High-performance liquid chromatography (HPLC) provides a rapid, sensitive, and reproducible means of separating and quantifying simultaneously a variety of sulfur-containing amino acids and related derivatives. The HPLC method described in this chapter is modified and is based on the initial formation of S-carboxy-methyl derivatives of free thiols followed by the conversion of free amino groups to 2,4-dinitrophenyl (DNP) derivatives. Following derivatization, nanomole levels of individual sulfur-containing amino acids are measured using UV detection at 365 nm after separation by reverse-phase ion-exchange HPLC. Because of the versatility of this HPLC method, biological specimen preparation as well as derivatization and HPLC analysis procedures are discussed. DNP derivatives of acidic amino acids (including thiol-containing compounds) are separated on a 3-aminopropyl column by reversed-phase ion-exchange HPLC. In the mobile phase, methanol is used to elute rapidly the excess 2,4-dinitrophenol and the DNP derivatives of basic and neutral amino acids. Acetic acid is present in the mobile phase to maintain the bonded-phase amino groups in the protonated form. By increasing the sodium acetate concentration of the mobile phase, selective elution of acidic DNP derivatives is accomplished. The eluted DNP derivatives are measured by detection at 365 nm.
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Lead is a very toxic and futile heavy metal for rice plants because of its injurious effects on plant growth and metabolic processes. Polyploidy or whole genome doubling increases the ability of plants to withstand biotic and abiotic stress. Considering the beneficial effects of nanoparticles and tetraploid rice, this research was conducted to examine the effectiveness of tetraploid and titanium dioxide nanoparticles (TiO2 NPs) in mitigating the toxic effects of lead. A diploid (E22-2x) and it's tetraploid (T-42) rice line were treated with Pb (200 μM) and TiO2 NPs (15 mg L−1). Lead toxicity dramatically reduced shoot length (16 % and 4 %) and root length (17 % and 9 %), biological yield (55 % and 36 %), and photosynthetic activity, as evidenced by lower levels of chlorophyll a and b (30 % and 9 %) in E-22 and T-42 rice cultivars compared to the control rice plants, respectively. Furthermore, lead toxicity amplified the levels of reactive oxygen species (ROS), such as malondialdehyde and H2O2, while decreasing activities of all antioxidant enzymes, such as superoxidase, peroxidase, and glutathione predominately in the diploid cultivar. Transmission electron microscopy and semi-thin section observations revealed that Pb-treated cells in E22-2x had more cell abnormalities than T-42, such as irregularly shaped mitochondria, cell wall, and reduced root cell size. Polyploidy and TiO2 reduced Pb uptake in rice cultivars and expression levels of metal transporter genes such as OsHMA9 and OsNRAMP5. According to the findings, genome doubling alleviates Pb toxicity by reducing Pb accumulation, ROS, and cell damage. Tetraploid rice can withstand the toxic effect of Pb better than diploid rice, and TiO2 NPs can alleviate the toxic impact of Pb. Our study findings act as a roadmap for future research endeavours, directing the focus toward risk management and assessing long-term impacts to balance environmental sustainability and agricultural growth.
The Cd toxicity causes severe perturbations to the plant’s growth and development. Here, polyploid and diploid rice lines were treated with zinc-oxide nanoparticles (ZnO-NPs) and Cd, and physiological, cytological and molecular changes were observed. The Cd toxicity significantly reduced plant's growth attributes (such as shoot length, biological yield, dry matter, and chlorophyll contents, which decreased by 19%, 18%, 16%, 19% in polyploid and 35%, 43%, 45% and 43% in diploid rice, respectively), and disturbed the sugar level through the production of electrolytes, hydrogen peroxide, and malondialdehyde. The application of ZnO-NPs significantly alleviated the Cd toxicity in both lines by improving the antioxidant enzymes activities and physiochemical attributes. Semi-thin sections and transmission electron microscope revealed more and different types of abnormalities in diploid rice compared to polyploid rice under Cd stress. Moreover, RNA-seq analysis identified several differentially expressed genes between polyploid and diploid rice, especially metal and sucrose transporter genes. The GO, COG, and KEGG analyses revealed ploidy-specific pathways associated with plant growth and development. In conclusion, ZnO-NPs application to both rice lines significantly improved plant growth and decreased Cd accumulation in plants. We inferred that polyploid rice is more resistant to Cd stress than diploid rice.
Abnormal cholesterol metabolism is linked to many neurodegenerative disorders. Here, we present a protocol for the production of a recombinant protein consisting of a Glutathione-S-Transferase tag fused with the Perfringolysin O (PFO). The GST-PFO tag enables analysis of the localization of cholesterol in subcellular membranes of human and mice brain and liver tissues. We have used this approach for samples from Niemann-Pick type C disease and non-alcoholic steatohepatitis models. The construct may also have applications for the diagnosis of cholesterol-accumulating disorders.
For complete details on the use and execution of this protocol, please refer to Kwiatkowska et al. (2014).
Thus, the detection of GSH is of great importance and has been widely investigated. So far, many strategies, such as liquid chromatography, electrochemical devices, Raman spectroscopy and optical sensors have been developed and used to detect GSH [5–16]. Among them, the optical sensors have attracted increasing attention, owing to their high sensitivity, simple operation and short analysis time.
Glutathione (GSH) is an important biothiol in biological systems and serves as a significant biomarker for the health of humans. Herein, red-emitting carbon dots (p-CDs) prepared by a simple one-step solvothermal process can exclusively, rapidly and sensitively respond to GSH owing to the high content of pyridinic N. Upon addition of GSH, p-CDs instantly give a significant color change from orange to bluish-purple. The absorbance ratio of 576 nm to 486 nm is linearly proportional to the logarithmic concentration of GSH in a wide range of 12.5−800 μM, which is the level of cellular GSH. Meanwhile, the fluorescence of p-CDs quenches obviously. The LOD of p-CDs towards GSH in fluorescence mode is 0.7 μM. Moreover, p-CDs have no response to Cys, Hcy, amino acids and ions existing in biological systems. Such a promising responsiveness offers a great opportunity to accurately, sensitively and selectively detect GSH by colorimetric and fluorescent dual modes. In addition, cellulose/p-CDs hydrogels and p-CDs-based test papers have been fabricated to successfully detect GSH according to the color-change and fluorescence quenching. The biocompatible, environment-friendly and disposable cellulose/p-CDs materials readily provide a simple and visual strategy to detect GSH levels in the biomedical fields.
α-Lipoic acid (LA) is a dietary supplement for maintaining energy balance, but well-controlled clinical trials in otherwise healthy, overweight adults using LA supplementation are lacking.
The primary objective was to evaluate whether LA supplementation decreases elevated plasma triglycerides in overweight or obese adults. Secondary aims examined if LA promotes weight loss and improves oxidative stress and inflammation.
Overweight adults [n = 81; 57% women; 21–60 y old; BMI (in kg/m2) ≥ 25] with elevated plasma triglycerides ≥100 mg/dL were enrolled in a 24-wk, randomized, double-blind, controlled trial, assigned to either (R)-α-lipoic acid (R-LA; 600 mg/d) or matching placebo, and advised not to change their diet or physical activity. Linear models were used to evaluate treatment effects from baseline for primary and secondary endpoints.
R-LA did not decrease triglyceride concentrations, but individuals on R-LA had a greater reduction in BMI at 24 wk than the placebo group (-0.8; P = 0.04). The effect of R-LA on BMI was correlated to changes in plasma triglycerides (r = +0.50, P = 0.004). Improvement in body weight was greater at 24 wk in R-LA subgroups than in placebo subgroups. Women and obese participants (BMI = 35) showed greater weight loss (-5.0% and -4.8%, respectively; both P < 0.001) and loss of body fat (-9.4% and -8.6%, respectively; both P < 0.005). Antioxidant gene expression in mononuclear cells at 24 wk was greater in the R-LA group (Heme oxygenase 1 [HMOX1] : +22%; P = 0.02) than in placebo. Less urinary F2-isoprostanes (-25%; P = 0.005), blood leukocytes (-10.1%; P = 0.01), blood thrombocytes (-5.1%; P = 0.03), and ICAM-1 (-7.4%; P = 0.04) at 24 wk were also observed in the R-LA group than in placebo.
Long-term LA supplementation results in BMI loss, greater antioxidant enzyme synthesis, and less potential for inflammation in overweight adults. Improved cellular bioenergetics is also evident in some individuals given R-LA. This trial was registered at clinicaltrials.gov as NCT00765310.
The consecutive annual use of a mixture of naturally occurring garlic compounds (GCs) as a dormancy-breaking treatment in table grape (Vitis vinifera L.) cultivars that did not yet meet their chilling requirement has led to an improvement in the productivity of the vines. This study tested the hypothesis that the sulfur molecules contained in the garlic compound mixture stimule an increase in glutathione content. Therefore, the reduced glutathione (GSH) and oxidized glutathione (GSSG) content in dormant buds of grapevine cv. Flame seedless from two consecutive winter seasons (2013/2014 and 2014/2015) were quantified following GCs and hydrogen cyanamide treatments. The GSH content was increased by both treatments, with similar patterns and timing but to different extents. Specifically, the buds of the 2013/2014 season, which accumulated less cold naturally, presented significant changes in GSH content, and consequently, a higher GSH:GSSG ratio was measured in the buds treated with GCs. Lower values of GSSG than GSH were quantified in the buds from both winters following GC treatment. The results suggest that the sulfur compounds contained in GCs have an important influence on the accumulation of the reduced form of glutathione (GSH), thus, maintaining a high GSH:GSSG ratio, which has been associated with plant growth and more specifically rapid cell growth.
