The apolipoprotein A‐I mimetic peptide,D‐4F,restrains neointimal formation through heme oxygenase‐1 up‐regulation

D‐4F, an apolipoprotein A‐I (apoA‐I) mimetic peptide, possesses distinctly anti‐atherogenic effects. However, the biological functions and mechanisms of D‐4F on the hyperplasia of vascular smooth muscle cells (VSMCs) remain unclear. This study aimed to determine its roles in the proliferation and migration of VSMCs. In vitro, D‐4F inhibited VSMC proliferation and migration induced by ox‐LDL in a dose‐dependent manner. D‐4F up‐regulated heme oxygenase‐1 (HO‐1) expression in VSMCs, and the PI3K/Akt/AMP‐activated protein kinase (AMPK) pathway was involved in these processes. HO‐1 down‐regulation with siRNA or inhibition with zinc protoporphyrin (Znpp) impaired the protective effects of D‐4F on the oxidative stress and the proliferation and migration of VSMCs. Moreover, down‐regulation of ATP‐binding cassette transporter A1 (ABCA1) abolished the activation of Akt and AMPK, the up‐regulation of HO‐1 and the anti‐oxidative effects of D‐4F. In vivo, D‐4F restrained neointimal formation and oxidative stress of carotid arteries in balloon‐injured Sprague Dawley rats. And inhibition of HO‐1 with Znpp decreased the inhibitory effects of D‐4F on neointimal formation and ROS production in arteries. In conclusion, D‐4F inhibited VSMC proliferation and migration in vitro and neointimal formation in vivo through HO‐1 up‐regulation, which provided a novel prophylactic and therapeutic strategy for anti‐restenosis of arteries.     

Role of a <Emphasis Type="Italic">Burkholderia pseudomallei</Emphasis> polyphosphate kinase in an oxidative stress response,motilities, and biofilm formation

Burkholderia pseudomallei, a motile and rod Gram-negative bacterium, is the causative agent of melioidosis. The bacterium is an intracellular pathogen and that motility is generally crucial for their survival in a natural environment and for systemic infection inside a host. We report here a role of B. pseudomallei polyphosphate kinase in virulence, such as an oxidative stress response, motilities and biofilm formation. The polyphosphate kinase (ppk) mutant is susceptible to hydrogen peroxide in an oxidative stress condition, unable to perform swimming, swarming motilities, and has lower density biofilm forming capacity than the wild-type strain. We also demonstrated that both polyphosphate kinase and motile flagella are essential and independently involved in biofilm formation. The B. pseudomallei flagellin (fliC) mutant and B. mallei, a nonmotile species, are shown to produce higher density biofilm formation than the ppk mutant, but less than wild type B. pseudomallei.     

A single‐nucleotide polymorphism of miR‐196a‐2 and vitiligo: an association study and functional analysis in a Han Chinese population

Recent evidence indicates that oxidative stress and genetic factors play an important role in the pathogenesis of vitiligo. SNPs in miRNAs involved in oxidative stress could potentially influence the development of vitiligo. In this case–control study, we investigated the association of a functional SNP of rs11614913 in miR‐196a‐2 with risk of vitiligo. A significantly lower risk of vitiligo was associated with the rs11614913 miR‐196a‐2 CC genotype (adjusted OR, 0.77; CI, 0.60–0.98). In addition, TYRP1 gene expression was considerably down‐regulated by the rs11614913 C allele in miR‐196a‐2, which lowered the levels of intracellular reactive oxygen species (ROS) and reduced the proportion of early apoptosis in human melanocytes in response to H₂O₂ treatment. Our data suggest that the rs11614913 C allele in miR‐196a‐2 confers potential protection against oxidative effects on human melanocytes through the modulation of the target gene, TYRP1, which may account for the decreased risk of vitiligo in this study population.     

N‐acetylcysteine counteracts oxidative stress and prevents hCG‐induced apoptosis in rat Leydig cells through down regulation of caspase‐8 and JNK

We have earlier reported that following persistent stimulation with hCG, oxidative stress‐induced apoptosis in rat Leydig cells was mainly achieved through the extrinsic pathway. In the present study, the role of N‐acetylcysteine (NAC) in counteracting the oxidative stress and the mechanisms of inhibition of apoptosis under such conditions were investigated. NAC (1 mM) intervention with repeated hCG stimulation (50 ng/ml, four times, each with 30 min challenge) prevented the decline in Leydig cell viability and the rise in lipid peroxidation and reactive oxygen species. Simultaneously, the activities of the enzymes glutathione‐S‐transferase, catalase, superoxide dismutase and the intracellular glutathione and antioxidant capacity of the treated cells improved significantly. Apoptotic markers Fas, FasL, and caspase‐8, up‐regulated following repeated hCG exposure, were significantly down‐regulated following NAC co‐incubation. While Bcl‐2 expression was fully restored, Bax and caspase‐9 remained unchanged. NAC treatment induced down‐regulation of upstream JNK/pJNK and down‐stream caspase‐3 in the target cells. Taken together, the above findings indicate that NAC counteracted the oxidative stress in Leydig cells induced as a result of repeated hCG stimulation, and inhibited apoptosis by mainly regulating the extrinsic and JNK pathways of metazoan apoptosis. Mol. Reprod. Dev. 77:900–909, 2010. © 2010 Wiley‐Liss, Inc.     

Characterization of Mitochondrial Proteomic Changes in Resistant Wheat Near‐Isogenic Line After Inoculation with Powdery Mildew

Wheat powdery mildew resistance mechanisms have been studied extensively at genomic level, however, infection induced mitochondrial proteomic changes in resistant line have not been fully characterized. Being critical organelles of chemical energy metabolism, mitochondria have also been suggested to be involved in the environmental stress response. Using proteomic approaches, we did comparative analysis of mitochondrial proteome in resistant wheat near‐isogenic line (NIL) (Brock × Jing411⁷) and its recurrent parent Jing 411 after infection of Blumeria graminis f.sp. tritici (Bgt). More than 50 down‐regulated mitochondrial protein spots were identified in NIL after 24‐h pathogen inoculation, and their abundance recovered to the levels prior to infection after extended inoculation (72‐h). We further analyzed a subgroup of down‐regulated proteins using mass spectrometry. MS/MS data analysis revealed the identities of nine protein spots and assigned them into three functional classes: synthesis of protein, disease resistance response and energy metabolism. For the first time we demonstrated pathogen stress induced mitochondrial proteomic changes and provided evidences that wheat powdery mildew resistance involves multiple biochemical events. Moreover, our results indicate that wheat mitochondrial proteome analysis can serve as a powerful tool to identify potential regulators of fungal invasion resistance.     

Acyl‐CoA‐binding protein 2 binds lysophospholipase 2 and lysoPC to promote tolerance to cadmium‐induced oxidative stress in transgenic Arabidopsis

Lysophospholipids are intermediates of phospholipid metabolism resulting from stress and lysophospholipases detoxify lysophosphatidylcholine (lysoPC). Many lysophospholipases have been characterized in mammals and bacteria, but few have been reported from plants. Arabidopsis thaliana lysophospholipase 2 (lysoPL2) (At1g52760) was identified as a protein interactor of acyl‐CoA‐binding protein 2 (ACBP2) in yeast two‐hybrid analysis and co‐immunoprecipitation assays. BLASTP analysis indicated that lysoPL2 showed ～35% amino acid identity to the lysoPL1 family. Co‐localization of autofluorescence‐tagged lysoPL2 and ACBP2 by confocal microscopy in agroinfiltrated tobacco suggests the plasma membrane as a site for their subcellular interaction. LysoPL2 mRNA was induced by zinc (Zn) and hydrogen peroxide (H₂O₂), and lysoPL2 knockout mutants showed enhanced sensitivity to Zn and H₂O₂ in comparison to wild type. LysoPL2‐overexpressing Arabidopsis was more tolerant to H₂O₂ and cadmium (Cd) than wild type, suggesting involvement of lysoPL2 in phospholipid repair following lipid peroxidation arising from metal‐induced stress. Lipid hydroperoxide (LOOH) contents in ACBP2‐overexpressors and lysoPL2‐overexpressors after Cd‐treatment were lower than wild type, indicating that ACBP2 and lysoPL2 confer protection during oxidative stress. A role for lysoPL2 in lysoPC detoxification was demonstrated when recombinant lysoPL2 was observed to degrade lysoPC in vitro. Filter‐binding assays and Lipidex competition assays showed that (His)₆‐ACBP2 binds lysoPC in vitro. Binding was disrupted in a (His)₆‐ACBP2 derivative lacking the acyl‐CoA‐binding domain, confirming that this domain confers lysoPC binding. These results suggest that ACBP2 can bind both lysoPC and lysoPL2 to promote the degradation of lysoPC in response to Cd‐induced oxidative stress.     

Non‐apoptotic function of caspases in a cellular model of hydrogen peroxide‐associated colitis

Oxidative stress, caused by reactive oxygen species (ROS), is a major contributor to inflammatory bowel disease (IBD)‐associated neoplasia. We mimicked ROS exposure of the epithelium in IBD using non‐tumour human colonic epithelial cells (HCEC) and hydrogen peroxide (H₂O₂). A population of HCEC survived H₂O₂‐induced oxidative stress via JNK‐dependent cell cycle arrests. Caspases, p21^WAF1 and γ‐H2AX were identified as JNK‐regulated proteins. Up‐regulation of caspases was linked to cell survival and not, as expected, to apoptosis. Inhibition using the pan‐caspase inhibitor Z‐VAD‐FMK caused up‐regulation of γ‐H2AX, a DNA‐damage sensor, indicating its negative regulation via caspases. Cell cycle analysis revealed an accumulation of HCEC in the G₁‐phase as first response to oxidative stress and increased S‐phase population and then apoptosis as second response following caspase inhibition. Thus, caspases execute a non‐apoptotic function by promoting cells through G₁‐ and S‐phase by overriding the G₁/S‐ and intra‐S checkpoints despite DNA‐damage. This led to the accumulation of cells in the G₂/M‐phase and decreased apoptosis. Caspases mediate survival of oxidatively damaged HCEC via γ‐H2AX suppression, although its direct proteolytic inactivation was excluded. Conversely, we found that oxidative stress led to caspase‐dependent proteolytic degradation of the DNA‐damage checkpoint protein ATM that is upstream of γ‐H2AX. As a consequence, undetected DNA‐damage and increased proliferation were found in repeatedly H₂O₂‐exposed HCEC. Such features have been associated with neoplastic transformation and appear here to be mediated by a non‐apoptotic function of caspases. Overexpression of upstream p‐JNK in active ulcerative colitis also suggests a potential importance of this pathway in vivo.     

Golgi/plastid‐type manganese superoxide dismutase involved in heat‐stress tolerance during grain filling of rice

Superoxide dismutase (SOD) is widely assumed to play a role in the detoxification of reactive oxygen species caused by environmental stresses. We found a characteristic expression of manganese SOD 1 (MSD1) in a heat‐stress‐tolerant cultivar of rice (Oryza sativa). The deduced amino acid sequence contains a signal sequence and an N‐glycosylation site. Confocal imaging analysis of rice and onion cells transiently expressing MSD1‐YFP showed MSD1‐YFP in the Golgi apparatus and plastids, indicating that MSD1 is a unique Golgi/plastid‐type SOD. To evaluate the involvement of MSD1 in heat‐stress tolerance, we generated transgenic rice plants with either constitutive high expression or suppression of MSD1. The grain quality of rice with constitutive high expression of MSD1 grown at 33/28 °C, 12/12 h, was significantly better than that of the wild type. In contrast, MSD1‐knock‐down rice was markedly susceptible to heat stress. Quantitative shotgun proteomic analysis indicated that the overexpression of MSD1 up‐regulated reactive oxygen scavenging, chaperone and quality control systems in rice grains under heat stress. We propose that the Golgi/plastid MSD1 plays an important role in adaptation to heat stress.     

Involvement of the MyD88‐independent pathway in controlling the intracellular fate of Burkholderia pseudomallei infection in the mouse macrophage cell line RAW 264.7

Burkholderia pseudomallei is a facultative intracellular Gram‐negative bacterium which is capable of surviving and multiplying inside macrophages. B. pseudomallei strain SRM117, a LPS mutant which lacks the O‐antigenic polysaccharide moiety, is more susceptible to macrophage killing during the early phase of infection than is its parental wild type strain (1026b). In this study, it was shown that the wild type is able to induce expression of genes downstream of the MyD88‐dependent (iκbζ, il‐6 and tnf‐α), but not of the MyD88‐independent (inos, ifn‐β and irg‐1), pathways in the mouse macrophage cell line RAW 264.7. In contrast, LPS mutant‐infected macrophages were able to express genes downstream of both pathways. To elucidate the significance of activation of the MyD88‐independent pathway in B. pseudomallei‐infected macrophages, the expression of TBK1, an essential protein in the MyD88‐independent pathway, was silenced prior to the infection. The results showed that silencing the tbk1 expression interferes with the gene expression profile in LPS mutant‐infected macrophages and allows the bacteria to replicate intracellularly, thus suggesting that the MyD88‐independent pathway plays an essential role in controlling intracellular survival of the LPS mutant. Moreover, exogenous IFN‐γ upregulated gene expression downstream of the MyD88‐independent pathway, and interfered with intracellular survival in both wild type and tbk1‐knockdown macrophages infected with either the wild type or the LPS mutant. These results suggest that gene expression downstream of the MyD88‐independent pathway is essential in regulating the intracellular fate of B. pseudomallei, and that IFN‐γ regulates gene expression through the TBK1‐independent pathway.     

<Emphasis Type="Italic">rpoS</Emphasis> Gene Expression in Carbon-Starved Cultures of the Polyhydroxyalkanoate-Accumulating Species <Emphasis Type="Italic">Pseudomonas oleovorans</Emphasis>

The expression of the rpoS gene during PHA depolymerization was monitored in Pseudomonas oleovorans GPo1 and its mutant defective in PHA degradation by analyzing the tolerance to oxidative and thermal stresses and the RpoS intracellular content. An increase in the tolerance to H₂O₂ and heat shock was observed coincidentally with PHA degradation. Western blotting experiments performed in carbon-starved cultures showed that the RpoS levels were higher in the wild type than in the mutant strain. Complementation of the phaZ mutation restores the wild-type RpoS levels. These results suggest a probable association between PHA depolymerization and the stress tolerance phenotype controlled by RpoS.