Nitric Oxide Accumulation Research Articles

AhGSNOR1 negatively regulates Al-induced programmed cell death by regulating intracellular NO and redox levels

The toxicity of aluminum (Al) in acidic soil inhibits plant development and reduces crop yields. Programmed cell death (PCD) is one of the important mechanisms in the plant response to Al toxicity. However, it is yet unknown if S-nitrosoglutathione reductase (GSNOR) provides Al-PCD. Here, transcription and protein expression of AhGSNOR1 were both induced by Al stress. AhGSNOR1-overexpressing transgenic tobacco plants reduced Al-induced nitric oxide (NO) and S-nitrosothiol accumulation, the inhibitory effect of Al stress on root elongation and the degree of cell death, and enhanced antioxidant enzyme activity to effectively remove hydrogen peroxide. In addition, AhGSNOR1 directly interacted with AhTRXh in vivo. Expression of Trxh3 in AhGSNOR1-overexpressing transgenic plants was significantly upregulated, indicating that AhGSNOR1 positively regulated the transcriptional level of Trxh3. Together, these results suggested that AhGSNOR1 was a negative regulatory factor of Al-induced PCD and improved plant Al-tolerance by modulating intracellular NO and redox homeostasis.

Mechanism of RSL3-induced ferroptotic cell death in HT22 cells: crucial role of protein disulfide isomerase.

Protein disulfide isomerase (PDI) was recently shown to be an upstream mediator of erastin-induced, glutathione depletion-associated ferroptosis through its catalysis of nitric oxide synthase (NOS) dimerization and nitric oxide (NO) accumulation. A recent study reported that RSL3, a known ferroptosis inducer and glutathione peroxidase 4 (GPX4) inhibitor, can inhibit thioredoxin reductase 1 (TrxR1). The present study seeks to test the hypothesis that RSL3 may, through its inhibition of TrxR1, facilitate PDI activation ( i. e., in a catalytically active, oxidized state), thereby enhancing RSL3-induced ferroptosis through NOS dimerization and NO accumulation. Using HT22 mouse neuronal cells as an in vitro model, we show that treatment of these cells with RSL3 strongly increases NOS protein levels and that PDI-mediated NOS dimerization is activated by RSL3, resulting in NO accumulation. Mechanistically, we find that PDI is activated in cells treated with RSL3 because of its inhibition of TrxR1, and the activated PDI then catalyzes NOS dimerization, which is followed by the accumulation of cellular NO, ROS and lipid-ROS and ultimately ferroptotic cell death. Genetic or pharmacological inhibition of PDI or TrxR1 partially abrogates RSL3-induced NOS activation and the subsequent accumulation of cellular NO, ROS/lipid-ROS, and ultimately ferroptosis in HT22 cells. The results of this study clearly show that PDI activation resulted from RSL3 inhibition of TrxR1 activity contributes crucially to RSL3-induced ferroptosis in a cell culture model through the PDI→NOS→NO→ROS/lipid-ROS pathway, in addition to its known inhibition of GPX4 activity.

Dissociation of the nuclear WWOX/TRAF2 switch renders UV/cold shock-mediated nuclear bubbling cell death at low temperatures

BackgroundNormal cells express functional tumor suppressor WW domain-containing oxidoreductase (WWOX), designated WWOXf. UV irradiation induces WWOXf cells to undergo bubbling cell death (BCD) — an event due to the accumulation of nuclear nitric oxide (NO) gas that forcefully pushes the nuclear and cell membranes to form one or two bubbles at room temperature (22 °C) and below. In contrast, when WWOX-deficient or -dysfunctional (WWOXd) cells are exposed to UV and/or cold shock, the cells undergo nuclear pop-out explosion death (POD). We aimed to determine the morphological and biochemical changes in WWOXf cells during BCD versus apoptosis.MethodsWWOXf and WWOXd cells were exposed to UV followed by measuring BCD or POD by time-lapse microscopy and/or time-lapse holographic microscopy at 4, 22, or 37 °C to visualize morphological changes. Live cell stains were used to measure the kinetics of nitric oxide (NO) production and Ca2+ influx. Extent of cell death was measured by uptake of propidium iodide and by internucleosomal DNA fragmentation using agarose gel electrophoresis.ResultsWWOXf cells were exposed to UV and then cold shock, or cold shock and then UV, and cultured at 4, 10, and 22 °C, respectively. Initially, UV induced calcium influx and NO production, which led to nuclear bubbling and final death. Cold shock pretreatment completely suppressed UV-mediated bubbling at 37 °C, so the UV/cold shock-treated cells underwent apoptosis. Without cold shock, UV only induced bubbling at all temperatures, whereas the efficiency of bubbling at 37 °C was reduced by greater than 50%. Morphologically, the WWOXf cell height or thickness was significantly increased during cell division or apoptosis, but the event did not occur in BCD. In comparison, when WWOXd cancer cells received UV or UV/cold shock, these cells underwent NO-independent POD. UV/cold shock effectively downregulated the expression of many proteins such as the housekeeping α-tubulin (> 70%) and β-actin (< 50%), and cortactin (> 70%) in WWOXf COS7 cells. UV/cold shock induced relocation of α-tubulin to the nucleus and nuclear bubbles in damaged cells. UV induced co-translocation of the WWOX/TRAF2 complex to the nuclei, in which the prosurvival TRAF2 blocked the proapoptotic WWOX via its zinc finger domain. Without WWOX, TRAF2 did not relocate to the nuclei. Cold shock caused the dissociation of the WWOX/TRAF2 complex in the nucleus needed for BCD. In contrast, the formation of the WWOX/TRAF2 complex, plus p53, was strengthened at 37 °C required for apoptosis.ConclusionsThe temperature-sensitive nuclear WWOX/TRAF2 complex acts as a molecular switch, whose dissociation favors BCD at low temperatures, and the association supports apoptosis at 37 °C in UV-treated WWOXf cells.

Protection of HT22 neuronal cells against chemically-induced ferroptosis by catechol estrogens: protein disulfide isomerase as a mechanistic target

Ferroptosis is a form of regulated cell death, characterized by excessive iron-dependent lipid peroxidation. Biochemically, ferroptosis can be selectively induced by erastin through glutathione depletion or through inhibition of glutathione peroxidase 4 by RSL3, which leads to accumulation of cytotoxic lipid reactive oxygen species (ROS). Protein disulfide isomerase (PDI) was recently shown to mediate erastin/RSL3-induced ferroptosis and thus also become a new target for protection against chemically-induced ferroptosis. The present study aims to identify endogenous compounds that can protect against erastin/RSL3-induced ferroptotic cell death. We find that 2-hydroxyestrone, 2-hydroxyestradiol, 4-hydroxyestrone and 4-hydroxyestradiol, four major endogenous catechol estrogens, are effective inhibitors of PDI, and can strongly protect against chemically-induced ferroptotic cell death in cultured HT22 mouse hippocampal neuronal cells. The CETSA assay showed that these catechol estrogens can bind to PDI in live cells. PDI knockdown attenuates the protective effect of these catechol estrogens against chemically-induced ferroptosis. Mechanistically, inhibition of PDI’s catalytic activity by catechol estrogens abrogates erastin/RSL3-induced dimerization of nitric oxide synthase, thereby preventing the subsequent accumulation of cellular nitric oxide, ROS and lipid-ROS, and ultimately ferroptotic cell death. In addition, joint treatment of cells with catechol estrogens also abrogates erastin/RSL3-induced upregulation of nitric oxide synthase protein levels, which also contributes to the cytoprotective effect of the catechol estrogens. In conclusion, the present study demonstrates that the catechol estrogens are protectors of HT22 neuronal cells against chemically-induced ferroptosis, and inhibition of PDI’s catalytic activity by these estrogens contributes to a novel, estrogen receptor-independent mechanism of cytoprotection.

Shade-induced ROS/NO reinforce COP1-mediated diffuse cell growth

Canopy shade enhances the activity of PHYTOCHROME INTERACTING FACTORs (PIFs) to boost auxin synthesis in the cotyledons. Auxin, together with local PIFs and their positive regulator CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), promotes hypocotyl growth to facilitate access to light. Whether shade alters the cellular redox status thereby affecting growth responses, remains unexplored. Here, we show that, under shade, high auxin levels increased reactive oxygen species and nitric oxide accumulation in the hypocotyl of Arabidopsis. This nitroxidative environment favored the promotion of hypocotyl growth by COP1 under shade. We demonstrate that COP1 is S-nitrosylated, particularly under shade. Impairing this redox regulation enhanced COP1 degradation by the proteasome and diminished the capacity of COP1 to interact with target proteins and to promote hypocotyl growth. Disabling this regulation also generated transversal asymmetries in hypocotyl growth, indicating poor coordination among different cells, which resulted in random hypocotyl bending and predictably low ability to compete with neighbors. These findings highlight the significance of redox signaling in the control of diffuse growth during shade avoidance.

Raloxifene Prevents Chemically-Induced Ferroptotic Neuronal Death In Vitro and In Vivo.

Ferroptosis, a regulated form of cell death characterized by excessive iron-dependent lipid peroxidation, can be readily induced in cultured cells by chemicals such as erastin and RSL3. Protein disulfide isomerase (PDI) has been identified as an upstream mediator of chemically induced ferroptosis and also a target for ferroptosis protection. In this study, we discovered that raloxifene (RAL), a selective estrogen receptor modulator known for its neuroprotective actions in humans, can effectively inhibit PDI function and provide robust protection against chemically induced ferroptosis in cultured HT22 neuronal cells. Specifically, RAL can bind directly to PDI both in vitro and in intact neuronal cells and inhibit its catalytic activity. Computational modeling analysis reveals that RAL can tightly bind to PDI through forming a hydrogen bond with its His256 residue, and biochemical analysis further shows that when PDI's His256 is mutated to Ala256, RAL loses its inhibition of PDI's catalytic activity. This inhibition of PDI by RAL significantly reduces the dimerization of both the inducible and neuronal nitric oxide synthases and the accumulation of nitric oxide, both of which have recently been shown to play a crucial role in mediating chemically induced ferroptosis through subsequent induction of ROS and lipid-ROS accumulation. In vivo behavioral analysis shows that mice treated with RAL are strongly protected against kainic acid-induced memory deficits and hippocampal neuronal damage. In conclusion, this study demonstrates that RAL is a potent inhibitor of PDI and can effectively prevent chemically induced ferroptosis in hippocampal neurons both in vitro and in vivo. These findings offer a novel estrogen receptor-independent mechanism for RAL's neuroprotective actions in animal models and humans.

Single von Willebrand factor C-domain protein-2 confers immune defense against bacterial infections in the silkworm, Bombyx mori

Single-domain von Willebrand factor type C proteins (SVWCs), primarily found in arthropods, responds to infections caused by various pathogens. Three SVWCs have been identified in the silkworm and BmSVWC2 might play a crucial role in the immune system. However, the regulatory mechanism of BmSVWC2 remains largely unknown. This study aimed to investigate the biochemical functions of BmSVWC2 in the immune system of B. mori comprehensively. Phylogenetic analysis revealed that BmSVWC1, BmSVWC3, and BmSVWC2 were distributed in diverse groups, suggesting distinct biochemical functions. The mRNA and protein levels of BmSVWC2 increased significantly in response to bacterial infection. BmSVWC2 exhibited clear binding activity to the polysaccharide pathogen-associated molecular patterns of bacteria and fungi, enhancing bacterial clearance in vivo but not in vitro. RNA-sequencing assays of the fat body and hemocytes showed that numerous immune genes were markedly up-regulated with higher level of BmSVWC2, primarily affecting recognition, signaling, and response production of the Toll and immune deficiency (IMD) signaling pathways. This led to the production of various antimicrobial peptides and significant antibacterial activities in the hemolymph. BmSVWC2 up-regulated phagocytosis-related genes in the fat body and hemocytes, and phagocytosis assays confirmed that BmSVWC2 improved the phagocytic ability of hemocytes against bacteria. Additionally, BmSVWC2 induced the expression of nitric oxide synthetase (NOS) in the fat body, and bioassays confirmed that BmSVWC2 increased NOS activity in the fat body and hemolymph, resulting in nitric oxide accumulation. However, BmSVWC2 did not affect phenoloxidase activity, despite it caused differential expression of a few serine proteases and serine protease inhibitors. Co-immunoprecipitation and mass spectrometry assays showed that BmSVWC2 interacted with 30 K proteins, such as 30 K protein 2, 30 K pBmHPC-19, 30 K 19G1-like, 30 K protein 8, 30 K protein 7, 30 K pBmHPC-23, and low molecular mass lipoprotein 4-like. Our study provides a comprehensive characterization of BmSVWC2 and elucidates the mechanism underlying its regulation of immune responses activation.

Hepatic BMAL1 and HIF1α regulate a time-dependent hypoxic response and prevent hepatopulmonary-like syndrome

The transcriptional response to hypoxia is temporally regulated, yet the molecular underpinnings and physiological implications are unknown. We examined the roles of hepatic Bmal1 and Hif1α in the circadian response to hypoxia in mice. We found that the majority of the transcriptional response to hypoxia is dependent on either Bmal1 or Hif1α, through shared and distinct roles that are daytime determined. We further show that hypoxia-inducible factor (HIF)1α accumulation upon hypoxia is temporally regulated and Bmal1 dependent. Unexpectedly, mice lacking both hepatic Bmal1 and Hif1α are hypoxemic and exhibit increased mortality upon hypoxic exposure in a daytime-dependent manner. These mice display mild liver dysfunction with pulmonary vasodilation likely due to extracellular signaling regulated kinase (ERK) activation, endothelial nitric oxide synthase, and nitric oxide accumulation in lungs, suggestive of hepatopulmonary syndrome. Our findings indicate that hepatic BMAL1 and HIF1α are key time-dependent regulators of the hypoxic response and can provide molecular insights into the pathophysiology of hepatopulmonary syndrome.

The role of pharmacological interventions in managing hyperlipidemia: A closer look at different drugs

Hyperlipidemia is a medical condition characterized by an increase in plasma lipids, including triglycerides, cholesterol, cholesterol esters, phospholipids, and plasma lipoproteins, which is a leading risk factor for cardiovascular diseases. The pathophysiology of hyperlipidemia is influenced by endothelial damage to blood vessels, leading to a loss of nitric oxide, increased inflammation, and lipid accumulation in the deepest layer of the endothelial wall. Lipid-lowering drugs like statins can reduce LDL levels by 25-60%, but they have negative impacts on clinical outcomes. Lipoprotein metabolism involves various enzymes, and atherosclerosis accumulates fat, cholesterol, and calcium in artery linings, creating fibrous plaques that reduce blood flow and oxygen supply, leading to heart damage and death. Lipoprotein metabolism involves various enzymes, including lipoprotein lipase (LPL), hepatic lipase (HL), lecithin cholesterol acyl transferase (LCAT), cholesterol ester transfer protein (CETP), microsomal triglyceride protein (MTP), and acyl Co-A transferase (ACAT). Atherosclerosis accumulates fat, cholesterol, and calcium in artery linings, creating fibrous plaques. This leads to reduced blood flow and oxygen supply, causing heart damage and death. Reducing LDL and total cholesterol can significantly lower the incidence of strokes. Reducing LDL and total cholesterol can significantly lower the incidence of strokes. Traditional antihyperlipidemic medicines have negative effects, and herbal treatments like onion, garlic, guggul, and Asparagus racemosus have been shown to have anti-diabetic and antihyperlipidemic properties.

Bitter Taste in the Treatment of Heart Diseases from Avicenna’s Point of View

The function of bitter taste due to the existence of too much of its receptor on many extra-oral tissues is not only related to the oral cavity, but is effective in many physiological functions. More than ten centuries ago, Avicenna (980–1032 CE), a Persian physician, pointed to the effects and functions of various tastes in the body. In this research, we examined the heart medicines mentioned by Avicenna, relying on their taste, especially the bitter taste. The books used in the case of Persian Medicine included the following: Qanun Fi al-Teb (Canon of Medicine), Manafe-al-Aghzieh va Daf-e-Mazareha and Treatise on Cardiac Drugs. In addition, articles published in English in the last 10 years were searched in PubMed, Google Scholar, Scopus and Embase. Vasodilation, inotropic effects, cardio-protection, digital like effect, reducing the accumulation of calcium and nitric oxide in the heart, antioxidant activity, improving heart metabolism and preserving mitochondrial function after MI are some of the cardiovascular effects of Avicenna`s bitter tasting heart medicines, which are also confirmed by clinical evidences in modern investigations. The findings of this research show that the function of bitter-tasting herbal drugs in the body can have beneficial cardiovascular effects, some of which have been proven in studies, and more researches is needed in this field.

S-nitrosylation of a receptor-like cytoplasmic kinase regulates plant immunity.

Perception of pathogen/microbial-associated molecular patterns (P/MAMPs) by plant cell surface receptors leads to a sustained burst of reactive oxygen species (ROS), a key feature of P/MAMP-triggered immunity (PTI). Here we report that P/MAMP recognition leads to a rapid nitrosative burst, initiating the accumulation of nitric oxide (NO), subsequently leading to S-nitrosylation of the receptor-like cytoplasmic kinase (RLCK), botrytis-induced kinase 1 (BIK1), at Cys80. This redox-based, posttranslational modification, promotes the phosphorylation of BIK1, subsequently resulting in BIK1 activation and stabilization. Further, BIK1 S-nitrosylation increases its physical interaction with RBOHD, the source of the apoplastic oxidative burst, promoting ROS formation. Our data identify mechanistic links between rapid NO accumulation and the expression of PTI, providing insights into plant immunity.

Kinetin-mediated reduction of cadmium accumulation in rice (Oryza sativa L.) via modulation of cell wall binding capacity in a NO-dependent manner

Cadmium (Cd) is a heavy metal that is toxic to both plant growth and human health. Kinetin, a precursor of cytokinin, plays crucial roles in the response of plants to abiotic stress. However, the mechanism underlying kinetin detoxification in Cd-induced stress remains poorly understood. In this study, we observed a significant increase in endogenous kinetin levels in both rice shoots and roots under Cd stress. Furthermore, the application of exogenous kinetin resulted in a reduction in hemicellulose content within cell walls, thereby reducing the cell wall's capacity to bind Cd and limit its entry into cells. In addition, exogenous kinetin downregulated the transcript levels of genes associated with Cd uptake and root-to-shoot translocation while simultaneously upregulating the transcript levels of genes involved in Cd efflux from cells and sequestration into vacuoles, consequently leading to reduced Cd accumulation in rice plants. Moreover, kinetin significantly decreased nitric oxide (NO) accumulation in response to Cd stress, potentially facilitating detoxification during Cd-induced stress through a NO-dependent mechanism. Overall, our findings suggest that kinetin enhances rice's tolerance to Cd by increasing the cell wall's ability to bind Cd and regulating gene expression related to Cd accumulation. These mechanisms may be NO-dependent and could potentially inform the development of new strategies for mitigating Cd toxicity in agricultural systems.

Denitrification in low oxic environments increases the accumulation of nitrogen oxide intermediates and modulates the evolutionary potential of microbial populations.

Denitrification in oxic environments occurs when a microorganism uses nitrogen oxides as terminal electron acceptors even though oxygen is available. While this phenomenon is well-established, its consequences on ecological and evolutionary processes remain poorly understood. We hypothesize here that denitrification in oxic environments can modify the accumulation profiles of nitrogen oxide intermediates with cascading effects on the evolutionary potentials of denitrifying microorganisms. To test this, we performed laboratory experiments with Paracoccus denitrificans and complemented them with individual-based computational modelling. We found that denitrification in low oxic environments significantly increases the accumulation of nitrite and nitric oxide. We further found that the increased accumulation of these intermediates has a negative effect on growth at low pH. Finally, we found that the increased negative effect at low pH increases the number of individuals that contribute to surface-associated growth. This increases the amount of genetic diversity that is preserved from the initial population, thus increasing the number of genetic targets for natural selection to act upon and resulting in higher evolutionary potentials. Together, our data highlight that denitrification in low oxic environments can affect the ecological processes and evolutionary potentials of denitrifying microorganisms by modifying the accumulation of nitrogen oxide intermediates.

Pseudomonas aeruginosa LasI-dependent plant growth promotion requires the host nitrate transceptor AtNRT1.1/CHL1 and the nitrate reductases NIA1 and NIA2.

In P. aeruginosa, mutation of the gene encoding N-acyl-L-homoserine lactone synthase LasI drives defense and plant growth promotion, and this latter trait requires adequate nitrate nutrition. Cross-kingdom communication with bacteria is crucial for plant growth and productivity. Here, we show a strong induction of genes for nitrate uptake and assimilation in Arabidopsis seedlings co-cultivated with P. aeruginosa WT (PAO1) or ΔlasI mutants defective on the synthesis of the quorum-sensing signaling molecule N-(3-oxododecanoyl)-L-homoserine lactone. Along with differential induction of defense-related genes, the change from plant growth repression to growth promotion upon bacterial QS disruption, correlated with upregulation of the dual-affinity nitrate transceptor CHL1/AtNRT1/NPF6.3 and the nitrate reductases NIA1 and NIA2. CHL1-GUS was induced in Arabidopsis primary root tips after transfer onto P. aeruginosa ΔlasI streaks at low and high N availability, whereas this bacterium required high concentrations of nitrogen to potentiate root and shoot biomass production and to improve root branching. Arabidopsis chl1-5 and chl1-12 mutants and double mutants in NIA1 and NIA2 nitrate reductases showed compromised growth under low nitrogen availability and failed to mount an effective growth promotion and root branching response even at high NH4NO3. WT P. aeruginosa PAO1 and P. aeruginosa ΔlasI mutant promoted the accumulation of nitric oxide (NO) in roots of both the WT and nia1nia2 double mutants, whereas NO donors SNP or SNAP did not improve growth or root branching in nia1nia2 double mutants with or without bacterial cocultivation. Thus, inoculation of Arabidopsis roots with P. aeruginosa drives gene expression for improved nitrogen acquisition and this macronutrient is critical for the plant growth-promoting effects upon disruption of the LasI quorum-sensing system.

Enhancing rhamnolipid production through a two-stage fermentation control strategy based on metabolic engineering and nitrate feeding

Nitrate plays a crucial role in the high-efficient fermentation production of rhamnolipids (RLs). However, the underlying mechanism remains unclear. Firstly, by knocking out the restriction endonuclease PaeKI and utilizatiing the endogenous CRISPR-Cas-mediated single-plasmid recombineering system, a genome editing system for P. aeruginosa KT1115 has been established. Secondly, an engineered strain KT1115ΔpaeKIΔnirS was obtained with a 87% of reduction in nitric oxide (NO) accumulation and a 93% of reduction in RLs production, revealing the crucial role of NO signaling molecule produced from nitrate metabolism in RLs production. Finally, by combining metabolic engineering of the nitrate metabolism pathway with nitrogen feeding, a new two-stage fermentation process was developed. The fermentation production period was reduced from 168 h to 120 h while achieving a high yield of 0.8 g/g, and the average productivity increased by 55%. In all, this study provides a novel insights in the RLs biosynthesis and fermentation control strategy.

Antioxidative Defense, Suppressed Nitric Oxide Accumulation, and Synthesis of Protective Proteins in Roots and Leaves Contribute to the Desiccation Tolerance of the Resurrection Plant Haberlea rhodopensis.

The desiccation tolerance of plants relies on defense mechanisms that enable the protection of macromolecules, biological structures, and metabolism. Although the defense of leaf tissues exposed to solar irradiation is challenging, mechanisms that protect the viability of the roots, yet largely unexplored, are equally important for survival. Although the photosynthetic apparatus in leaves contributes to the generation of oxidative stress under drought stress, we hypothesized that oxidative stress and thus antioxidative defense is also predominant in the roots. Thus, we aimed for a comparative analysis of the protective mechanisms in leaves and roots during the desiccation of Haberlea rhodopensis. Consequently, a high content of non-enzymatic antioxidants and high activity of antioxidant enzymes together with the activation of specific isoenzymes were found in both leaves and roots during the final stages of desiccation of H. rhodopensis. Among others, catalase and glutathione reductase activity showed a similar tendency of changes in roots and leaves, whereas, unlike that in the leaves, superoxide dismutase activity was enhanced under severe but not under medium desiccation in roots. Nitric oxide accumulation in the root tips was found to be sensitive to water restriction but suppressed under severe desiccation. In addition to the antioxidative defense, desiccation induced an enhanced abundance of dehydrins, ELIPs, and sHSP 17.7 in leaves, but this was significantly better in roots. In contrast to leaf cells, starch remained in the cells of the central cylinder of desiccated roots. Taken together, protective compounds and antioxidative defense mechanisms are equally important in protecting the roots to survive desiccation. Since drought-induced damage to the root system fundamentally affects the survival of plants, a better understanding of root desiccation tolerance mechanisms is essential to compensate for the challenges of prolonged dry periods.

Biochemical mechanism of erastin-induced ferroptotic cell death in neuronal cells.

Ferroptosis is a new form of nonapoptotic cell death closely associated with glutathione (GSH) peroxidase 4 inhibition and/or GSH depletion, resulting in the accumulation of cellular iron and lipid peroxides. The exact mechanism by which GSH depletion causes the accumulation of reactive oxygen species (ROS) and lipid-ROS and subsequent ferroptotic cell death in neuronal cells remains unclear. In the present study, using immortalized HT22 mouse hippocampal neuronal cells as a model, we show that nitric oxide (NO) accumulation via protein disulfide isomerase (PDI)-mediated neuronal nitric oxide synthase (nNOS) activation plays a critical role in chemically-induced ferroptosis. Mechanistically, we find that erastin-induced GSH depletion leads to activation of PDI, which then mediates ferroptosis by catalyzing nNOS dimerization, followed by accumulation of cellular NO, ROS and lipid ROS and ultimately ferroptotic cell death. Pharmacological inhibition of PDI enzymatic activity or selective PDI knockdown can effectively abrogate erastin-induced ferroptosis in HT22 cells. The results of this study reveal an important role of PDI in mediating chemically induced ferroptosis in a neuronal cell model, and PDI may serve as a potential drug target for protection against GSH depletion-associated ferroptotic neuronal cell death.

Plasma-Generated Nitric Oxide Water Mediates Environmentally Transmitted Pathogenic Bacterial Inactivation via Intracellular Nitrosative Stress.

Over time, the proportion of resistant bacteria will increase. This is a major concern. Therefore, effective and biocompatible therapeutic strategies against these bacteria are urgently needed. Non-thermal plasma has been exhaustively characterized for its antibacterial activity. This study aims to investigate the inactivation efficiency and mechanisms of plasma-generated nitric oxide water (PG-NOW) on pathogenic water, air, soil, and foodborne Gram-negative and Gram-positive bacteria. Using a colony-forming unit assay, we found that PG-NOW treatment effectively inhibited the growth of bacteria. Moreover, the intracellular nitric oxide (NO) accumulation was evaluated by 4-amino-5-methylamino-2',7'-dichlorofluorescein diacetate (DAF-FM DA) staining. The reduction of viable cells unambiguously indicates the anti-microbial effect of PG-NOW. The soxR and soxS genes are associated with nitrosative stress, and oxyR regulation corresponds to oxidative stress in bacterial cells. To support the nitrosative effect mediated by PG-NOW, we have further assessed the soxRS and oxyR gene expressions after treatment. Accordingly, soxRS expression was enhanced, whereas the oxyR expression was decreased following PG-NOW treatment. The disruption of cell morphology was observed using scanning electron microscopy (SEM) analysis. In conclusion, our findings furnish evidence of an initiation point for the further progress and development of PG-NOW-based antibacterial treatments.

Absence of Arabidopsis Polyamine Oxidase 5 Influences the Cytokinin-Induced Shoot Meristem Formation from Lateral Root Primordia.

Lateral root primordia (LRPs) of Arabidopsis can be directly converted to shoot meristems (SMs) by the application of exogenous cytokinin. Here, we report that Arabidopsis POLYAMINE OXIDASE 5 (AtPAO5) contributes to this process, since the rate of SM formation from LRPs was significantly lower in the pao5-2 knockout mutant. Furthermore, the presented experiments showed that AtPAO5 influences SM formation via controlling the thermospermine (T-Spm) level. Gene expression analyses supported the view that the pao5-2 mutation as well as exogenous T-Spm downregulate the expression of the class 3 haemoglobin coding genes AtGLB1 and AtGLB2. AtGLB1 and 2 have been reported to augment cytokinin sensitivity, indirectly inhibiting the expression of type-A ARABIDOPSIS RESPONSE REGULATORs (ARRs). In agreement, the same ARR-coding genes were found to be upregulated in the pao5-2 mutant. Although GLB proteins might also control cytokinin-induced nitric oxide (NO) accumulation, we could not find experimental evidence for it. Rather, the negative effect of NO-donor treatment on AtPAO5 gene expression and SM formation was seen. Nevertheless, a hypothetical pathway is set up explaining how AtPAO5 may affect direct shoot meristem formation, controlling cytokinin sensitivity through T-Spm and GLBs.

Specificity in root domain accumulation of Phytoglobin1 and nitric oxide (NO) determines meristematic viability in water-stressed Brassica napus roots.

Drought reduces plant productivity, especially in the susceptible species Brassica napus. Water stress, mimicked by applications of 10 % polyethylene glycol (PEG), elevates nitric oxide (NO) in root cells after a few hours, contributing to degradation of the root apical meristems (RAMs), the function of which relies on auxin and brassinosteroids (BRs). Phytoglobins (Pgbs) are effective NO scavengers induced by this stress. This study examines the effects of BnPgb1 dysregulation in dehydrating B. napus roots, and the spatiotemporal relationship between Pgb1 and activities of auxin and BRs in the regulation of the RAM. Brassica napus lines over-expressing [BnPgb1(S)] or down-regulating [BnPgb1(RNAi)] BnPgb1 were exposed to PEG-induced water stress. The localization of BnPgb1, NO, auxin and PIN1 were analysed during the first 48 h, while the expression level of biosynthetic auxin and BR genes was measured during the first 24 h. Pharmacological treatments were conducted to assess the requirement of auxin and BR in dehydrating roots. During PEG stress, BnPgb1 protein accumulated preferentially in the peripheral domains of the root elongation zone, exposing the meristem to NO, which inhibits polar auxin transport (PAT), probably by interfering with PIN1 localization and the synthesis of auxin. Diminished auxin at the root tip depressed the synthesis of BR and caused the degradation of the RAMs. The strength of BnPgb1 signal in the elongation zone was increased in BnPgb1(S) roots, where NO was confined to the most apical cells. Consequently, PAT and auxin synthesis were retained, and the definition of RAMs was maintained. Auxin preservation of the RAM required BRs, although BRs alone was not sufficient to fully rescue drought-damaged RAMs in auxin-depleted environments. The tissue-specific localization of BnPgb1 and NO determine B. napus root responses to water stress. A model is proposed in which auxin and BRs act as downstream components of BnPgb1 signalling in the preservation of RAMs in dehydrating roots.