Influence of rutin-ammonium complex on the physiological condition of pine seedlings

In the plant body, phenolic compounds nonspecifically affect the processes of morphogenesis and perform a wide range of regulatory and protective functions. Of particular interest are the processes involved in the complexation of flavonoids as a result of their interaction with ammonium forms of nitrogen. Polar compounds, which are formed in tissues as a result of chemical transformation, are quite mobile in soil solutions and show high biological activity. The properties of phenol-ammonium complexes are of considerable interest in terms of morphogenesis, physiology of stability, as well as in the system of interaction of plants with soil microorganisms. Studies of the effect of phenol-ammonium complex were performed on seeds and seedlings of Scots pine. Quantitative indicators of germination energy and germination were determined by seed germination. Biochemical profiling of seedling tissue extracts was performed by high-performance thin layer chromatography. It has been experimentally confirmed that rutin (quercetin-3-O-rutinoside) after interaction with 10% aqueous ammonia solution forms a complex of substances, among which the chromatography revealed polar products that potentially affect the regulation of growth. At a total concentration of 15 mg/l, these substances significantly increased germination energy and seed germination. In pine seedlings, they stimulated the growth of roots and shoots. The effect of the complex of organic compounds on seedlings depended on the concentration, duration of seed treatment and had a prolonged effect. The obtained phenol-ammonium complex at a concentration of 10-15 mg/l contributed to an increase in the amount of chlorophylls, carotenoids in the tissues of seedlings, and at 20-40 mg/l increased the content of phenolic synthesis products.

A Review on Various Analytical Methodologies for Etoricoxib

Etoricoxib belongs to the class of highly selective COX-2 inhibitor NSAIDs. It is mostly used for the treatment of pain, arthritic conditions including rheumatoid arthritis and osteoarthritis. The current study focuses primarily on analytical and bioanalytical method development methodologies, as well as numerous methods established for the estimation of etoricoxib, whether in pharmaceutical dose form or in bulk. Analytical procedures are critical for determining compositions, as they allow us to obtain both qualitative and quantitative results utilising advanced analytical tools. The analytical method for Etoricoxib may be chromatographic, electrochemical, spectral or hyphenated. These methods aid in the comprehension of critical process parameters as well as the minimization of their impact on precision and accuracy. Analytical method development is required to sustain high commercial product quality standards and to meet regulatory requirements. Following the reference, regulatory organisations in several nations have established standards and procedures for providing approval, authentication, and registration. Bioanalytical methods are designed to quantify the concentration of drug, metabolite, or typical biomarkers from various biological fluids including serum, urine, saliva and tissue extracts.

Total Starch Enzymatic Digestion v1

Enzymatic digestion of total soluble starch to glucose in plant tissue extracts for preparation for quantification via the GOD-POD Method (NZYtech).

Total Soluble Sugar Quantification from Ethanolic Plant Extracts v1

Quantification of total soluble sugars (as glucose) in plant tissue extracts via the sulfuric phenol method adapted for 96 well plates.

Heregulin Activity Assays for Residual Testing of Cell Therapy Products

Background Heregulin is a ligand for the protooncogene product ErbB/HER that acts as a key mitogenic factor for human Schwann cells (hSCs). Heregulin is required for sustained hSC growth in vitro but must be thoroughly removed before cell collection for transplantation due to potential safety concerns. The goal of this study was to develop simple cell-based assays to assess the effectiveness of heregulin addition to and removal from aliquots of hSC culture medium. These bioassays were based on the capacity of a β1-heregulin peptide to elicit ErbB/HER receptor signaling in adherent ErbB2+/ErbB3+ cells. Results Western blotting was used to measure the activity of three different β1-heregulin/ErbB-activated kinases (ErbB3/HER3, ERK/MAPK and Akt/PKB) using phospho-specific antibodies against key activating residues. The duration, dose-dependency and specificity of β1-heregulin-initiated kinase phosphorylation were investigated, and controls were implemented for assay optimization and reproducibility to detect β1-heregulin activity in the nanomolar range. Results from these assays showed that the culture medium from transplantable hSCs elicited no detectable activation of the aforementioned kinases in independent rounds of testing, indicating that the implemented measures can ensure that the final hSC product is devoid of bioactive β1-heregulin molecules prior to transplantation. Conclusions These assays may be valuable to detect impurities such as undefined soluble factors or factors for which other biochemical or biological assays are not yet available. Our workflow can be modified as necessary to determine the presence of ErbB/HER, ERK, and Akt activators other than β1-heregulin using native samples, such as fresh isolates from cell- or tissue extracts in addition to culture medium.

ROS Metabolism Perturbation as an Element of Mode of Action of Allelochemicals

The allelopathic interaction between plants is one of the elements that influences plant communities. It has been commonly studied by applying tissue extracts onto the acceptors or by treating them with isolated allelotoxins. Despite descriptive observations useful for agricultural practice, data describing the molecular mode of action of allelotoxins cannot be found. Due to the development of -omic techniques, we have an opportunity to investigate specific reactive oxygen species (ROS)-dependent changes in proteome or transcriptome that are induced by allelochemicals. The aim of our review is to summarize data on the ROS-induced modification in acceptor plants in response to allelopathic plants or isolated allelochemicals. We present the idea of how ROS are involved in the hormesis and plant autotoxicity phenomena. As an example of an -omic approach in studies of the mode of action of allelopatic compounds, we describe the influence of meta-tyrosine, an allelochemical exudated from roots of fescues, on nitration—one of nitro-oxidative posttranslational protein modification in the roots of tomato plants. We conclude that ROS overproduction and an induction of oxidative stress are general plants’ responses to various allelochemicals, thus modification in ROS metabolisms is regarded as an indirect mode of action of allelochemicals.

Insulin-Degrading Enzyme: Paradoxes and Possibilities

More than seven decades have passed since the discovery of a proteolytic activity within crude tissue extracts that would become known as insulin-degrading enzyme (IDE). Certainly much has been learned about this atypical zinc-metallopeptidase; at the same time, however, many quite fundamental gaps in our understanding remain. Herein, I outline what I consider to be among the most critical unresolved questions within the field, many presenting as intriguing paradoxes. For instance, where does IDE, a predominantly cytosolic protein with no signal peptide or clearly identified secretion mechanism, interact with insulin and other extracellular substrates? Where precisely is IDE localized within the cell, and what are its functional roles in these compartments? How does IDE, a bowl-shaped protein that completely encapsulates its substrates, manage to avoid getting “clogged” and thus rendered inactive virtually immediately? Although these paradoxes are by definition unresolved, I offer herein my personal insights and informed speculations based on two decades working on the biology and pharmacology of IDE and suggest specific experimental strategies for addressing these conundrums. I also offer what I believe to be especially fruitful avenues for investigation made possible by the development of new technologies and IDE-specific reagents. It is my hope that these thoughts will contribute to continued progress elucidating the physiology and pathophysiology of this important peptidase.

Methods for simultaneous determination of legacy and insensitive munition (IM) constituents in aqueous, soil/sediment, and tissue matrices

Currently, no standard method exists for analyzing insensitive munition (IM) compounds in environmental matrices, with or without concurrent legacy munition compounds, resulting in potentially inaccurate determinations. The primary objective of this work was to develop new methods of extraction, pre-concentration, and analytical separation/quantitation of 17 legacy munition compounds along with several additional IM compounds, IM breakdown products, and other munition compounds that are not currently included in U.S. Environmental Protection Agency (EPA) Method 8330B. Analytical methods were developed to enable sensitive, simultaneous detection and quantitation of the 24 IM and legacy compounds, including two orthogonal high-performance liquid chromatography (HPLC) column separations with either ultraviolet (UV) or mass spectrometric (MS) detection. Procedures were developed for simultaneous extraction of all 24 analytes and two surrogates (1,2-dinitrobenzene, 1,2-DNB; o-NBA) from high- and low-level aqueous matrices and solid matrices, using acidification, solid phase extraction (SPE), or solvent extraction (SE), respectively. The majority of compounds were recovered from four tissue types within current limits for solids, with generally low recovery only for Tetryl (from 4 to 62%). A preparatory chromatographic interference removal procedure was adapted for tissue extracts, as various analytical interferences were observed for all studied tissue types.

A robust method of extraction and GC-MS analysis of Monophenols exhibited UV-B mediated accumulation in Arabidopsis

Studies on specialised metabolites like phenolics are of immense interest owing to their significance to agriculture, nutrition and health. In plants, phenolics accumulate and exhibits spatial and temporal regulations in response to growth conditions. Robust methodologies aimed at efficient extraction of plant phenolics, their qualitative and quantitative analysis is desired. We optimised the analytical and experimental bottlenecks that captured free, ester, glycoside and wall-bound phenolics after acid or alkali treatments of the tissue extracts and subsequent GC-MS analysis. Higher recovery of phenolics from the methanolic extracts was achieved by through a) Ultrasonication assisted extraction along with Methyl tert-butyl ether (MTBE) enrichment b) nitrogen gas drying and c) their derivatisation using MSTFA for GC-MS analysis. The optimised protocol was tested on Arabidopsis rosette exposed to UV-B radiation (280-315 nm) which triggered enhanced levels of 11 monophenols and might be attributed to photoprotection and other physiological roles. Interestingly, coumaric acid (308 m/z) and caffeic acid (396 m/z) levels were enhanced by 12-14 folds under UV-B. Other phenolics such as cinnamic acid (220 m/z), hydroxybenzoic acid (282 m/z), vanillic acid (312 m/z, gallic acid (458 m/z), ferulic acid (338 m/z), benzoic acid (194 m/z), hydroxycinnamic acid (368 m/z) and protocatechuic acid (370 m/z) also showed elevated levels by about 1 to 4 folds. Notably, vanillin (253 m/z) was detected only in the UV-B exposed tissues. The protocol also comprehensively captured the variations in the levels of ester, glycoside and wall-bounded phenolics with high reproducibility and sensitivity. The robust method of extraction and GC-MS analysis can readily be adopted for studying phenolics in plant systems.