O(2)-stable membrane-bound [NiFe]hydrogenase from a newly isolated Citrobacter sp. S-77

Hydrogenases are of great interest due to their potential use in H(2)-based technology. However, most hydrogenases are highly sensitive to O(2), which have been the major bottleneck in hydrogenase studies. Here we report an O(2)-stable membrane-bound [NiFe]hydrogenase (MBH) purified from a newly isolated strain, S-77. According to the 16S rRNA gene sequence and phylogenetic analysis of the strain S-77, it belongs to the genus of Citrobacter. In vitro experiments using the cytoplasmic membrane of strain S-77 suggested that a cytochrome b acts as the physiological electron acceptor of the MBH. The purified MBH was composed of a dimer of heterodimers, consisting of two distinct subunits with the molecular weights of 58.5 and 38.5?kDa. The enzyme showed a specific activity for H(2)-oxidation of 661U/mg, which is 35-fold greater than that for H(2)-production of 18.7U/mg. Notably, the MBH showed a remarkable O(2)-stability, maintaining almost 95% of its original activity even after incubation for 30?h in air at 4°C. These results suggest that the O(2)-stable MBH may play an important role in the H(2)-metabolic pathway under the aerobic conditions of Citrobacter sp. S-77. This is the first report of the purification and biochemical characterization of an O(2)-stable MBH from the genus of Citrobacter.     

Protective effect of whey protein hydrolysates on H2O2-induced PC12 cells oxidative stress via a mitochondria-mediated pathway

Whey protein hydrolysates (WPHs) were prepared with pepsin and trypsin. A PC12 cell model was built to observe the protective effect of WPHs against H₂O₂-induced oxidative stress. The results indicated that WPHs reduced apoptosis by 14% and increased antioxidant enzyme activities. Flow cytometry was used to assess the accumulation of reactive oxygen species (ROS), Ca²⁺ levels and the mitochondrial membrane potential (MMP). The results showed that WPHs suppressed ROS elevation and Ca²⁺ levels and stabilised MMP by 16%. The anti-apoptosis/pro-apoptosis proteins Bcl-2/Bax and poly (ADP-ribose) polymerase (PARP) were investigated by Western-blot analysis, which indicated that WPHs increased the expression of Bcl-2 while inhibiting the expression of Bax and the degradation of PARP. WPHs also blocked Caspase-3 activation by 62%. The results demonstrate that WPHs can significantly protect PC12 cells against oxidative stress via a mitochondria-mediated pathway. These findings indicate the potential benefits of WPHs as valuable food antioxidative additives.     

Free radical scavenging activity and comparative proteomic analysis of antioxidative protein against H2O2-induced oxidative stress in neuronal cells

Artemisia annua was enzymatically hydrolyzed by five proteases and seven carbohydrases. All enzymatic extracts scavenged DPPH, hydroxyl and alkyl radicals. Especially, the Protamex among the various proteases and Maltogenase among the various carbohydrases extracts exhibited the highest scavenging activity on hydroxyl radical. The extracts of A. annua clearly reduced neuronal cell death from H₂O₂-induced damage. In addition, a proteomic analysis, two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption ionisation-time of flight/time of flight (MALDI-TOF/TOF) was used to identify the proteins of the neuronal cells whose expressions were or were not altered by the treatment of the Maltogenase extracts which showed the highest hydroxyl radical scavenging activity among all enzymatic extracts for 24 h. The protein characterisation revealed that translation elongation factor Tu (EF-Tu), Immunoglobulin E (IgE) and voltage-dependent anion channel 1 (VDAC-1) were involved in the cell survival effects against H₂O₂-induced apoptosis. These results suggest that EF-Tu, IgE and VDAC-1 have an important role in the reduction of neuronal apoptosis by oxidative stress, and the enzymatic extracts of A. annua shows potent antioxidative activities by regulating EF-Tu, IgE and VDAC-1.     

牡丹花蕊醇提物对H2O2诱导HUVEC细胞损伤的保护作用

采用高效液相色谱分析牡丹花蕊醇提物的主要成分,并通过H_2O_2诱导HUVEC细胞建立损伤模型,研究牡丹花蕊醇提物对其保护作用。结果表明:牡丹花蕊醇提物主要成分为芦丁、槲皮素和芍药苷3种单体,含量分别为44.25%,15.50%,17.00%;牡丹花蕊醇提物能够降低细胞及其培养液中MDA含量,提高细胞内SOD和GPX活性以及GSH的含量。牡丹花蕊醇提物能够提高HUVEC细胞的抗氧化能力。     

Protective effects of triterpenoids from Ganoderma resinaceum on H2O2-induced toxicity in HepG2 cells

Ganoderma resinaceum Boud. (Polyporeseae) has long been used for antioxidant, immunoregulation and liver protection. From the fruiting bodies of G. resinaceum, eight new lanostanoids, lucidones D–G (1–4), 7-oxo-ganoderic acid Z₂ (5), 7-oxo-ganoderic acid Z₃ (6), ganoderesin A (7), and ganoderesin B (8), together with six known lanostanoids (9–14) were isolated. The structures of new compounds were elucidated through extensive spectroscopic analysis. In an in vitro model, ganoderesin B (8), ganoderol B (10) and lucidone A (11) showed inhibitory effects against the increase of ALT and AST levels in HepG2 cells induced by H₂O₂ compared to a control group in the range of their maximum non-toxic concentration (MNTC). However, compounds 8, 10 and 11 displayed no anti-oxidant activities by DPPH assay. Meanwhile, activation for PXR (Pregnane X Receptor) of ganoderesin B (8), ganoderol B (10) and lucidone A (11) was evaluated; ganoderol (10) exhibited a vital activation for PXR-induced CYP3A4 expression. These results suggested that GTs (Ganoderma triterpenoids) exhibited hepatoprotective activities by lowering ALT and AST levels.     

Comparative formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in creatinine/phenylalanine and creatinine/phenylalanine/4-oxo-2-nonenal reaction mixtures

The comparative formation of the heterocyclic aromatic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in both creatinine/phenylalanine (CRN/Phe) and creatinine/phenylalanine/4-oxo-2-nonenal (CRN/Phe/ON) systems was studied to analyse the ability of lipid-derived reactive carbonyls to promote PhIP formation. Although PhIP was produced to some extent in the CRN/Phe system, the presence of the oxidized lipid increased considerably the amount of PhIP produced. This increase seemed to be a consequence of the decrease in the E_a of the reaction when the lipid was present, which diminished from 112.9 to 80.9 kJ/mol. On the other hand, the addition of the lipid did not seem to produce PhIP by an alternative mechanism because PhIP was formed analogously in both CRN/Phe and CRN/Phe/ON systems as a function of pH, creatinine concentration, phenylalanine concentration, time, temperature, oxygen concentration in the reaction atmosphere, and the addition of different amounts of ammonia. All these results suggest that the ability of lipid oxidation products to produce PhIP is related to their capacity to induce the Strecker degradation of phenylalanine to phenylacetaldehyde. Therefore, any other reactive carbonyl compound that can produce the Strecker degradation of phenylalanine should also be considered as a potential inducer of PhIP formation under appropriate conditions.