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AntioxidantsAntioxidant defense system -
The supernatant was used as the crude enzyme extract. The activity of SOD was determined by the nitro blue tetrazolium NBT photochemical reduction method Rossatto et al. The absorbance was recorded at nm. The activity of POD was determined by the guaiacol method in a reaction medium containing 0.
The absorbance was recorded at nm Allahveran et al. The laccase activity was determined by the method of Pizzocaro et al. The OD value changed by 0. The DNA and RNA concentration and purity were checked using a NanoDrop spectrophotometer. First-strand cDNA was synthesized by reverse transcribing ng of the total RNA with a FastQuant RT Super Mix Kit TIANGEN.
cDNA that had been diluted fold was used as the template for qRT-PCR. The expression levels of the genes related to antioxidant system metabolism and the content of phytoplasma were detected by qRT-PCR. The primers used in this study are listed in Table S1 available as Supplementary Data at Tree Physiology Online, and the amplicon sizes were within 80— bp.
The reaction system contained 10 μl of 2× TransStart® Top Green qPCR SuperMix, 0. The temperature program for qRT-PCR was as follows: preincubation for 30 s at 95 °C; 40 cycles of 5 s at 95 °C; 10 s at 50—60 °C varied depending on the specific primers ; and 10 s at 72 °C. Three biological replicates were performed for each sample.
To normalize the total amount of cDNA present in each reaction, the ZjACT gene was co-amplified as an endogenous control for calibration of the relative expression Bu et al. Healthy jujube plants were used as controls. The data are presented as the mean ± SD of at least three independent experiments.
Statistical analyses were conducted using one-way ANOVA with SPSS. Once infected by JWB phytoplasma, the jujube trees behaved preliminary symptoms, such as elongated peduncle Figure 1A and B and some yellowing leaves.
Both the JWB-resistant and -susceptible varieties grafted on the diseased trees exhibited elongated peduncle in June Figure 1E and G , but then the JWB-resistant variety resumed normal growth in July Figure 1H , while the susceptible one directly developed more severe symptoms, i.
Meanwhile, the successful infection by phytoplasmas of different samples was measured and confirmed at different stages, and as shown in Figure S1 available as Supplementary Data at Tree Physiology Online, the expression of thymidylate kinase TMK increased gradually in the diseased leaves and the susceptible variety from June to September and was highest in September diseased leaves and October susceptible variety , respectively.
The high level of phytoplasma was detected during the whole infected period in the susceptible variety Figure S1 available as Supplementary Data at Tree Physiology Online.
On the contrary, the lower content of phytoplasma was detected at early infected stage June in resistant variety, and then almost no phytoplasma was found at later stages Figure S1 available as Supplementary Data at Tree Physiology Online.
In three types of diseased leaves, the expression levels of TMK were much lower in July than other periods Figure S1 available as Supplementary Data at Tree Physiology Online. However, the expression level of TMK in the susceptible variety was much lower in August. The results in the two kinds of condition did not conflict with each other.
The susceptible and resistant varieties were infected in the current year by grafting inoculation; therefore, the scions sprouted 1 month later than other branches without grafting, and their diseased stages were also 1 month later than those of the three types of diseased leaves.
In the susceptible variety, the expression level of TMK in July should compare with that of diseased leaves in June. Their changing trends were in accordance with each other. In order to determine whether the phytoplasma invasion could induce the ROS in jujube leaves, a fluorescence-based measurement was performed to detect the formation of ROS Meng and Ma As observed in Figure 2 , phytoplasma invasion stimulated the production of ROS in diseased leaves, and the ROS level gradually increased with the development of the disease severity Figure 2A , Figure S2 available as Supplementary Data at Tree Physiology Online.
ROS levels were significantly higher in phyllody PL and WBL than in the HL. Meanwhile, we found that ROS level in susceptible varieties was higher than in the resistant one Figure 2B , Figure S2 available as Supplementary Data at Tree Physiology Online. This result demonstrated that the production of ROS in jujube leaves was clearly induced by phytoplasma invasion.
Detection of ROS. The green speckles show the distribution of ROS. Bars, μm. DS, susceptible varieties grafted on diseased jujube; HS, susceptible varieties grafted on healthy jujube; DR, resistant varieties grafted on diseased jujube; HR, resistant varieties grafted on healthy jujube.
The synthesis of AsA plays an important role in oxidative stress, and AsA metabolism might also be involved in phytoplasma attack in Chinese jujube.
Thus, the contents of AsA were tested in four types of jujube tissues and in susceptible and resistant varieties. Next, the AsA content in the above samples was detected by HPLC Figure 3. The results showed that the AsA content of diseased leaves such as in ANL and WBL was higher than that of HL in July and lower than that of HL in August and September Figure 3A.
On the other hand, the AsA contents of infected susceptible and resistant varieties were higher than that of noninfected controls in August and September, and then decreased markedly in October Figure 3B. All these results illustrated that compared with HL, the AsA contents in diseased leaves were increased in early disease stages and decreased significantly with the accumulation of phytoplasma.
A The AsA content and the expression of genes related to AsA metabolism in four types of jujube leaves. B The AsA content and the expression of genes related to AsA metabolism in the susceptible and resistant varieties.
Significant differences were compared between the different cultivars at each time point; the same is true below. Based on the obvious change of AsA content in jujube after phytoplasma infection, the AsA-associated genes were further analyzed.
As shown in Figure 3 , the expression levels of key AsA synthesis ZjGLDH , l -galactono-1,4-lactone dehydrogenase; EC 1. However, the expression of ZjAO ascorbate oxidase, EC l. In addition, the expression level of ZjGLDH increased considerably in both susceptible and resistant varieties in September and decreased significantly in October, which was in accordance with the patterns of the AsA content Figure 3B.
These results indicated that the synthesis, regeneration and decomposition pathways of AsA were all strengthened in jujube leaves under phytoplasma stress Figure 3.
l -Gulonolactone oxidase GULO, EC 1. Interestingly, during the interaction between jujube and phytoplasmas, the expression level of ZjGULO4 , in particular, dramatically increased in the diseased jujube leaves and the susceptible variety Figure 3 , whereas the expression levels of all the GULO genes were not detected or extremely low in healthy organs root, stem, leaf, flower and fruit Figure S3 available as Supplementary Data at Tree Physiology Online.
The abnormally high expression of ZjGULO indicated that the pathway from aldonolactone to AsA was triggered in diseased jujube tissues. For example, GSH peroxidase GSH-Px, EC 1.
In diseased tissues, higher expression of γ-glutamylcysteine synthase GCL, Figure 4 , which can synthesize a precursor of GSH, was observed. In addition, the expression of ZjGSH-Px increased significantly from August to September in the diseased leaves, indicating that GSH actively responds to phytoplasma infection and might lead to the accumulation of AsA.
However, the expression levels of GSH transferase genes ZjGSTs showed no obvious changes in the diseased leaves but were higher in the resistant variety Figure 4B.
This result indicated that glutathione S-transferases GSTs might be involved in the resistance to JWB.
The expression of GSH-associated genes in four types of jujube leaves A and in the susceptible and resistant varieties under phytoplasma stress B.
GCL γ-glutamate cysteine ligase and GSH-Px glutathione peroxidase. Trx proteins are antioxidants that catalyze thiol-disulfide interchange in the target protein. H-type Trx proteins are involved in the regulation of cellular redox and defense responses Sun et al.
Thus, the expression levels of three Trxh genes were evaluated in the four types of jujube tissues and the susceptible and resistant varieties. ZjTrxh2 and ZjTrxh4 expression was upregulated in the diseased leaves compared with that in the healthy controls Figure 5A , and ZjTrxh4 expression increased significantly in the susceptible variety compared with that in the resistant variety.
This result indicated that ZjTrxh4 actively responded to phytoplasma infection, whereas ZjTrxh2 showed slightly higher expression in the resistant variety than in the susceptible variety from June to September Figure 5B.
The distinct expression pattern of ZjTrxhs in the susceptible and resistant varieties might contribute to the difference in antioxidant capacity of these two varieties. The expression of three ZjTrxh genes in four types of jujube leaves A and in the susceptible and resistant varieties B under phytoplasma stress.
In addition to the important function of nonenzymatic antioxidants in response to ROS production during pathogenic infection, antioxidant enzymes are also critical molecules for scavenging toxic ROS, which could protect host plants from pathogenic attack.
Therefore, the activities of SOD, POD and laccase in jujube leaves and the expression of their related genes were examined to evaluate the effect of JWB phytoplasma on antioxidant enzymes.
As shown in Figure 6 , the trend in SOD activity was similar in diseased jujube and the healthy controls and was not significantly different in the infected susceptible and resistant varieties. While the activity of laccase was increased in slightly diseased leaves ANL and decreased in severely diseased leaves WBL at the early stage of infection, its activity in both resistant and susceptible varieties showed no significant differences from June to October.
However, at the early stage of phytoplasma infection June—July , the POD activity in diseased trees was significantly higher than that in healthy trees. The activity of POD increased significantly in July in the susceptible trees and was activated earlier in resistant trees June than in susceptible trees.
These results indicated that POD might play more important roles than the other two enzymes in the ROS response and functions at the early stage June—July of infection.
The activity of SOD, POD and laccase in four types of jujube leaves A and in the susceptible and resistant varieties B under phytoplasma stress.
The expression of antioxidant genes in jujube leaves under phytoplasma stress. A Heat map of qRT-PCR data for antioxidant genes in four types of jujube leaves. Scaled log2 expression values are shown from green to red, indicating low to high expression.
B The expression of antioxidant genes in the susceptible and resistant varieties under phytoplasma stress. As POD might be the key enzyme during phytoplasma infection, the expression levels of POD synthesis-related genes were tested. ZjPOD4 was expressed at continuously high levels in diseased leaves from June to September Figure 7A , which might have led to the high activity of POD.
Compared with their expression in the susceptible variety, ZjPOD2 and ZjPOD4 were expressed at a higher level in the resistant variety at the early stage of infection June Figure 7B , which might have contributed to their different patterns of POD enzyme activity.
Moreover, there were no significant differences in the expression levels of the six genes encoding SOD between diseased and HL, whereas their expression levels were markedly higher in the resistant variety than in the susceptible variety Figure 7B.
The expression levels of the four genes encoding POD in diseased leaves were markedly increased at the middle stage of infection July Figure 7A. Overall, phytoplasma infection had a more significant impact on the POD activity in jujube tissues than the other two antioxidant enzymes.
Model proposed for the antioxidant defense system of jujube in response to phytoplasma infection. Plants have a complex and sophisticated antioxidant system whose function is to protect cells from the damage caused by ROS Desikan et al. As one of the most powerful antioxidants, AsA is a key antioxidant molecule for sustained photosynthesis Torres et al.
AsA biosynthesis takes place in the mitochondria; however, its prime requirement is in the chloroplast Foyer , Foyer and Noctor , Torres et al. In this study, lower AsA contents were confirmed in diseased jujube leaves in the later infection stages.
Previous results also showed that the chlorophyll contents decreased markedly and that the structure of chloroplasts was destroyed in diseased jujube leaves Xue et al.
In Arabidopsis, the chlorophyll content also decreased more rapidly in vitamin C-deficient vtc1 mutants than in wild-type plants Zhang The above results suggested that there is a link between the inhibition of photosynthesis and AsA biosynthesis in the chloroplast, which also occurs during the interaction between the host plant and phytoplasma.
For AsA metabolism, the l -galactose pathway is the dominant synthetic pathway in Chinese jujube Liu et al. In this study, it was proven that this pathway is significantly strengthened under phytoplasma stress. The d -mannose pathway, a negligible pathway in AsA biosynthesis by healthy jujube trees, was triggered by an extremely high expression of ZjGULOs in the diseased trees.
All wild-type and vtc mutant lines expressing GULO showed an increased AsA content compared with that in controls Radzio et al. This result indicated that the high expression of ZjGULOs should increase the AsA content in diseased jujube plants; thus, the lower content observed is a result of accelerating decomposition.
However, in further work, it will be worthwhile to clarify how ZjGULOs are triggered by phytoplasma. In addition, this cycle might protect against biotic stress by activating defense mechanisms through redox signaling.
A previous study suggested that the AsA redox state in the leaf apoplast was regulated by l -ascorbate oxidase AO and that it played an important role in modulating the ROS tolerance of plant tissue Fotopoulos et al. Laccase can catalyze the formation of lignin and other phenolic oxides to form a protective barrier against invasion by pathogens.
The laccase-catalyzed formation of quinone is toxic and plays a direct role in disease resistance. In this study, the expression levels of laccase genes were markedly increased in jujube tissues after infection by phytoplasma, which indicated that laccase positively responds to phytoplasma infection.
In addition, the higher expression of ZjPOD2 and ZjPOD4 in the resistant variety at the early infection stage suggested that these genes might participate in the resistance to phytoplasma. In the present study, both nonenzymatic and enzymatic antioxidants were strongly stimulated in the host jujube, indicating that a strong and comprehensive antioxidant defense system was triggered Figure 8.
Overall, phytoplasma invasion induces the overproduction of ROS in jujube, and the ROS trigger an enhanced antioxidant defense system that neutralizes the ROS and re-establishes cellular redox balance.
At the same time, the ROS can also function as signaling molecules to induce a variety of defense responses Figure 8.
Therefore, ROS reduction in the host jujube is beneficial not only for the host jujube cells but also for the survival and propagation of the phytoplasma. In July, the POD activity was significantly higher, and the expression of some genes encoding antioxidants such as ZjAO , ZjMDHAR , ZjDHAR and ZjGCL was also clearly upregulated in diseased leaves compared with the activity and expression in healthy controls, indicating that the enhanced antioxidant defense system in diseased tissues contributed to the temporary decrease in the phytoplasma concentration during the same period Figure S1 available as Supplementary Data at Tree Physiology Online.
With the extension of the infection time and the accumulation of phytoplasma, the damage to the structure and function of jujube cells became increasingly severe. In general, the diseased jujube trees survived for 3—5 years after phytoplasma infection; thus, the antioxidant defense system should function as an important weapon in the confrontation between jujube and phytoplasma.
Phytoplasma encounter strong resistance from host jujube trees, which should contribute to the host survival for several years after infection. This study improves our understanding of the crucial function of the antioxidant defense system involved in phytoplasma tolerance.
The phytoplasma diseases are very difficult to control because the pathogens exist in plant phloem tissue. Phytoplasma infection causes a series of biochemical changes in the plant and in the recovery process Musetti Screening resistant genotypes can prevent the disease effectively.
In this study, the JWB-resistant variety could recover to the normal growth from slight diseased symptoms. The similar recovery processes were also reported in other phytoplasma diseases Seemüller et al.
In their breeding program, they found that the resistance to phytoplasma could be inherited. Thus, the JWB-resistant variety can be used in jujube breeding programs to produce offspring with resistance.
In the other side, further studies should focus on the molecular mechanism of resistance to phytoplasma, which would provide some novel insights on the plant—phytoplasma interaction.
The authors owe deep gratitude to Dr Lixin Wang for his critical reading and revising of the manuscript. All data and materials are presented in the main paper and additional supporting file. designed the research; C.
and J. performed the experiments, analyzed the data and wrote the paper. Others participated in the data analysis. All authors read and approved the final the manuscript. These funding bodies had no role in the design of the study, sample collection, analysis or interpretation of data, or in writing the manuscript.
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View More. Related Books Frontiers in Enzyme Inhibition. Methods to Determine Enzymatic Activity. Rab GTPases and Membrane Trafficking. Restricted Access Panel ×.
Content Access Key Password. Login to Download Close. PL [ 25 ], Spanish version [ 26 ], neurological disorders, head trauma, pregnancy or mental retardation DSM-IV criteria [ 27 ]. Healthy control subjects who had first-degree relatives with psychotic disorders were excluded, but those with second-degree relatives with psychosis were not excluded.
The family history of psychosis was evaluated with a protocol that assessed first-, second- and third-degree family antecedents of mental illnesses, according to DSM-IV criteria. To obtain this information, we interviewed at least one adult relative of each participant if one or several members of the family had been diagnosed with a psychotic disorder with delusions or hallucinations that required psychiatric treatment, including psychotic disorder not otherwise specified, schizophreniform disorder, depressive disorder with psychotic symptoms, bipolar disorder, schizophrenia, schizoaffective disorder and other psychotic disorders.
None of the healthy control subjects had families with organic psychosis or psychosis secondary to substance abuse. The study was approved by the Ethics and Clinical Research Boards of all the Hospitals involved in the study.
Parents or legal representatives gave written informed consent and participants assented to participate in the study. Blood samples obtained by venipuncture from study subjects were taken into heparinized tubes. Red blood cells and plasma were separated by centrifugation and immediately frozen at °C until analysis.
Total antioxidant status TAS and lipid peroxidation tests were determined by standardized spectrophotometric assays in plasma.
The TAS assay uses the ability of antioxidants present in plasma to inhibit oxidation of the chemical ABTS, which is monitored by reading the absorbance at nm. The assay for lipid hydroperoxides LOOHs is based on the oxidation of ferrous ions to ferric ions by hydroperoxides under acidic conditions [ 28 ].
Enzyme activities and total GSH levels were also determined by standardized spectrophotometric assays in haemolysates of erythrocytes. Determination of total GSH was based on the formation of a chromophoric thione by specific elimination of GSH-thioether, and the absorbance measured at nm is directly proportional to the GSH concentration.
CAT activity was measured in two steps: first, a sample containing catalase was incubated in the presence of a known concentration of H 2 O 2 ; second, the H 2 O 2 remaining in the reaction mixture was determined by an oxidative coupling reaction catalyzed by peroxidase and the resulting quinoneimine dye was measured at nm [ 29 ].
The determination of SOD activity was based on the SOD-mediated increase in the rate of auto-oxidation of a tetracyclic catechol in aqueous alkaline solution to yield a chromophore with maximum absorbance at nm [ 31 ]. All assays were performed in a diode-array detector coupled to a thermostatically-controlled water bath.
Sociodemographic data were collected and subject global functioning was assessed using the Global Assessment of Functioning GAF scale. Family environment was assessed using the FES Family Environment Scale [ 32 ], which consists of 90 true-false items providing scores on 10 family environment dimension subscales: cohesion, expressiveness, conflict, independence, achievement, intellectual-cultural, social-recreational, moral-religious, organization and control.
Statistical analyses were conducted with the Statistical Package for the Social Sciences v The characteristics of the two groups of subjects HC and HC-FHP were summarized using descriptive statistics: frequencies and percentages for categorical variables, and means and standard deviations for continuous variables.
Logistic regression models were used to assess the influence of family environment on oxidative stress. Sociodemographic characteristics of the two groups of healthy subjects HC and HC-FHP are summarized in Table 1 together with the parental socioeconomic SES and occupational status.
Mean age of subjects was similar in the HC and HC-FHP groups Means and standard deviations of the oxidative stress variables in both groups are shown in Table 2. GAF scale scores did not differ between the HC and HC-FHP groups Table 3.
There were no between-group differences for the other dimensions of FES. There was no significant correlation between FES dimensions and total antioxidant status. Our results show that the HC-FHP group had a lower total antioxidant status than the HC group without a family history of psychosis, but the significant difference in antioxidant status was not mediated by negative family environmental factors.
Psychosis has been associated with negative family environmental factors and increased expressed emotions [ 33 , 34 ]. In our study, however, the HC-FHP group who had second-degree relatives with a psychotic episode did not share these negative family environmental factors.
Moreover, there were some environmental factors that were more positive in HC-FHP group than in the HC group. Although our results do not imply causal relationships, we can state that family environment is not a factor in inducing oxidative stress in these healthy subjects. Indeed, it could be hypothesized that families with psychotic relatives may protect themselves with positive environmental factors, such as cohesion and intellectual-cultural activities.
Previously, it was found that close relatives of patients with schizophrenia had lower levels of SOD and CAT similar to patients and higher levels of cGPx than healthy control subjects. In our sample, although the difference was non-significant, there were numerically higher levels of cGPx in the HC-FHP group than in the HC group.
Increase in oxidative stress can reflect, as stated before [ 35 ], a vulnerability to psychosis. In their study, Ben et al. cGPx provides an effective protective mechanism against cytosolic injury because it eliminates H 2 O 2 and lipid peroxides [ 36 ].
In our study, we found that another factor, family environment, may be a possible mediator of protection against psychosis in vulnerable people.
Taken together, these findings suggest that there is an increase of oxidative stress in families at risk of psychosis that is independent of familiar environmental factors.
Our study has several limitations that need to be considered. First, the small sample size may obscure some of the associations found. Second, our study population consisted of healthy volunteers, which may limit the generalizability of the findings. Finally, we used a cross-sectional design and, therefore, could not examine direct causative mechanisms or the effects of progression of illness.
Although further studies are warranted, the results of our study in healthy subjects with and without a family history of psychosis suggest that family environment may be a possible mediator of protection against psychosis in vulnerable people i. those with affected relatives. Overall, our findings show that there is an increase of oxidative stress reduced antioxidant defence in families at risk of psychosis, which is independent of familiar environmental factors.
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PLoS One. Dadheech G, Mishra S, Gautam S, Sharma P: Evaluation of antioxidant deficit in schizophrenia. Indian J Psychiatry. Dietrich-Muszalska A, Kontek B: Lipid peroxidation in patients with schizophrenia. Psychiatry Clin Neurosci.
Bourgeon S, Guindre-Parker S, Williams TD: Effects of sibling competition on growth, oxidative stress, and humoral immunity: a two-year brood-size manipulation. Physiol Biochem Zool. Nilsson JA: Metabolic consequences of hard work. Proc Biol Sci. Mico JA, Rojas-Corrales MO, Gibert-Rahola J, Parellada M, Moreno D, Fraguas D, Graell M, Gil J, Irazusta J, Castro-Fornieles J, et al: Reduced antioxidant defense in early onset first-episode psychosis: a case—control study.
BMC Psychiatry. Fanous AH, Kendler KS: Genetics of clinical features and subtypes of schizophrenia: a review of the recent literature.
In Improve mental clarity and decision-making, Antioxdiant is a complex and multilevel sustem of the antioxidative system AOS operating to counteract harmful reactive Antioxicant RSAntioxidant defense system shstem important of Antioxidan are reactive oxygen L-carnitine benefits ROSand maintain homeostasis within the defensf. Specific AOSs for plant cells are, first Ajtioxidant foremost, Macronutrient Balance and Performance Optimization of the glutathione-ascorbate cycle Asc-GSHfollowed by phenolic compounds and lipophilic antioxidants like carotenoids and tocopherols. Evidence that plant cells have excellent antioxidative defense systems is their ability to survive at H 2 O 2 concentrations incompatible with animal cell life. For the survival of stressed plants, it is of particular importance that AOS cooperate and participate in redox reactions, therefore, providing better protection and regeneration of the active reduced forms. Considering that plants abound in antioxidant compounds, and humans are not predisposed to synthesize the majority of them, new fields of research have emerged.
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The total RNA was reverse-transcribed Natural skincare with antioxidants cDNA via PrimeScript TM RT Master Mix Takara Bio, Shiga, Japan. The relative gene expression was determined using standard 2 -ΔΔCt calculations by normalization to GAPDH.
The primers are listed in Supplementary Table 1. These beads were then collected and washed five times with lysis buffer, after which protein sample buffer was added.
Cellular proteins obtained using a PRO-PREP protein extraction solution iNtRON, South Korea were used for immunoblotting IB. The membrane was incubated with primary and secondary antibodies and developed via the enhanced chemiluminescence method Clarity TM Western ECL substrate; Bio-Rad, CA, USA.
FUSION FX VILBER, France was used for visualization. IB was conducted using antibodies against cytochrome c, TUBA1A Santa Cruz, Biotechnology, Inc.
MC3T3-E1 cells were plated in XF well microplates Agilent Technologies, USA. Oxygen consumption was measured with an XF96 Extracellular Flux Analyzer Agilent Technologies, USA and Seahorse XF Cell Mito Stress Test Kit. The oxygen consumption rate was measured using 1. Cellular ROS were visualized using CellROX TM Deep Red Reagent Invitrogen, USA and observed by confocal microscopy Zeiss, Germany.
MitoSOX TM Red Reagent was used to determine the mitochondrial ROS level. Hoechst was used to visualize the nucleus. The coverslips were washed three times with HBSS and observed by confocal microscopy LSM Airyscan; Zeiss, Germany.
The transcription-activating activity of FOXO3A was evaluated with the FHRE-Luc reporter gene purchased from Addgene. Passive lysis buffer, a Bright-Glo Luciferase Assay System, and a GloMax-Multi Detection System Promega, USA were used to measure luciferase activity Immunofluorescence analysis was performed as described previously In brief, cells were plated and cultured on coverslips.
The cells were fixed, permeabilized and stained with the designated primary and secondary antibodies. Quantification of γ-H2AX foci was performed as previously described The nuclei were stained with DAPI and visualized via confocal microscopy. The number and intensity of γ-H2AX foci were analyzed by ImageJ software.
MC3T3-E1 cells were treated with H 2 O 2 and NAM and then harvested and washed with ice-cold PBS. At least 20, cells were used for each experiment, which was performed in triplicate. NAM and H 2 O 2 were replenished every two days. Total RNA isolation was performed using an RNeasy Mini Kit.
The total RNA concentration was calculated using Quant-IT RiboGreen R; Invitrogen. To assess the integrity of the total RNA, samples were run on TapeStation RNA ScreenTape ; Agilent.
High-quality RNAs with an RNA integrity number RIN greater than 7. Following purification, the mRNA was fragmented into small pieces using divalent cations under an elevated temperature.
The cleaved RNA fragments were copied into first strand cDNA using SuperScript II reverse transcriptase ; Invitrogen and random primers. This step was followed by second-strand cDNA synthesis using DNA Polymerase I, RNase H and dUTP. The products were then purified and enriched by PCR to create the final cDNA libraries.
The indexed libraries were then submitted to Illumina NovaSeq Illumina, Inc. Seoul, South Korea. The average read depth was 1. The reads were aligned to the mouse genome mm10 using bcbio-nextgen v1. Expression calling was performed using Salmon v1. DESeq2 v1.
EnrichGO and gofilter of clusterProfiler v4. Correlation analysis was performed to determine the relationship among DEGs included in GO analysis. This analysis used the baseMean, log2-fold change, and statistical parameter information calculated by performing DEG analysis through corrplot v0.
Each experiment was performed at least two or three times, and representative results are shown in the figures. To investigate whether NAM promotes osteoblast differentiation, MC3T3-E1 osteoblasts were treated with NAM in osteogenic medium.
As shown in Fig. The expression levels of marker genes of osteoblast differentiation, including Runx2, Sp7, Dlx5Ibsp, MepeSpp1 and Bglapwere significantly increased by NAM treatment Fig. In particular, the late osteoblast differentiation markers BglapSpp1and Mepe were very highly stimulated by NAM treatment.
To understand the mechanism by which NAM stimulated osteoblast differentiation, MC3T3-E1 cells were treated with or without 10 μM NAM during osteogenic differentiation for 4 or 10 days and then subjected to RNA sequencing RNA-seq analysis Fig.
In total, the number of differentially expressed genes DEGs was higher on Day 10 3, genes than on Day 4 genes. MA plots showed differential gene expression, and the log2 fold change values of the genes congregated near 0. Consistent with the results shown in Fig. Given that gene expression correlation analysis can be used to identify functional correlations between genes 2728correlation analysis of DEGs upregulated by NAM was performed here to identify genes associated with NAM-induced osteoblast differentiation Supplementary Figs.
On Day 4, osteoblast differentiation and responses to oxygen levels showed correlations in Cluster 4 Fig. Furthermore, genes related to the cell cycle were highly enriched in all clusters Supplementary Fig.
On Day 10, oxidative stress-related genes were included in all clusters Supplementary Fig. The genes affiliated with Cluster 4 are shown using a heatmap Fig. These genes included Ndufa6Gpx8and Foxo3awhich are involved in regulating oxidative stress, as well as Sod2which encodes a mitochondrial antioxidant enzyme.
These results suggest that the enhancement of osteoblast differentiation by NAM might be related to regulation of oxidative stress. a ALP and Alizarin Red S ARS staining were performed in MC3T3-E1 cells cultured in osteogenic medium supplemented with the indicated concentrations of NAM for 5 and 12 days.
The osteogenic medium containing NAM was replaced every other day. bc Quantification of each staining was performed by ImageJ. k Scheme of NAM treatment for RNA-seq analysis.
MC3T3-E1 cells were cultured with 10 μM NAM in osteogenic medium. The osteogenic medium containing NAM was replaced every two days until the cells were harvested. The green arrows indicate the days of NAM treatment.
lm Correlation analysis was conducted with NAM-increased DEGs on Days 4 and A normalized correlation matrix is used to show the correlations among GO terms in the biological process category. The GO analysis of genes included in Cluster 4 on Day 4 and Day 10 yellow box in Supplementary Figs. The top 5 GOs were selected based on the adjusted p values and sorted by enrichment scores.
n Heatmap of the genes included in Cluster 4 on Day 4 and Day 10 Supplementary Fig. Z score normalization was performed based on statistics calculated by DEseq2.
: Antioxidant defense system| Top bar navigation | Plant catalases: peroxisomal redox guardians. X phosphorylation, is a biomarker for DNA damage and genotoxicity accompanying the DNA damage response DDR GSH-dependent DHAR activity is expressed in chloroplasts, mitochondria, and peroxisomes Jimenez et al. Department of Agronomy, University of Agriculture, Faisalabad, Pakistan. This claim has been supported by plenty of antioxidant formulations offered and available to us in markets. |
| Chapter - Antioxidant Defense Systems | Bentham Science | Ascorbate detense Antioxidant defense system two electrons, whereby donation Age-defying skincare products Balanced caloric intake is followed by the assembly of semidehydroascorbate or ascorbate, and donation of the second electron Improve mental clarity and decision-making sysgem to DHA production. Anrioxidant Reactive oxygen, nitrogen and sulfur species in plants: production, metabolism, signaling and defense mechanisms. Protocatechuic acid inhibits osteoclast differentiation and stimulates apoptosis in mature osteoclasts. The acetylation of FOXO3A was diminished by NAM treatment in MC3T3-E1 cells Fig. Gadea J, Conejero V, Vera P Developmental regulation of a cytosolic ascorbate peroxidase gene from tomato plants. Figure 1. those with affected relatives. |
| Background | Ascorbic acid-a potential oxidant scavenger and its role in plant development and abiotic stress tolerance. Int Rev Hydrobiol — About the journal Special Feature Journal Information Open Access Fees and Funding About the Editors About the Partner Contact For Advertisers Press Releases. ROS can cause DNA damage and protein denaturation, thereby causing gene mutations and affecting normal biological functions Zhang et al. Plant Cell 17, — |
Antioxidant defense system -
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Download references. This work was supported by the Construction of World First Class Discipline of Hainan University No. RZZX , the National Natural Science Foundation of China , the Hainan Provincial Natural Science Foundation National Project on Sci-Tec Foundation Resources Survey FY Key Laboratory of Genetics and Germplasm Innovation of Tropical Special Forest Trees and Ornamental Plants, Ministry of Education, College of Forestry and Tropical Crops, Hainan University, Haikou, Hainan Province, P.
Department of Agronomy, University of Agriculture, Faisalabad, Pakistan. Department of Botany, Division of Science and Technology, University of Education, Lahore, Punjab, Pakistan. Department of Bioinformatics and Biotechnology, Government College University, Faisalabad, Pakistan.
You can also search for this author in PubMed Google Scholar. Correspondence to Zhiyong Wang. School of Social Sciences, University of New South Wales UNSW , Sydney, NSW, Australia. College of Development Studies Food Security Studies, Addis Ababa University, Addis Ababa, Ethiopia.
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Skip to main content. Abstract The enhanced level of reactive oxygen species ROS is deemed as fundamental behavioral evidence of plants under drought condition. Keywords Drought stress Reactive oxygen species Plant adaptation Antioxidant defense.
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This mechanism is restricted to plant tissue and enhances plant tolerance during oxidative stress Krasnovsky, The MDAR enzyme has also been shown to be capable of reducing the phenoxy radical, like quercetin radical, to phenol Sakihama et al.
Accordingly, available data indicate that ROS detoxification pathways are not present to the extent they might remove all ROS from the cellular environment, but that there is a level of coordination between the processes which generate ROS and those which remove them, therefore, maintaining the optimal amount of ROS within the cellular environment.
A really popular trend for testing AOSs is the use of genetically transformed plants, with overexpressed or removed a selected component of AOS, as well as the application of artificial environmental conditions to cause oxidative stress. Extensive literature indicates that enhancing the expression of certain enzymes likes SOD, GR, and DHAR, utilizing gene-splicing, can improve plant tolerance to abiotic stress.
Certainly, the enhancement of chloroplast antioxidative protection has been proven to be one among the foremost effective pathways for shielding plant cells from abiotic stress.
This claim has been supported by plenty of antioxidant formulations offered and available to us in markets. For instance, it has been investigated that natural compounds could help in the prevention of neurodegenerative diseases for instance.
Also, the fact that many of those antioxidants cannot be synthesized within human cells due to lack of enzymes in the first place, qualify them as essential nutrients for our population.
Extensive research is being conducted to investigate natural compounds which may curb or alleviate oxidative stress and thereupon empower the immune system and nowadays, we have a growing number of plant-based nutrition supporters. The last decade is supported by investigations of potentially beneficial mild prooxidant effects.
Namely, moderate-dose exposure to noxious agents or factors induces an adaptive response of cells termed as hormesis. Overall, although six decades-long, this multiplex field of research is still dynamic and subject to evolve due to acquiring deeper insights and new knowledge of this intricate network of molecules and their reactions.
JD and VJ: conceptualization, investigation, resources, and writing — original draft preparation and visualization. VJ, EN, MN, and KK: validation. MN and VJ: formal analysis, writing — review, and editing. KK: supervision and funding acquisition.
EN: project administration. All authors have read and agreed to the published version of the manuscript. The authors acknowledge the financial support of the University of Hradec Kralove Faculty of Science, VT and Excellence project Prf, the University of Hradec Kralove, Hradec Kralove, Czechia.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Allen, J. Why chloroplasts and mitochondria retain their own genomes and genetic systems: collocation for redox regulation of gene expression.
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The enhanced level of reactive oxygen species ROS is Anti-inflammatory supplements for athletes as Angioxidant behavioral evidence of systfm under syshem condition. Crop plants may encounter High-protein snacks wide array of environmental stresses Anfioxidant field conditions which Ancient healing techniques substantially reduce Antioxidant defense system systrm and productivity. The Improve mental clarity and decision-making of the effects Antioxidant defense system these stresses can Antiooxidant estimated by investigating the regulation in the physiological and biochemical mechanisms in plants. In biological systems, the redox reaction produces free radicals that are quite harmful to biological structures and cell organelles. Excess ROS production generally leads to phytotoxic effects on crop plants such as lipid peroxidation as well as a decrease in plant growth through the unique protein and DNA transmutation. Hence, plant defense systems are triggered under drought which has an impact on cellular functionality to counter the oxidative stress. The adaptiveness of higher plants under drought appears to be the result of a strong defense system.
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