Microplate spectroscopic methods for determination of the organophosphate soman.

Two microplate spectroscopic methods for determination of organophosphates, based on inhibition of acetylcholinesterase activity, have been elaborated and evaluated for determination of the chemical weapon agent soman. The principal difference between the methods is that one measures reaction substrate concentration (elaborated from Hestrin), while the other measures reaction product (elaborated from Ellman). The linear ranges of the two methods were found to be similar. Although the limit of quantification was lower for the Ellman method (110 pM), the sensitivity coefficient was in favor of the Hestrin method (1.55-fold higher). The effects of the main soman hydrolysis products were consistent for the two methods: both methylphosphonic acid and pinacolyl methylphosphonic acid did not inhibit acetylcholinesterase activity. The main components of decontaminating solutions showed differential effects: while monoethanolamine had no influence upon results obtained by either method, hydrogen peroxide interfered with the Ellman method at far lower concentrations than with the Hestrin method. In practical applications involving samples containing hydrogen peroxide, the method based on Hestrin should be regarded as much more specific for OP determination than the Ellman method

Proteome Map of Pea (<i>Pisum sativum</i> L.) Embryos Containing Different Amounts of Residual Chlorophylls

Due to low culturing costs and high seed protein contents, legumes represent the main global source of food protein. Pea (Pisum sativum L.) is one of the major legume crops, impacting both animal feed and human nutrition. Therefore, the quality of pea seeds needs to be ensured in the context of sustainable crop production and nutritional efficiency. Apparently, changes in seed protein patterns might directly affect both of these aspects. Thus, here, we address the pea seed proteome in detail and provide, to the best of our knowledge, the most comprehensive annotation of the functions and intracellular localization of pea seed proteins. To address possible intercultivar differences, we compared seed proteomes of yellow- and green-seeded pea cultivars in a comprehensive case study. The analysis revealed totally 1938 and 1989 nonredundant proteins, respectively. Only 35 and 44 proteins, respectively, could be additionally identified after protamine sulfate precipitation (PSP), potentially indicating the high efficiency of our experimental workflow. Totally 981 protein groups were assigned to 34 functional classes, which were to a large extent differentially represented in yellow and green seeds. Closer analysis of these differences by processing of the data in KEGG and String databases revealed their possible relation to a higher metabolic status and reduced longevity of green seeds

First Insight into the Neuroprotective and Antibacterial Effects of Phlorotannins Isolated from the Cell Walls of Brown Algae Fucus vesiculosus and Pelvetia canaliculata

Phaeophyceae (brown algae) essentially contribute to biotopes of cold and temperate seas. Their thalli are rich in biologically active natural products, which are strongly and universally dominated with phlorotannins-polyphenols of complex and diverse structure based on multiple differently arranged phloroglucinol units and well known as strong antioxidants with a broad spectrum of biological activities. In the algal cells, phlorotannins can either accumulate in the cytoplasm or can be secreted into the cell wall (CW). The biological activities of extractable intracellular phlorotannins have been comprehensively characterized, whereas the properties of the CW-bound polyphenol fraction are still mostly unknown. Recently, we identified dibenzodioxin bonding as the principal structural feature of the CW-bound phlorotannins in fucoid algae, whereas soluble intracellular phlorotannins rely on aryl and ether bonds. However, profiles of biological activity associated with these structural differences are still unknown. Therefore, to the best of our knowledge, for the first time we address the antioxidant, cytotoxic, neuroprotective, and antibacterial properties of the CW-bound phlorotannin fractions isolated from two representatives of the order Fucales-Fucus vesiculosus and Pelvetia canaliculata. The CW-bound phlorotannins appeared to be softer antioxidants, stronger antibacterial agents and were featured with essentially less cytotoxicity in comparison to the intracellular fraction. However, the neuroprotective effects of both sub-cellular phlorotannin fractions of F. vesiculosus and P. canaliculata were similar. Thus, due to their lower cytotoxicity, CW-bound phlorotannins can be considered as promising antioxidants and neuroprotectors

Neuroprotective and antibacterial effects of phlorotannins isolated from the cell walls of brown algae Fucus vesiculosus and Pelvetia canaliculata

The Phaeophyceae (brown algae) essentially contribute to biotopes of cold and temperate seas. Their thalli are rich in biologically active natural products which are strongly and universally dominated with phlorotannins – polyphenols of complex and diverse structure based on multiple differently arranged phloroglucinol units. These electron-rich compounds are strong antioxidants with antimicrobial, anti-inflammatory and neuroprotective activities. In the algal cells phlorotannins can either accumulate in cytoplasm or can be secreted into the cell wall (CW) with subsequent covalent binding to the alginate network. The biological activities of easily extractable intracellular phlorotannins were comprehensively characterized, whereas the properties of the CW-bound polyphenol fraction are still mostly unknown. Recently, we identified dibenzodioxin bonding as the principal structural feature of the CW-bound phlorotannins of fucoid algae, whereas soluble intracellular phlorotannins relied on aryl- and ether bonds. However, profiles of biological activity associated with these structural differences are still unknown. Therefore, here, to the best of our knowledge, for the first time, we address the antioxidant, cytotoxic, neuroprotective, and antibacterial properties of the CW-bound phlorotannin fractions isolated from two representatives of the order Fucales - Fucus vesiculosus and Pelvetia canaliculata. The CW-bound phlorotannins appeared to be softer antioxidants, stronger antibacterial agents and were featured with essentially less cytotoxicity in comparison to the intracellular fraction. However, the neuroprotective effects of both sub-cellular phlorotannin fractions of F. vesiculosus and P. canaliculata were essentially similar. Thus, due to their lower cytotoxicity, CW-bound phlorotannins can be considered as promising antioxidants and neuroprotectors

PHOS-Select Iron Affinity Beads Enrich Peptides for the Detection of Organophosphorus Adducts on Albumin

Albumin is covalently modified by organophosphorus toxicants (OP) on tyrosine 411, but less than 1% of albumin is modified in humans by lethal OP doses that inhibit 95% of plasma butyrylcholinesterase. A method that enriches OP-modified albumin peptides could aid analysis of low dose exposures. Soman or chlorpyrifos oxon treated human plasma was digested with pepsin. Albumin peptides were enriched by binding to Fe³⁺ beads at pH 11 and eluted with pH 2.6 buffer. Similarly, mouse and guinea pig albumin modified by chlorpyrifos oxon were digested with pepsin and enriched by binding to Fe³⁺ beads. Peptides were identified by MALDI-TOF/TOF mass spectrometry. PHOS-select iron affinity beads specifically enriched albumin peptides VRY₄₁₁TKKVPQVST and LVRY₄₁₁TKKVPQVST in a pepsin digest of human plasma. The unmodified as well as OP-modified peptides bound to the beads. The binding capacity of 500 μL of beads was the pepsin digest of 2.1 μL of human plasma. The limit of detection was 0.2% of OP-modified albumin peptide in 0.43 μL of plasma. Enrichment of OP-modified albumin peptides by binding to Fe³⁺ beads is a method with potential application to diagnosis of OP pesticide and nerve agent exposure in humans, mice, and guinea pigs

Does Protein Glycation Impact on the Drought-Related Changes in Metabolism and Nutritional Properties of Mature Pea (Pisum sativum L.) Seeds?

Protein glycation is usually referred to as an array of non-enzymatic post-translational modifications formed by reducing sugars and carbonyl products of their degradation. The resulting advanced glycation end products (AGEs) represent a heterogeneous group of covalent adducts, known for their pro-inflammatory effects in mammals, and impacting on pathogenesis of metabolic diseases and ageing. In plants, AGEs are the markers of tissue ageing and response to environmental stressors, the most prominent of which is drought. Although water deficit enhances protein glycation in leaves, its effect on seed glycation profiles is still unknown. Moreover, the effect of drought on biological activities of seed protein in mammalian systems is still unstudied with respect to glycation. Therefore, here we address the effects of a short-term drought on the patterns of seed protein-bound AGEs and accompanying alterations in pro-inflammatory properties of seed protein in the context of seed metabolome dynamics. A short-term drought, simulated as polyethylene glycol-induced osmotic stress and applied at the stage of seed filling, resulted in the dramatic suppression of primary seed metabolism, although the secondary metabolome was minimally affected. This was accompanied with significant suppression of NF-kB activation in human SH-SY5Y neuroblastoma cells after a treatment with protein hydrolyzates, isolated from the mature seeds of drought-treated plants. This effect could not be attributed to formation of known AGEs. Most likely, the prospective anti-inflammatory effect of short-term drought is related to antioxidant effect of unknown secondary metabolite protein adducts, or down-regulation of unknown plant-specific AGEs due to suppression of energy metabolism during seed filling