and expression of Mn-SOD in patients with Parkinson disease (PD) and with dementia with Lewy bodies (DLB) in comparison with those of normal healthy controls. Brain cortex and mitochondrial O₂ uptake and complex I activity were significantly lower in PD and DLB, whereas mtNOS activity, cytochrome content, expression of Mn-SOD, mitochondrial mass, and oxidative damage were significantly higher in the frontal cortex in PD and DLB. The decreases in tissue and mitochondrial O₂ uptake and in complex I activity are considered the consequences of mitochondrial oxidative damage. The increases in mtNOS activity and in mitochondrial mass are interpreted as an adaptive response of the frontal cortex that involves increased NO signaling for mitochondrial biogenesis. The adaptive response would partially compensate for mitochondrial dysfunction in these neurodegenerative diseases and would afford a human evolutionary response to shortage of ATP in the frontal cortex.     

Pulsatile shear stress increased mitochondrial membrane potential: Implication of Mn-SOD

Mitochondrial dysfunction is intimately involved in cardiovascular diseases. Mitochondrial membrane potential (ΔΨ_m) is coupled with oxidative phosphorylation to drive ATP synthesis. In this study, we examined the effect of physiological pulsatile shear stress (PSS) on ΔΨ_m and the role of Mn-SOD expression on ΔΨ_m. Confluent human aortic endothelial cells (HAEC) were exposed to PSS, and ΔΨ_m was monitored using tetramethylrhodamine methyl ester (TMRM⁺), a mitochondrial membrane potential probe. PSS significantly increased ΔΨ_m and the change in ΔΨ_m was a dynamic process. ΔΨ_m returned to baseline level after PSS for 2 h followed by static state for 4 h. Mitochondrial Mn-SOD expression and activities were also significantly up-regulated in response to PSS. Silencing Mn-SOD attenuated PSS-mediated ΔΨ_m increase while adding Mn-SOD mimetic, MnTMPyP, increased ΔΨ_m to the similar extent as induced by PSS. Our findings suggest that PSS-increased mitochondrial ΔΨ_m, in part, via Mn-SOD up-regulation.     

Comparison of Pharmacokinetic and Cell Binding Properties of Turkey Cu-Sod and E. Coli Mn-Sod

The pharmacokinetics in rats and cell fixation properties of coli Mn-SOD are compared with those of Cu-SODs extracted from turkey blood. Despite similarities in molecular weight and pi the different enzymes show different characteristics. The results are discussed with respect to the mechanism of antiinflammatory activity of superoxide dismutase.     

Mitochondria-derived Hydrogen Peroxide Selectively Enhances T Cell Receptor-initiated Signal Transduction

T cell receptor (TCR)-initiated signal transduction is reported to increase production of intracellular reactive oxygen species, such as superoxide (O₂^⨪) and hydrogen peroxide (H₂O₂), as second messengers. Although H₂O₂ can modulate signal transduction by inactivating protein phosphatases, the mechanism and the subcellular localization of intracellular H₂O₂ as a second messenger of the TCR are not known. The antioxidant enzyme superoxide dismutase (SOD) catalyzes the dismutation of highly reactive O₂^⨪ into H₂O₂ and thus acts as an intracellular generator of H₂O₂. As charged O₂^⨪ is unable to diffuse through intracellular membranes, cells express distinct SOD isoforms in the cytosol (Cu,Zn-SOD) and mitochondria (Mn-SOD), where they locally scavenge O₂^⨪ leading to production of H₂O₂. A 2-fold organelle-specific overexpression of either SOD in Jurkat T cell lines increases intracellular production of H₂O₂ but does not alter the levels of intracellular H₂O₂ scavenging enzymes such as catalase, membrane-bound peroxiredoxin1 (Prx1), and cytosolic Prx2. We report that overexpression of Mn-SOD enhances tyrosine phosphorylation of TCR-associated membrane proximal signal transduction molecules Lck, LAT, ZAP70, PLCγ1, and SLP76 within 1 min of TCR cross-linking. This increase in mitochondrial H₂O₂ specifically modulates MAPK signaling through the JNK/cJun pathway, whereas overexpressing Cu,Zn-SOD had no effect on any of these TCR-mediated signaling molecules. As mitochondria translocate to the immunological synapse during TCR activation, we hypothesize this translocation provides the effective concentration of H₂O₂ required to selectively modulate downstream signal transduction pathways.