Role of Phytochelatins in Redox Caused Stress in Plants and Animals

Varied environmental compartments (such as soil and water) potentially contaminated with different metals/metalloids can impact the health of both plants and animals/humans. Trace amounts of Cu, Mn, Mo, Ni and Zn are beneficial for higher plants, whereas, Cr, Cu, Co, Mn, Mo, Se, V and Zn are known as the micronutrient metal/metalloids for animals/humans. However, elevated levels of the metals/metalloids can cause severe toxic consequences in both plants and animals/humans. Common in plants and animals/humans, phytochelatins (PCs), the principal non-protein, S-rich, thiolate peptides, protect (through different mechanisms) cellular functions and metal/metalloid homeostasis by performing their chelation and/or detoxification. With the major aim of broadening the current knowledge on the subject, this chapter (a) overviews PCs’ role and modulation separately in metal/metalloid-exposed plants and animals/humans; (b) discusses major methods for determination of PCs and bioassays for enzymes involved in PC synthesis; (c) evaluates the connection of PCs with bionanoparticles; and finally (d) highlights so far unexplored aspects in the present context

Anticarcinogenic effect of spices due to phenolic and flavonoid compounds-in vitro evaluation on prostate cells

This study shows the effects of spices, and their phenolic and flavonoid compounds, on prostate cell lines (PNT1A, 22RV1 and PC3). The results of an MTT assay on extracts from eight spices revealed the strongest inhibitory effects were from black pepper and caraway seed extracts. The strongest inhibitory effect on prostatic cells was observed after the application of extracts of spices in concentration of 12.5 mg.mL- 1. An LC/MS analysis identified that the most abundant phenolic and flavonoid compounds in black pepper are 3,4-dihydroxybenzaldehyde and naringenin chalcone, while the most abundant phenolic and flavonoid compounds in caraway seeds are neochlorogenic acid and apigenin. Using an MTT assay for the phenolic and flavonoid compounds from spices, we identified the IC50 value of ~1 mmol.L- 1 PNT1A. The scratch test demonstrated that the most potent inhibitory effect on PNT1A, 22RV1 and PC3 cells is from the naringenin chalcone contained in black pepper. From the spectrum of compounds assessed, the naringenin chalcone contained in black pepper was identified as the most potent inhibitor of the growth of prostate cells.O

The Application of Curve Fitting on the Voltammograms of Various Isoforms of Metallothioneins–Metal Complexes

The translation of metallothioneins (MTs) is one of the defense strategies by which organisms protect themselves from metal-induced toxicity. MTs belong to a family of proteins comprising MT-1, MT-2, MT-3, and MT-4 classes, with multiple isoforms within each class. The main aim of this study was to determine the behavior of MT in dependence on various externally modelled environments, using electrochemistry. In our study, the mass distribution of MTs was characterized using MALDI-TOF. After that, adsorptive transfer stripping technique with differential pulse voltammetry was selected for optimization of electrochemical detection of MTs with regard to accumulation time and pH effects. Our results show that utilization of 0.5 M NaCl, pH 6.4, as the supporting electrolyte provides a highly complicated fingerprint, showing a number of non-resolved voltammograms. Hence, we further resolved the voltammograms exhibiting the broad and overlapping signals using curve fitting. The separated signals were assigned to the electrochemical responses of several MT complexes with zinc(II), cadmium(II), and copper(II), respectively. Our results show that electrochemistry could serve as a great tool for metalloproteomic applications to determine the ratio of metal ion bonds within the target protein structure, however, it provides highly complicated signals, which require further resolution using a proper statistical method, such as curve fitting.The work has been supported by the Agency for the Czech Republic Health Research (AZV) project no. 15-28334A. The presented research was financed by the Czech Ministry of Education in frame of the National Sustainability Program, the grant LO1401 INWITE

Bio-Sensing of Cadmium(II) Ions Using Staphylococcus aureus†

Cadmium, as a hazardous pollutant commonly present in the living environment, represents an important risk to human health due to its undesirable effects (oxidative stress, changes in activities of many enzymes, interactions with biomolecules including DNA and RNA) and consequent potential risk, making its detection very important. New and unique technological and biotechnological approaches for solving this problems are intensely sought. In this study, we used the commonly occurring potential pathogenic microorganism Staphylococcus aureus for the determination of markers which could be used for sensing of cadmium(II) ions. We were focused on monitoring the effects of different cadmium(II) ion concentrations (0, 1.25, 2.5, 5, 10, 15, 25 and 50 μg mL−1) on the growth and energetic metabolism of Staphylococcus aureus. Highly significant changes have been detected in the metabolism of thiol compounds—specifically the protein metallothionein (0.79–26.82 mmol/mg of protein), the enzyme glutathione S-transferase (190–5,827 μmol/min/mg of protein), and sulfhydryl groups (9.6–274.3 μmol cysteine/mg of protein). The ratio of reduced and oxidized glutathione indicated marked oxidative stress. In addition, dramatic changes in urease activity, which is connected with resistance of bacteria, were determined. Further, the effects of cadmium(II) ions on the metabolic pathways of arginine, β-glucosidase, phosphatase, N-acetyl β-d-glucosamine, sucrose, trehalose, mannitol, maltose, lactose, fructose and total proteins were demonstrated. A metabolomic profile of Staphylococcus aureus under cadmium(II) ion treatment conditions was completed seeking data about the possibility of cadmium(II) ion accumulation in cells. The results demonstrate potential in the application of microorganisms as modern biosensor systems based on biological components

Bio-Sensing of Cadmium(II) Ions Using Staphylococcus aureus†

Cadmium, as a hazardous pollutant commonly present in the living environment, represents an important risk to human health due to its undesirable effects (oxidative stress, changes in activities of many enzymes, interactions with biomolecules including DNA and RNA) and consequent potential risk, making its detection very important. New and unique technological and biotechnological approaches for solving this problems are intensely sought. In this study, we used the commonly occurring potential pathogenic microorganism Staphylococcus aureus for the determination of markers which could be used for sensing of cadmium(II) ions. We were focused on monitoring the effects of different cadmium(II) ion concentrations (0, 1.25, 2.5, 5, 10, 15, 25 and 50 μg mL−1) on the growth and energetic metabolism of Staphylococcus aureus. Highly significant changes have been detected in the metabolism of thiol compounds—specifically the protein metallothionein (0.79–26.82 mmol/mg of protein), the enzyme glutathione S-transferase (190–5,827 μmol/min/mg of protein), and sulfhydryl groups (9.6–274.3 μmol cysteine/mg of protein). The ratio of reduced and oxidized glutathione indicated marked oxidative stress. In addition, dramatic changes in urease activity, which is connected with resistance of bacteria, were determined. Further, the effects of cadmium(II) ions on the metabolic pathways of arginine, β-glucosidase, phosphatase, N-acetyl β-d-glucosamine, sucrose, trehalose, mannitol, maltose, lactose, fructose and total proteins were demonstrated. A metabolomic profile of Staphylococcus aureus under cadmium(II) ion treatment conditions was completed seeking data about the possibility of cadmium(II) ion accumulation in cells. The results demonstrate potential in the application of microorganisms as modern biosensor systems based on biological components

Are early somatic embryos of the norway spruce (Picea abies (L.) Karst.) organised?

Background Somatic embryogenesis in conifer species has great potential for the forestry industry. Hence, a number of methods have been developed for their efficient and rapid propagation through somatic embryogenesis. Although information is available regarding the previous process-mediated generation of embryogenic cells to form somatic embryos, there is a dearth of information in the literature on the detailed structure of these clusters. Methodology/Principal Findings The main aim of this study was to provide a more detailed structure of the embryogenic tissue clusters obtained through the in vitro propagation of the Norway spruce (Picea abies (L.) Karst.). We primarily focused on the growth of early somatic embryos (ESEs). The data on ESE growth suggested that there may be clear distinctions between their inner and outer regions. Therefore, we selected ESEs collected on the 56th day after sub-cultivation to dissect the homogeneity of the ESE clusters. Two colourimetric assays (acetocarmine and fluorescein diacetate/propidium iodide staining) and one metabolic assay based on the use of 2,3,5-triphenyltetrazolium chloride uncovered large differences in the metabolic activity inside the cluster. Next, we performed nuclear magnetic resonance measurements. The ESE cluster seemed to be compactly aggregated during the first four weeks of cultivation; thereafter, the difference between the 1H nuclei concentration in the inner and outer clusters was more evident. There were clear differences in the visual appearance of embryos from the outer and inner regions. Finally, a cluster was divided into six parts (three each from the inner and the outer regions of the embryo) to determine their growth and viability. The innermost embryos (centripetally towards the cluster centre) could grow after sub-cultivation but exhibited the slowest rate and required the longest time to reach the common growth rate. To confirm our hypothesis on the organisation of the ESE cluster, we investigated the effect of cluster orientation on the cultivation medium and the influence of the change of the cluster’s three-dimensional orientation on its development. Maintaining the same position when transferring ESEs into new cultivation medium seemed to be necessary because changes in the orientation significantly affected ESE growth. Conclusions and Significance This work illustrated the possible inner organisation of ESEs. The outer layer of ESEs is formed by individual somatic embryos with high metabolic activity (and with high demands for nutrients, oxygen and water), while an embryonal group is directed outside of the ESE cluster. Somatic embryos with depressed metabolic activity were localised in the inner regions, where these embryonic tissues probably have a very important transport function

An Investigation of Glutathione-Platinum(II) Interactions by Means of the Flow Injection Analysis Using Glassy Carbon Electrode

Despite very intensive research in the synthesising of new cytostatics, cisplatin isstill one of the most commonly used anticancer drugs. Therefore, an investigation ofinteractions of cisplatin with different biologically important amino acids, peptides andproteins is very topical. In the present paper, we utilized flow injection analysis coupledwith electrochemical detection to study and characterize the behaviour of various forms ofglutathione (reduced glutathione – GSH, oxidized glutathione – GSSG and S-nitrosoglutathione – GSNO). The optimized conditions were as follows: mobile phase consistedof acetate buffer (pH 3) with a flow rate of 1 mL min-1. Based on results obtained we chose850 mV as the optimal potential for detection of GSH and 1,100 mV as the optimalpotential for detection of GSSG and GSNO. The detection limits of GSH, GSSG andGSNO were 100 pg mL-1, 50 ng mL-1 and 300 pg mL-1, respectively. Further, the optimized technique was used for investigation of interactions between cisplatin and GSH. We were able to observe the interaction between GSH and cisplatin via decrease in the signal corresponding to glutathione. Moreover, we evaluated the formation of the complex by spectrometry. The spectrometric results obtained were in good agreement with electrochemical ones

Capillary Blood GSH Level Monitoring, Using an Electrochemical Method Adapted for Micro Volumes

Glutathione (γ-glutamyl-cysteinyl-glycine; also known as GSH) is an endogenous antioxidant that plays a crucial role in cell defense mechanisms against oxidative stress. It is thus not surprising that this molecule can serve as a biomarker for oxidative stress monitoring. As capillary blood is a highly accessible target for biomarking, it is a valuable bodily fluid for diagnosing human GSH levels. This study focused on the optimization of GSH measurements from micro volumes of capillary blood prior to using electrochemical detection. The optimization of experimental parameters, including the sample volume and its stability, was performed and evaluated. Moreover, we tested the optimized method as part of a short-term study. The study consisted of examining 10 subjects within 96 h of their consumption of high amounts of antioxidants, attained from a daily dose of 2 g/150 mL of green tea. The subjects’ capillary blood (5 μL) was taken at 0 h, 48 h, and 96 h for subsequent analysis. The short-term supplementation of diet with green tea showed an increase of GSH pool by approximately 38% (between 0 and 48 h) within all subjects

Liquid Chromatographic/Mass Spectrometric Study on the Role of Beech (Fagus sylvatica L.) Wood Polyphenols in Red Heartwood Formation

The present research focused on the analysis of European beech (Fagus sylvatica L.) wood polyphenols in respect to red heartwood formation, which is a significant color and technological defect of the species. For the first time, high-performance liquid chromatography/tandem mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) were applied for the investigation of the structure and the radial distribution of polyphenols. Altogether 125 compounds were characterized by their MSn spectra, of which 71 were tentatively identified, including procyanidins (dimers to pentamers), flavonoids (taxifolin, naringenin, isorhamnetin, (epi)afzelechin), (+)-catechin, (-)-epicatechin) and their glycoside conjugates, phenolic acids (gallic acid, vanillic acid, syringic acid) and their glycosides as well as gallic acid derivatives, many of which were identified for the first time in beech wood. It was found that the concentration of many compounds increased at the color boundary. In situ polyphenol synthesis and metabolism were clearly evidenced at the color boundary. Red heartwood contained only free aglycones (syringic acid, taxifolin, naringenin, isorhamnetin, naringenin, syringic acid). Contrary to earlier assumptions, the MALDI-TOF analysis did not indicate the presence of oxidized high-molecular-weight polymeric polyphenols in the red heartwood tissues. The role of individual compounds in the formation of the red heartwood chromophores are discussed