H2O2 Induction Research Articles

148 - Pharmacological ascorbate enhances expression of dual oxidase which mediates sustained toxicity via alterations in cellular metabolism

Pancreatic Ductal Adenocarcinoma is a leading cause of cancer related death in the United States that is in critical need of adjuvant therapies to enhance patient survival. One such adjuvant is pharmacological ascorbate treatment (P-AscH-, high-dose, intravenous vitamin C). P-AscH- treatment results in a short-term increased flux of H2O2 that is preferentially cytotoxic to cancer cells vs. normal cells. Treatment with P-AscH- results in sustained increases in oxygen consumption (25-30 attomoles cell-1 sec-1) and reactive oxygen species production (1.5 fold as DCFH-DA oxidation), with no change in glucose metabolism in tumor cells compared to non-tumorigenic pancreatic ductal epithelial cells. These results lead to the hypothesis that there is a sustained effect (> 24 h) of P-AscH- that contributes to cytotoxicity. Seahorse XF96 mito stress test (48 h) demonstrated that P-AscH- results in a significant and sustained increase in non-mitochondrial OCR (~15 attomoles cell-1 sec-1). A potential source of this sustained non-mitochondrial OCR is the NADPH oxidase family of enzymes Dual Oxidase 1 and 2 (DUOX) which are up-regulated after P-AscH-. P-AscH- treatment of PDAC cell lines resulted in a dose (1-10 mM; 4-6 fold) and duration (24-72 h; 5-20 fold) dependent increase in DUOX. Catalase pretreatment reverses the P-AscH--induced increases in DUOX, while DUOX inhibition partially rescues P-AscH- toxicity. Additionally, nutritional ascorbate (100 mm; Ascorbate-2-Phosphate) is unable to mediate an increase in DUOX expression. P-AscH- also induces DUOX protein expression in pancreatic tumor xenografts. These results suggest that P-AscH--induced toxicity may be enhanced by a sustained metabolic shift in tumor cells that is related to the H2O2 produced from P-AscH-. We suggest that P-AscH- results in a feed-forward mechanism of H2O2 generation and induction of metabolic stress via enhanced DUOX expression and OCR.

A18652 Kallikrein-1 Overexpression Alleviates Age-associated Cardiac Fibrosis Through Alternatively Regulating Activated Macrophage Polarization

Objectives: The study was designed to determine the effect of KLK1 on cardiac aging and the mechanism of macrophage polarization in cardiac fibrosis Methods: Experiments are carried out using 3 and 24-month-old SD and hKLK1 overexpression rats. Echocardiography were performed to determine the cardiac function. Morphological staining were used to evaluate cardiac fibrosis and electron microscope to detect mitochondria ultrastructure. The speciemen were also used to detect expression of NAPDH subunits, anti-oxidative stress enzymes and ROS level by DHE staining. Immunofluorescence co-localization of CD68 and CD163, Th1 and Th2 cytokines examinations were used to measure macrophage polarization level. In the in vitro study migration capacity of Raw264.7 was detected by different dose of H2O2 administration with or without bradykinin or HOE-140 pre-treatment. Flow cytometry was used to examine the differentiation capacity of M0 towards M2 macrophage under the induction of H2O2 before IL-4 induction Results: hKLK1 overexpression protected against aging-induced cardiac hypertrophy and fibrosis, decreased expression of MCP-1, suppressed mitochondrial dysfunction and excess oxidative stress, decreased recruitment and retention of M2 macrophages and reduced secretion of pro-fibrotic cytokines mediated by TGF-β1/Smad3 signaling pathway. H2O2-induced senescence caused differentiation of RAW264.7 cells into M2-type cells and increased the secretion of Th2-type cytokines. In addition, bradykinin treatments reverse the enhanced migration capacity of Raw264.7induced by H2O2 treatments while HOE-140 administration recover the migration ability of macrophage. Conclusion: hKLK1 overexpression alleviated cardiac aging by alternatively regulating M2 recruitment and polarization mediated by p53-PGC-1α axis and TGF-β1/Smad3 pathway.

Effect of Temperature on Symptoms Expression and Viral RNA Accumulation in Groundnut Bud Necrosis Virus Infected Vigna unguiculata.

BackgroundGroundnut bud necrosis virus (GBNV) (Tospovirus genus, Bunyaviridae family) infects the major crops of solanaceae, leguminosae and cucurbitaceae in India. Temperature is an important factor which influences the plant growth and development under diseased conditions.ObjectiveIn the present study, we evaluated the effect of four different temperatures on the symptoms expression and viral RNA accumulation in the GBNV inoculated cowpea plants.Material and MethodsFor the evaluation of viral symptoms and RNA accumulation, we used 2–3 leaf stage cowpea plants grown in the controlled conditions. GBNV was mechanically inoculated by sap method to the cowpea plants and inoculated plants were incubated at four different temperatures (30, 25, 20 and 15 °C).ResultsThe first visible symptom of GBNV infection at the inoculated site was observed in the form of chlorotic spots which were converted into the necrotic spots as the infections succeeded. Some yellow mosaic symptoms were also observed at the systemic site during viral infection cycle. Plants incubated at higher (30 and 25 °C) temperatures showed a severe necrosis and a higher viral RNA accumulation at the inoculated site and facilitated the viral spread at the systemic site. However, viral RNA accumulation was less at the systemic site than the inoculated site. In contrast, symptoms’ expression and viral RNA accumulation were decreased at the inoculated site at low (20 and 15 °C) temperatures, no viral symptoms were observed at the systemic site (15 °C); in addition to viral RNA accumulation suppression at this site. GBNV infection at the inoculated site induced the higher accumulation of H2O2 followed by the induction of cell death at higher temperatures (30 and 25 °C) than the lower (20 and 15 °C) temperatures.ConclusionThis study suggests that viral RNA accumulation parallels with the H2O2 production and induction of cell death by GBNV infection in cowpea plants is temperature dependent.

Effect of Temperature on Symptoms Expression and Viral RNA Accumulation in Groundnut Bud Necrosis Virus Infected Vigna unguiculata

Background: Groundnut bud necrosis virus (GBNV) (Tospovirus genus, Bunyaviridae family) infects the major crops of solanaceae, leguminosae and cucurbitaceae in India. Temperature is an important factor which influences the plant growth and development under diseased conditions.Objective: In the present study, we evaluated the effect of four different temperatures on the symptoms expression and viral RNA accumulation in the GBNV inoculated cowpea plants.Material and Methods: For the evaluation of viral symptoms and RNA accumulation, we used 2-3 leaf stage cowpea plants grown in the controlled conditions. GBNV was mechanically inoculated by sap method to the cowpea plants and inoculated plants were incubated at four different temperatures (30, 25, 20 and 15 °C).Results: The first visible symptom of GBNV infection at the inoculated site was observed in the form of chlorotic spots which were converted into the necrotic spots as the infections succeeded. Some yellow mosaic symptoms were also observed at the systemic site during viral infection cycle. Plants incubated at higher (30 and 25 °C) temperatures showed a severe necrosis and a higher viral RNA accumulation at the inoculated site and facilitated the viral spread at the systemic site. However, viral RNA accumulation was less at the systemic site than the inoculated site. In contrast, symptoms’ expression and viral RNA accumulation were decreased at the inoculated site at low (20 and 15 °C) temperatures, no viral symptoms were observed at the systemic site (15 °C); in addition to viral RNA accumulation suppression at this site. GBNV infection at the inoculated site induced the higher accumulation of H2O2 followed by the induction of cell death at higher temperatures (30 and 25 °C) than the lower (20 and 15 °C) temperatures.Conclusion: This study suggests that viral RNA accumulation parallels with the H2O2 production and induction of cell death by GBNV infection in cowpea plants is temperature dependent.

Engineering bacterial motility towards hydrogen-peroxide.

Synthetic biologists construct innovative genetic/biological systems to treat environmental, energy, and health problems. Many systems employ rewired cells for non-native product synthesis, while a few have employed the rewired cells as ‘smart’ devices with programmable function. Building on the latter, we developed a genetic construct to control and direct bacterial motility towards hydrogen peroxide, one of the body’s immune response signaling molecules. A motivation for this work is the creation of cells that can target and autonomously treat disease, the latter signaled by hydrogen peroxide release. Bacteria naturally move towards a variety of molecular cues (e.g., nutrients) in the process of chemotaxis. In this work, we engineered bacteria to recognize and move towards hydrogen peroxide, a non-native chemoattractant and potential toxin. Our system exploits oxyRS, the native oxidative stress regulon of E. coli. We first demonstrated H2O2-mediated upregulation motility regulator, CheZ. Using transwell assays, we showed a two-fold increase in net motility towards H2O2. Then, using a 2D cell tracking system, we quantified bacterial motility descriptors including velocity, % running (of tumble/run motions), and a dynamic net directionality towards the molecular cue. In CheZ mutants, we found that increased H2O2 concentration (0–200 μM) and induction time resulted in increased running speeds, ultimately reaching the native E. coli wild-type speed of ~22 μm/s with a ~45–65% ratio of running to tumbling. Finally, using a microfluidic device with stable H2O2 gradients, we characterized responses and the potential for “programmed” directionality towards H2O2 in quiescent fluids. Overall, the synthetic biology framework and tracking analysis in this work will provide a framework for investigating controlled motility of E. coli and other ‘smart’ probiotics for signal-directed treatment.

Role of abscisic acid in strigolactone-induced salt stress tolerance in arbuscular mycorrhizal Sesbania cannabina seedlings

BackgroundStrigolactones (SLs) are considered to be a novel class of phytohormone involved in plant defense responses. Currently, their relationships with other plant hormones, such as abscisic acid (ABA), during responses to salinity stress are largely unknown.ResultsIn this study, the relationship between SL and ABA during the induction of H2O2 – mediated tolerance to salt stress were studied in arbuscular mycorrhizal (AM) Sesbania cannabina seedlings. The SL levels increased after ABA treatments and decreased when ABA biosynthesis was inhibited in AM plants. Additionally, the expression levels of SL-biosynthesis genes in AM plants increased following treatments with exogenous ABA and H2O2. Furthermore, ABA-induced SL production was blocked by a pre-treatment with dimethylthiourea, which scavenges H2O2. In contrast, ABA production was unaffected by dimethylthiourea. Abscisic acid induced only partial and transient increases in the salt tolerance of TIS108 (a SL synthesis inhibitor) treated AM plants, whereas SL induced considerable and prolonged increases in salt tolerance after a pre-treatment with tungstate.ConclusionsThese results strongly suggest that ABA is regulating the induction of salt tolerance by SL in AM S. cannabina seedlings.

Vanadium toxicity in chickpea (Cicer arietinum L.) grown in red soil: Effects on cell death, ROS and antioxidative systems

The agricultural soil contaminated with heavy metals induces toxic effects on plant growth. The present study was conducted to evaluate the effects of vanadium (V) on growth, H2O2 and enzyme activities, cell death, ion leakage, and at which concentration; V induces the toxic effects in chickpea plants grown in red soil. The obtained results indicated that the biomass (fresh and dry) and lengths of roots and shoots were significantly decreased by V application, and roots accumulated more V than shoots. The enzyme activities (SOD, CAT, and POD) and ion leakage were increased linearly with increasing V concentrations. However, the protein contents, and tolerance indices were significantly declined with the increasing levels of V. The results about the cell death indicated that the cell viability was badly damaged when plants were exposed to higher V, and induction of H2O2 might be involved in this cell death. In conclusion, all the applied V levels affected the enzymatic activities, and induced the cell death of chickpea plants. Furthermore, our results also confirmed that vanadium ≥ 130 mg kg−1 induced detrimental effects on chickpea plants. Additional investigation is needed to clarify the mechanistic explanations of V toxicity at the molecular level and gene expression involved in plant cell death.

High temperature effects on Pi54 conferred resistance to Magnaporthe oryzae in two genetic backgrounds of Oryza sativa

The global temperatures are predicted to rise due to climate change. However, knowledge on the mechanisms underlying the effect of high temperature (HT) on plant pathogen interaction is limited. We investigated the effect of elevated temperature on host phenotypic, biochemical and gene expression patterns in the rice-Magnaporthe oryzae (Mo) pathosystem using two genetic backgrounds, Co39 (Oryzae sativa-indica) and LTH (O. sativa-japonica) with (CO and LT) and without (Co39 and LTH) R gene (Pi54). After exposure to 28°C and 35°C the two genetic backgrounds showed contrasting responses to Mo. At 28°C, CO, Co39 and LTH displayed a more severe disease phenotype than LT. Surprisingly, CO became resistant to Mo after exposure to 35°C. CO and LT were used for further analysis to determine the defence related biochemical and transcriptome changes associated with HT induced resistance. Pre-exposure to 35°C triggered intense callose deposits and cell wall fluorescence of the attacked epidermal cells, as well as, increased hydrogen peroxide (H2O2) and salicylic acid (SA) levels. Transcriptional changes due to combined stress (35°C+Mo) were largely overridden by pathogen infection in both backgrounds, suggesting that the plants tended to shift their response to the pathogen. However, significant differences in global gene expression patterns occurred between CO and LT in response to both single (35°C and Mo) and double stress (35°C+Mo). Collectively, our results suggest that rice lines carrying Pi54 respond to Mo by rapid induction of callose and H2O2, and that these resistance mechanisms are amplified at HT. The relative difference in disease severity between CO and LT at 28°C suggests that the genetic background of japonica rice facilitates the function of Pi54 more than the background of indica rice. The phenotypic plasticity and gene expression differences between CO and LT reveal the presence of intricate background specific molecular signatures that may potentially influence adaptation to plant stresses.

Bacillus amyloliquefaciens SC06 alleviates the oxidative stress of IPEC-1 via modulating Nrf2/Keap1 signaling pathway and decreasing ROS production

Oxidative stress (OS) plays a major role in the gastrointestinal disorders. Although probiotics were reported to repress OS, few researches compared the antioxidant ability of different Bacillus strains and deciphered the mechanisms. To select a Bacillus strain with higher antioxidant capacity, we used H2O2 to induce intestinal porcine epithelial cell 1 (IPEC-1) OS model. The most suitable H2O2 concentration and incubation time were determined by the half lethal dose and methyl thiazolyl tetrazolium. Correlation analysis was performed to choose a sensitive indicator for OS. As for the comparison of Bacillus, cells were divided into control, Bacillus treatment, H2O2 treatment, and Bacillus pre-protection + H2O2 treatment. Bacillus were co-cultured with IPEC-1 for 3h in Bacillus and Bacillus pre-protection + H2O2 treatments. Then, based on OS model, 300μmol/L H2O2 was added into medium of H2O2 and Bacillus pre-protection + H2O2 treatments for another 12h. Antioxidant and apoptosis gene expressions were detected to screen the target strain. Nuclear factor erythroid-derived 2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein1 (Keap1) pathway, reactive oxygen species (ROS) production, mitochondrial membrane potential (Δψm), apoptosis, and necrosis were analyzed. Results revealed that heme oxygenase-1 (HO-1) gene expression had a positive correlation with H2O2 induction. Moreover, Bacillus amyloliquefaciens SC06 (SC06)-meditated IPEC-1 showed the best antioxidant capacity though modulating Nrf2 phosphorylation. Δψm was elevated, while ROS generation was reduced with SC06 pre-protection, resulting in decreased apoptosis and necrosis. Altogether, HO-1 expression could be regarded as an OS indicator. The regulation of Nrf2/Keap1 pathway and ROS production by SC06 are involved in alleviating OS of IPEC-1.

Analysis of early events in barley (Hordeum vulgare L.) roots in response to Fusarium culmorum infection

Fusarium culmorum is able to cause devastating crown rot disease, particularly in barley and wheat worldwide. The aim of this study was to investigate the early physiological and molecular changes in barley roots in response to F. culmorum infection. Therefore, we have infected 3-day old barley roots with a highly pathogenic F. culmorum isolate (F16). The root length and shoot length were significantly reduced at 7 days after infection in six widely cultivated Turkish barley cultivars. Based on the disease index values, Marti (six-rowed) and Tokak 157/37 (two-rowed) were selected. Defense response was comparatively assessed with measures including H2O2 production and induction of stress-induced genes at six-time points after infection (0–96 h). Fungal infection did not affect the membrane integrity of root cells while osmolality decreased and H2O2 production increased. At the molecular level, antioxidant-related genes, HvCu/ZnSOD, HvGST6, HvAPX and HvBAS1 were constitutively and strongly expressed unlikely to HvCAT2 in which transcript accumulation was slightly detected upon infection. Differential expression of HvMT2, HvLOX1 and HvWRKY12 has been observed following the infection. Importantly, pathogenesis related (PR) genes HvPR1, HvPR3, HvPR4, HvPR5 and HvPR10 were induced at different time points of infection. The transcript accumulation of HvPR4 was the highest while HvPR10 expressed in minimal levels. Our results showed unexpected cellular responses such as disruption of osmotic adjustment in barley roots and the role of PR genes in initial response under F. culmorum attack.

Diabetic levels of glucose increase cellular reducing equivalents but reduce survival in three models of 661W photoreceptor-like cell injury.

BackgroundThe effect of excess glucose on retinal cellular health remains controversial, and cellular reducing equivalents, as indicators of cellular energy production, are widely used as substitute indicators of retinal cellular health. These investigations hypothesised that excess energy substrate availability, as occurs in the diabetic retina, increases the susceptibility of retinal neurons to injury in the presence of increased cellular reducing equivalents.MethodsThe response of 661W cells to phototoxicity, oxidative stress induced by H2O2 and apoptosis induction by staurosporine was characterised in the presence of 5mM glucose and B27 defined media without insulin. Cellular insult was produced by phototoxicity, H2O2 and the apoptosis induction agent staurosporine. The effect of physiologically relevant alterations in environmental glucose on cellular reducing equivalents was assessed by MTT dye reduction and NAD(P)H assays, and cell survival was assessed via caspase 3/7 activation and Annexin V/PI flow cytometry.Results661W photoreceptor-like cells underwent dose dependent cell death primarily by apoptosis in response to phototoxic insult, H2O2, and staurosporine by all measures of cellular viability. Exposure of cells to 25mM glucose (diabetic-type conditions) increased cell death in response to all insults as measured by caspase 3/7 activation and Annexin V/PI flow cytometry. Cellular reducing equivalents were nonetheless increased in all models of injury in the presence of excess glucose. The mechanism of this increase was partly due to increased NADPH but not NADH levels in the presence of 25mM glucose.ConclusionsAcute exposure to 25mM glucose decreased the resilience of 661W photoreceptor-like cells to a range of cellular stressors whilst maintaining or increasing cellular reducing equivalents, partly be increasing NADPH levels. This shows that in 661W cells, diabetic levels of glucose decrease cellular resilience to injury. The decoupling of cellular reducing equivalents levels from cell survival has important implications when investigating the mechanisms of neuronal damage in diabetic retinal neuropathy.

Parathyroid hormone-related peptide protects cardiomyocytes from oxidative stress-induced cell death: First evidence of a novel endocrine–cardiovascular interaction

Although there is a growing interest in the molecular cross-talk between the endocrine and cardiovascular systems, the cardiac effects of calcium-regulating hormones (i.e., parathyroid hormone-related peptide (PTHrP)) have not been explored. In this study, we examined the effect of PTHrP on the viability of isolated adult mouse cardiomyocytes subjected to oxidative stress. Myocytes from 19 to 22 week old male 129J/C57BL6 mice were exposed to oxidative insult in the form of H2O2 which led to more than 70% loss of cell viability. Herein we demonstrate, for the first time, that pretreatment with 100 nM PTHrP prior to 100 μM H2O2 incubation prevents H2O2 -induced cell death by more than 50%. Immunoblot analysis revealed H2O2 induction of MKP-1 protein expression while PTHrP decreased MKP-1 expression. Moreover, myocytes derived from MKP1 KO mice were resistant to oxidative injury. No added benefit of PTHrP treatment was noted in MKP-1 null cardiomyocytes. Using specific pharmacological inhibitors we demonstrated that P-p38, P-ERK and P-AKT mediated PTHrP's cardioprotective action. These data provide novel evidence that: i) down-regulation of MKP1 affords profound protection against oxidative stress; and ii) PTHrP is cardioprotective, possibly via down-regulation of MKP-1 and activation of MAPK and PI3K/AKT signaling.

(1→3)-β-d-Glucan oligosaccharides monomers purification and its H2O2 induction effect study

In order to produce highly purified (1→3)-β-d-glucan oligosaccharides ((1→3)-β-d-GOS) monomers, a hydrophilic interaction liquid chromatography (HILIC) system with X-Amide stationary phase was performed. Nine (1→3)-β-d-GOS monomers with degree of polymerization (DP) from 2 to 10 were successfully separated. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) demonstrated that these monomers were with high purity. Furthermore, a hydrogen peroxide (H2O2) online detection method was established to monitor H2O2 releases in tobacco cells. This is the first report on nine consecutive (1→3)-β-d-GOS monomers purification and its effect upon H2O2-releasing in plants. It was found that (1→3)-β-d-GOS monomers with higher DP induced stronger defense responses in plants, which will pave the way for elucidating the relationship between (1→3)-β-d-GOS and biological activities.

Role of metal speciation in lead-induced oxidative stress to Vicia faba roots

Chemical speciation of metals in soil/solution plays an important role in determining their biogeochemical behavior in soil-plant system. The current study evaluated the influence of applied form of Pb (metal speciation) on its toxicity to metal sensitive Vicia faba L. roots. Lead was applied to young V. faba seedlings alone or chelated by organic ligands (citric acid and ethylenediaminetetraacetic acid). Plants were exposed to all treatments for 1, 4, 8, 12, and 24 h in nutrient solution, and contents of H2O2 and thiobarbituric-acid-reactive substances (TBARS) production were analyzed in V. faba roots. The results showed that Pb toxicity to V. faba roots depended on its applied chemical form and duration of exposure. Lead alone caused two burst of lipid peroxidation and H2O2 induction at 1 h and 12 h. Addition of EDTA dose-dependently inhibited Pb-induced H2O2 and TBARS production, indicating a protective role of this chelator against Pb toxicity during the first 24 h. In contrast, citric acid did not show significant effects on Pb-induced H2O2 and TBARS production, but delayed the induction of these effects. This study suggested that Pb toxicity to V. faba roots varies with Pb speciation in growth medium.

Icariin promotes angiogenic differentiation and prevents oxidative stress-induced autophagy in endothelial progenitor cells.

Reduced tissue levels of endothelial progenitor cells (EPCs) and functional impairment of endothelium are frequently observed in patients with diabetes and cardiovascular disease. The vascular endothelium is specifically sensitive to oxidative stress, and this is one of the mechanisms that causes widespread endothelial dysfunction in most cardiovascular diseases and disorders. Hence attention has increasingly been paid to enhance mobilization and differentiation of EPCs for therapeutic purposes. The aim of this study was to investigate whether Icariin, a natural bioactive component known from traditional Chinese Medicine, can induce angiogenic differentiation and inhibit oxidative stress-induced cell dysfunction in bone marrow-derived EPCs (BM-EPCs), and, if so, through what mechanisms. We observed that treatment of BM-EPCs with Icariin significantly promoted cell migration and capillary tube formation, substantially abrogated hydrogen peroxide (H2 O2 )-induced apoptotic and autophagic programmed cell death that was linked to the reduced intracellular reactive oxygen species levels and restored mitochondrial membrane potential. Icariin downregulated endothelial nitric oxide synthase 3, as well as nicotinamide-adenine dinucleotide phosphate-oxidase expression upon H2 O2 induction. These antiapoptotic and antiautophagic effects of Icariin are possibly mediated by restoring the loss of mammalian target of rapamycin /p70S6K/4EBP1 phosphorylation as well as attenuation of ATF2 and ERK1/2 protein levels after H2 O2 treatment. In summary, favorable modulation of the angiogenesis and redox states in BM-EPCs make Icariin a promising proangiogenic agent both enhancing vasculogenesis and protecting against endothelial dysfunction.

Roles of specific serines phosphorylation of αA crystallin on human lens epithelial cells

To investigate the impacts of specific serines phosphorylation of αA Crystallin on anti-oxidation and anti-apoptotic ability of human lens epithelial cells (HLEC). Experimental Study. Using site-directed mutagenesis, serines on specific sites (Ser45, Ser66, Ser122) were replaced respectively by aspartic acid (D) to mimic phosphorylation. Target genes were inserted into pEGFP-C1 plasmid and transfected into HLEC by Lipofectamine™ 2000. HLEC transfected with blank plasmid and plasmid over-expressing wide-type (WT) αA Crystallin were defined as control group and WT group. After H2O2 induction, Dihydroethidium (DHE) and Annexin V-PE/7AAD staining were used to detect the reactive oxygen species level and apoptotic rate. T-test, Wilcoxon-two sample test, analysis of variance and Kruskal-Wallis test were used for statistical analysis. The DHE intensity of three phosphorylation groups were 167.53±18.21, 143.54±4.78 and 143.40±30.19, which reduced significantly comparing to control group (274.42±3.71, t=9.963, P=0.001; t=34.961, P<0.01; Z=1.993, P=0.046); The same tendency were found when compared with cell group (WT) over-expressing wide-type αA Crystallin (192.67±1.47, Z=1.964, P=0.049; t=17.887, P<0.01; Z=1.964, P=0.049). After H2O2 induction for apoptosis, early apoptotic rates for three phosphorylation groups were 15.08%±0.90%, 16.59%±5.37% and 15.36%±2.62% respectively, which significantly decreased comparing with control group (41.28%±2.70%; t=21.632, P<0.01; t=8.855, t=15.450, P<0.01) and WT group (31.40%±1.64%, t=15.117, P<0.01; t=4.524, P=0.006; t=8.988, P=0.001). No significant differences were found in both measurements between the three phosphorylation groups (F=1.08, P=0.407; x2=4.345, P=0.114). The Ser45, Ser66 and Ser122 phosphorylation enhanced the anti-oxidative stress and anti-apoptotic capability of HLEC.

The antisenescence effect of trans-cinnamaldehyde on adipose-derived stem cells.

As assuring cell quality is an essential parameter for the success of stem cell therapy, the impact of various senescence-inducing stress signals, and strategies to circumvent them, has been an important area of focus in stem cell research. The aim of this study was to demonstrate the capacity of Trans-cinnamaldehyde (TC) in reversing stress-induced senescence and maintaining the quality of stem cells in a chemically (H2O2)-induced cell senescence model. Because of the availability and the promising application potential in regenerative medicine, adipose-derived stem cells (ADSCs) were chosen for the study. We found that H2O2 treatment resulted in the expression of senescence characteristics in the ADSCs, including decreased proliferation rate, increased senescence-associated β-galactosidase (SA-β-gal) activity, decreased silent mating type information regulation 2 homolog (SIRT1) expression, and decreased telomerase activity. However, TC treatment was sufficient to rescue or reduce the effects of H2O2 induction, ultimately leading to an increased proliferation rate, a decrease in the percentage of SA-β-gal-positive cells, upregulation of SIRT1 expression, and increased telomerase activity of the senescent ADSCs at the cellular level. Moreover, a chemically induced liver fibrosis animal model was used to evaluate the functionality of these rescued cells in vivo. Liver dysfunction was established by injecting 200 mg/kg thioacetamide (TAA) intraperitoneally into Wistar rats every third day for 60 days. The experimental rats were separated into groups: normal group (rats without TAA induction), sham group (without ADSC transplantation), positive control group (transplanted with normal ADSCs), H2O2 group (transplanted with H2O2-induced senescent ADSCs), and H2O2 + TC group (transplanted with ADSCs pretreated with H2O2 and then further treated with TC). In the transplantation group, 1 × 10(6) human ADSCs were introduced into each rat via direct liver injection. Based on the biochemical analysis and immunohistochemical staining results, it was determined that the therapeutic effects on liver fibrosis by the induced senescent ADSCs (H2O2 group) were not as significant as those exerted by the normal ADSCs (the positive control group). However, the H2O2 + TC group showed significant reversal of liver damage when compared to the H2O2 group 1 week posttransplantation. These data confirmed that the TC treatment had the potential to reduce the effects of H2O2-induced senescence and to restore in vivo functionality of the induced senescent ADSCs. It is therefore suggested that TC has potential applications in maintaining the quality of stem cells and could aid in treating senescence-related disorders.

Preconditioning treatment induces chilling tolerance in zucchini fruit improving different physiological mechanisms against cold injury

Zucchini fruit is susceptible to develop chilling injuries (CI) when stored at low temperature. In this study, the effects of a preconditioning treatment during cold storage and its relation with the physiological response to chilling tolerance have been investigated. The commercial variety Sinatra, whose fruit are very sensitive to cold storage, has been used. After harvest, fruit were kept at 4°C or preconditioned during 48 h at 15°C before cold storage. Weight loss, electrolyte leakage and lipid peroxidation were lower in preconditioned at the end of storage time, and CI index was significantly reduced in preconditioned compared to control fruit. The preconditioning treatment improved the energy status of the fruit increasing the pool of ATP, and maintaining the energy charge. The preconditioned fruit improved their antioxidant status with lower H2O2 content and induction of ascorbate peroxidase (APX) and catalase (CAT) activities. A reduction in putrescine was detected in preconditioned fruit along with a lower expression of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) and a rise in activity of diamine oxidase (DAO). The concentrations of glutamate and γ-aminobutyrate (GABA) were lower during preconditioning, while that of proline was higher. In summary, preconditioning treatment induces chilling tolerance in zucchini fruit triggering a defence-response against oxidative stress and increasing ATP pool and proline content.

A Subpopulation of Blasts with Attenuated p38MAPK Response Is Seen in Virtually All AML Patients and AML Cell Lines and Is Defined By Cells with Augmented Lipid-Associated Anti-Oxidant Defense

The serine/ threonine kinase, p38MAPK is activated by phosphorylation in response to a variety of cellular stresses including oxidative stress. Prolonged p38MAPK activation drives cell-cycle arrest and apoptosis; and in HSC activation of p38MAPK leads to a loss of reconstituting capacity (Ito et al, Nat.Med. 2006;12:446-451). In cancer, p38MAPK responses are often attenuated and cancer models suggest that this is a necessary adaptation for transformation (Dolado et al, Cancer Cell 2007;11:191-205). Previously we have shown that 60% of acute myeloid leukemia (AML) patients constitutively generate significantly more extracellular reactive oxygen species (ROS) than normal hematopoietic CD34+ cells (Hole et al, Blood 2013;122:3322-3330). Despite this, AML blasts showed low or absent p38MAPK phosphorylation; even in patients generating high levels of ROS. Here we examine p38MAPK activation at the single cell level in primary AML blasts using flow cytometry.We challenged AML blasts with a dose of hydrogen peroxide (H2O2) sufficient to completely activate p38MAPK in normal CD34+ cells (1 mM for 30 min), where the threshold for activation was defined as the 95th percentile of basal p38MAPK activation in unstimulated cells. Attenuated responses to H2O2 were seen in 14/15 (93%) of patients; where 16-95% of the total blast population failed to activate p38MAPK. These non-responding cells are hereafter termed “Δpp38MAPK cells” and were absent in normal CD34+ cells (p < 0.01; Figure 1). Examination of a panel of 6 AML cell lines showed that each of the lines contained Δpp38MAPK cells at different frequencies: MV4-11 (10%); HL60 (10%); KG-1 (15%), U937 (30%), NB4 (50%), THP-1 (65%). Further analysis showed that Δpp38MAPK cells were not distinguished by cell cycle phase, immunophenotype or reduced viability in either cell lines or AML blasts. These data suggest that nearly all AML patients harbor a population of blasts which have developed resistance to p38MAPK activation. We reasoned that failure to respond could arise either through defective p38MAPK signaling or because of enhanced anti-oxidative protection in a subpopulation of cells. To investigate the latter, we labelled cells with the lipophilic oxidation probe, C11 -BODIPY or the cytosolic oxidant probe, CM-DCFDA and monitored the oxidative response to H2O2 at the single cell level in the AML cell lines: KG-1, MV4-11 and THP-1. In each case C11 -BODIPY oxidation exactly matched the heterogeneous profile of p38MAPK activation in these cells, whereas CM-DCFDA showed only homogeneous responses to H2O2 induction. These data show that Δpp38MAPK cells are defined by an enhanced membrane-associated anti-oxidant capacity and we are currently analyzing this resistant subpopulation to identify the molecules responsible.To examine whether p38MAPK responsiveness influenced responsiveness to pro-oxidant drugs, we selected KG-1 and THP-1 cells (as representative examples of strong and weak p38MAPK responses respectively) and tested their sensitivity to the pro-oxidant drugs, phenethyl isothiocyanate (PEITC) and buthionine sulfoximine (BSO). We found that the IC50 was higher for THP-1 for both PEITC (KG-1 = 0.6µM; THP-1 = 7.5µM) and BSO (KG-1 = 50µM; THP-1 = 70µM), indicating that the p38MAPK responsiveness limits the effectiveness of pro-oxidant drugs. We next examined whether promoting p38MAPK activation could augment the effects of these pro-oxidants. We used the p38MAPK activator 2-benzylidene-3-(cyclohexylamino)-1-indanone (BCI), which promotes activation of p38MAPK via inhibition of a p38MAPK phosphatase, DUSP1. This compound weakly promoted phosphorylation of p38MAPK in THP-1 cells and consistent with this, had no effect on the efficacy of these compounds in these cells. However, BCI potently activated p38MAPK in KG-1 cells and showed synergy with BSO in this context (CI = 0.3; Figure 2) indicating that where BCI is effective in activating p38MAPK it can promote the effectiveness of pro-oxidant drugs.In summary, we show for the first time that AML patients almost universally display attenuated p38MAPK responses in all or part of the blast population and we suggest that this trait may be selected for to maintain self-renewing potential under the pro-oxidative conditions found in the leukemic marrow. Further we show that by manipulating p38MAPK activity, we can augment the potency of the pro-oxidant compound BSO. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pepper pathogenesis-related protein 4c is a plasma membrane-localized cysteine protease inhibitor that is required for plant cell death and defense signaling.

Xanthomonas campestris pv.vesicatoria (Xcv) type III effector AvrBsT triggers programmed cell death (PCD) and activates the hypersensitive response (HR) in plants. Here, we isolated and identified the plasma membrane localized pathogenesis-related (PR) protein 4c gene (CaPR4c) from pepper (Capsicum annuum) leaves undergoing AvrBsT-triggered HR cell death. CaPR4c encodes a protein with a signal peptide and a Barwin domain. Recombinant CaPR4c protein expressed in Escherichia coli exhibited cysteine protease-inhibitor activity and ribonuclease (RNase) activity. Subcellular localization analyses revealed that CaPR4c localized to the plasma membrane in plant cells. CaPR4c expression was rapidly and specifically induced by avirulent Xcv (avrBsT) infection. Transient expression of CaPR4c caused HR cell death in pepper leaves, which was accompanied by enhanced accumulation of H2 O2 and significant induction of some defense-response genes. Deletion of the signal peptide from CaPR4c abolished the induction of HR cell death, indicating a requirement for plasma membrane localization of CaPR4c for HR cell death. CaPR4c silencing in pepper disrupted both basal and AvrBsT-triggered resistance responses, and enabled Xcv proliferation in infected leaves. H2 O2 accumulation, cell-death induction, and defense-response gene expression were distinctly reduced in CaPR4c-silenced pepper. CaPR4c overexpression in transgenic Arabidopsis plants conferred greater resistance against infection by Pseudomonas syringae pv.tomato and Hyaloperonospora arabidopsidis. These results collectively suggest that CaPR4c plays an important role in plant cell death and defense signaling.