The present study was conducted to investigate the immunomodulatory and anti-inflammatory effects of Elettaria cardamomum essential oil (ECEO) for the control of acute Toxoplasma gondii infection. The effect of ECEO on T. gondii tachyzoites was measured by the tetrazolium bromide method. Mice received ECEO orally at doses of 1-4 mg/kg/day for 14 days. Once acute toxoplasmosis was induced in mice, their mortality rate and parasite load were recorded. The level of liver antioxidant/oxidant enzymes and the level of mRNA expression of interleukin-1 beta and interferongamma were also investigated. ECEO particularly at a concentration of 150 µg/ml has promising in vitro anti-Toxoplasma effects (p<0.001). After treatment with ECEO, the mortality rate (9th day) and parasite load decreased (p<0.001) in the infected mice. ECEO markedly (p < 0.05) restored hepatic oxidant and antioxidant enzyme levels, as well as increased cytokines. These results report a significant inhibitory effect of ECEO mainly at a dose of 4 mg/mL, against the T. gondii Rh strain through strengthening the immune system and reducing inflammation and oxidative stress; however, further research is needed to verify these results.
Eukaryotic cells typically respond to stress conditions by inhibiting global protein synthesis. The initiation phase is the main target of regulation and represents a key control point for eukaryotic gene expression. In Saccharomyces cerevisiae and mammalian cells this is achieved by phosphorylation of eukaryotic initiation factor 2 (eIF2α). We have examined how the fungal pathogen Candida albicans responds to oxidative stress conditions and show that oxidants including hydrogen peroxide, the heavy metal cadmium and the thiol oxidant diamide inhibit translation initiation. The inhibition in response to hydrogen peroxide and cadmium largely depends on phosphorylation of eIF2α since minimal inhibition is observed in a gcn2 mutant. In contrast, translation initiation is inhibited in a Gcn2-independent manner in response to diamide. Our data indicate that all three oxidants inhibit growth of C. albicans in a dose-dependent manner, however, loss of GCN2 does not improve growth in the presence of hydrogen peroxide or cadmium. Examination of translational activity indicates that these oxidants inhibit translation at a post-initiation phase which may account for the growth inhibition in a gcn2 mutant. As well as inhibiting global translation initiation, phosphorylation of eIF2α also enhances expression of the GCN4 mRNA in yeast via a well-known translational control mechanism. We show that C. albicans GCN4 is similarly induced in response to oxidative stress conditions and Gcn4 is specifically required for hydrogen peroxide tolerance. Thus, the response of C. albicans to oxidative stress is mediated by oxidant-specific regulation of translation initiation and we discuss our findings in comparison to other eukaryotes including the yeast S. cerevisiae.
Amylase is one of the most important enzymes in the world due to its wide application in various industries and biotechnological processes. In this study, amylase enzyme from Hylocereus polyrhizus was encapsulated for the first time in an Arabic gum-chitosan matrix using freeze drying. The encapsulated amylase retained complete biocatalytic activity and exhibited a shift in the optimum temperature and considerable increase in the pH and temperature stabilities compared to the free enzyme. Encapsulation of the enzyme protected the activity in the presence of ionic and non-ionic surfactants and oxidizing agents (H₂O₂) and enhanced the shelf life. The storage stability of amylase is found to markedly increase after immobilization and the freeze dried amylase exhibited maximum encapsulation efficiency value (96.2%) after the encapsulation process. Therefore, the present study demonstrated that the encapsulation of the enzyme in a coating agent using freeze drying is an efficient method to keep the enzyme active and stable until required in industry.
The efficiency of phenol degradation via Fenton reaction using mixture of heterogeneous goethite catalyst with homogeneous ferrous ion was analyzed as a function of three independent variables, initial concentration of phenol (60 to 100 mg /L), weight ratio of initial concentration of phenol to that of H2O2 (1: 6 to 1: 14) and, weight ratio of initial concentration of goethite catalyst to that of H2O2 (1: 0.3 to 1: 0.7). More than 90 % of phenol removal and more than 40% of TOC removal were achieved within 60 minutes of reaction. Two separate models were developed using artificial neural networks to predict degradation percentage by a combination of Fe3+ and Fe2+ catalyst. Five operational parameters were employed as inputs while phenol degradation and TOC removal were considered as outputs of the developed models. Satisfactory agreement was observed between testing data and the predicted values (R2Phenol = 0.9214 and R2TOC= 0.9082).
In order to investigate the role of roasting conditions in antioxidant formation, methanol and hot water extracts from Robusta coffee beans roasted for various lengths of time and at various temperatures were analyzed for total phenolic acid, chlorogenic acid, and caffeine content, as well as for their antioxidant activities using 1,1-diphenyl-2-picryhydrazyl (DPPH), thiobarbituric acid (TBA), and malonaldehyde/gas chromatography (MA/GC) assays. The amount of total phenolics in methanol extracts decreased linearly over the roasting temperature from 63.51 ± 0.77 mg chlorogenic acid equivalent (CAE)/g coffee beans (roasted at 200°C) to 42.56 ± 0.33 mg CAE/g coffee beans (roasted at 240°C). The total chlorogenic acid content decreased when the roasting time was increased from 78.33 ± 1.41 mg/g (green coffee beans) to 4.31 ± 0.23 mg/g (roasted for 16 min at 250°C). All methanol extracts from roasted coffee beans possessed over 90% antioxidant activities in the DPPH assay. The antioxidant activity of methanol extracts ranged from 41.38 ± 1.77% (roasted at 250°C for 10 min) to 98.20 ± 1.49% (roasted at 230°C for 16 min) as tested by the TBA assay. The antioxidant activity of methanol extracts of green coffee beans and roasted coffee beans ranged from 93.01% (green coffee beans) to 98.62 ± 1.32% (roasted at 250°C for 14 min) in the MA/GC assays. All hot water extracts exhibited moderate pro-oxidant activities in TBA and MA/GC assays. The results indicated that roasting conditions of coffee beans play an important role in the formation of antioxidants in brewed coffee, which can be dietary supplements having beneficial effect to human health.
The neuroprotective and antioxidative effects of germinated brown rice (GBR), brown rice (BR) and commercially available γ-aminobutyric acid (GABA) against cell death induced by hydrogen peroxide (H(2)O(2)) in human neuroblastoma SH-SY5Y cells have been investigated. Results show that GBR suppressed H(2)O(2)-mediated cytotoxicity and induced G0/G1 phase cell cycle arrest in SH-SY5Y cells. Moreover, GBR reduced mitochondrial membrane potential (MMP) and prevented phosphatidylserine (PS) translocation in SH-SY5Y cells, key features of apoptosis, and subsequent cell death. GBR exhibited better neuroprotective and antioxidative activities as compared to BR and GABA. These results indicate that GBR possesses high antioxidative activities and suppressed cell death in SH-SY5Y cells by blocking the cell cycle re-entry and apoptotic mechanisms. Therefore, GBR could be developed as a value added functional food to prevent neurodegenerative diseases caused by oxidative stress and apoptosis.
Several genes have been used as housekeeping genes and choosing an appropriate reference gene is important for accurate quantitative RNA expression in real time RT-PCR technique. The expression levels of reference genes should remain constant between the cells of different tissues and under different experimental conditions. The purpose of this study was to determine the effect of different experimental treatments on the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA so that the reliability of GAPDH as reference gene for quantitative real time RT-PCR in human diploid fibroblasts (HDFs) can be validated. HDFs in 4 different treatment groups viz; young (passage 4), senescent (passage 30), H2O2-induced oxidative stress and gamma-tocotrienol (GTT)-treated groups were harvested for total RNA extraction. Total RNA concentration and purity were determined prior to GAPDH mRNA quantification. Standard curve of GAPDH expression in serial diluted total RNA, melting curve analysis and agarose gel electrophoresis were used to determine the reliability of GAPDH as reference gene.
Besides acting as potent free radical scavengers, tocopherols and tocotrienols have been known to have non-antioxidant properties such as the involvement of alpha-tocopherol (alphaT) in PKC pathway and the anti-cancer properties of gamma-tocotrienol (gammaT3). This study aims to elucidate whether protective effects shown by alphaT and gammaT3 in H(2)O(2)-induced neuron cultures have anti-apoptotic or pro-apoptotic tendency toward the initiation of neuronal apoptosis. H(2)O(2) is used to induce apoptosis in primary cerebellar neuron cultures which is attenuated by pretreatment of alphaT or gammaT3 at concentrations < or =10 microM. Similar to our previous work, gammaT3 was found to be neurotoxic at concentrations > or =100 microM, whereas alphaT showed no neurotoxicity. Cellular uptake of gammaT3 was higher than that of alphaT. Treating cells simultaneously with either gammaT3 or alphaT and with then H(2)O(2) led to higher expression of Bax and Bcl-2 than in neurons exposed to H(2)O(2) alone. Analysis of Bcl-2/Bax ratio as 'survival index' showed that both pretreatment of gammaT3 and alphaT followed by H(2)O(2) increase the 'survival index' of Bcl-2/Bax ratio compared to H(2)O(2)-treated cells, while treatment of gammaT3 alone decrease the ratio compared to unchanged Bcl2/Bax ratio of similar treatment with alphaT alone. Similar treatment of gammaT3 decreased p53 expression and activates p38 MAPK phosphorylation, whereas alphaT did not alter its expression compared to H(2)O(2)-treated cells. Treating neurons with only gammaT3 or alphaT increased the expression of Bax, Bcl-2, p53, and p38 MAPK compared to control with gammaT3 exerting stronger expression for proteins involved than alphaT. In conclusion, low doses of gammaT3 and alphaT confer neuroprotection to H(2)O(2)-treated neurons via their antioxidant mechanism but gammaT3 has stronger pro-apoptosis tendency than alphaT by activating molecules involved in the neuronal apoptotic pathway in the absence of H(2)O(2).
The present study represents the first report of the effect of hydrogen peroxide (H(2)O(2)) on the growth, development and quality of the wax apple fruit, a widely cultivated fruit tree in South East Asia. The wax apple trees were spray treated with 0, 5, 20 and 50 mM H(2)O(2) under field conditions. Photosynthetic rates, stomatal conductance, transpiration, chlorophyll and dry matter content of the leaves and total soluble solids and total sugar content of the fruits of wax apple (Syzygium samarangense, var. jambu madu) were significantly increased after treatment with 5 mM H(2)O(2). The application of 20 mM H(2)O(2) significantly reduced bud drop and enhanced fruit growth, resulting in larger fruit size, increased fruit set, fruit number, fruit biomass and yield compared to the control. In addition, the endogenous level of H(2)O(2) in wax apple leaves increased significantly with H(2)O(2) treatments. With regard to fruit quality, 20 mM H(2)O(2) treatment increased the K(+), anthocyanin and carotene contents of the fruits by 65%, 67%, and 41%, respectively. In addition, higher flavonoid, phenol and soluble protein content, sucrose phosphate synthase (SPS), phenylalanine ammonia lyase (PAL) and antioxidant activities were recorded in the treated fruits. There was a positive correlation between peel colour (hue) and TSS, between net photosynthesis and SPS activity and between phenol and flavonoid content with antioxidant activity in H(2)O(2)-treated fruits. It is concluded that spraying with 5 and 20 mM H(2)O(2) once a week produced better fruit growth, maximising the yield and quality of wax apple fruits under field conditions.
We investigated the biomechanical relationship between intraluminal pressure within small mesenteric resistance arteries, oxidant activation of PKG, Ca2+ sparks, and BK channel vasoregulation. Mesenteric resistance arteries from wild type (WT) and genetically modified mice with PKG resistance to oxidative activation were studied using wire and pressure myography. Ca2+ sparks and Ca2+ transients within vascular smooth muscle cells of intact arteries were characterized using high-speed confocal microscopy of intact arteries. Arteries were studied under conditions of varying intraluminal pressure and oxidation. Intraluminal pressure specifically, rather than the generic stretch of the artery, was necessary to activate the oxidative pathway. We demonstrated a graded step activation profile for the generation of Ca2+ sparks and also a functional "ceiling" for this pressure --sensitive oxidative pathway. During steady state pressure - induced constriction, any additional Ca2+ sensitive-K+ channel functional availability was independent of oxidant activated PKG. There was an increase in the amplitude, but not the Area under the Curve (AUC) of the caffeine-induced Ca2+ transient in pressurized arteries from mice with oxidant-resistant PKG compared with wild type. Overall, we surmise that intraluminal pressure within resistance arteries controls Ca2+ spark vasoregulation through a tightly controlled pathway with a graded onset switch. The pathway, underpinned by oxidant activation of PKG, cannot be further boosted by additional pressure or oxidation once active. We propose that these restrictive characteristics of pressure-induced Ca2+ spark vasoregulation confer stability for the artery in order to provide a constant flow independent of additional pressure fluctuations or exogenous oxidants.
The intracellular level of bilirubin (BR), an endogenous antioxidant that is cytotoxic at high concentrations, is tightly controlled within the optimal therapeutic range. We have recently described a concerted intracellular BR regulation by two microsomal enzymes: heme oxygenase 1 (HMOX1), essential for BR production and cytochrome P450 2A5 (CYP2A5), a BR oxidase. Herein, we describe targeting of these enzymes to hepatic mitochondria during oxidative stress. The kinetics of microsomal and mitochondrial BR oxidation were compared. Treatment of DBA/2J mice with 200mgpyrazole/kg/day for 3days increased hepatic intracellular protein carbonyl content and induced nucleo-translocation of Nrf2. HMOX1 and CYP2A5 proteins and activities were elevated in microsomes and mitoplasts but not the UGT1A1, a catalyst of BR glucuronidation. A CYP2A5 antibody inhibited 75% of microsomal BR oxidation. The inhibition was absent in control mitoplasts but elevated to 50% after treatment. An adrenodoxin reductase antibody did not inhibit microsomal BR oxidation but inhibited 50% of mitochondrial BR oxidation. Ascorbic acid inhibited 5% and 22% of the reaction in control and treated microsomes, respectively. In control mitoplasts the inhibition was 100%, which was reduced to 50% after treatment. Bilirubin affinity to mitochondrial and microsomal CYP2A5 enzyme is equally high. Lastly, the treatment neither released cytochrome c into cytoplasm nor dissipated membrane potential, indicating the absence of mitochondrial membrane damage. Collectively, the observations suggest that BR regulatory enzymes are recruited to mitochondria during oxidative stress and BR oxidation by mitochondrial CYP2A5 is supported by mitochondrial mono-oxygenase system. The induced recruitment potentially confers membrane protection.