Ilva Nakurte

Development and Validation of a Reversed-Phase Liquid Chromatography Method for the Simultaneous Determination of Indole-3-Acetic Acid, Indole-3-Pyruvic Acid, and Abscisic Acid in Barley (Hordeum vulgare L.)

A simple, sensitive, precise, and specific reverse HPLC method was developed and validated for the determination of plant hormones in barley (Hordeum vulgare L.). The method includes extraction in aqueous organic solvent followed by solid-phase extraction, sample evaporation, and reversed-phase HPLC analysis in a general purpose UV-visible (abscisic acid (ABA)) and fluorescence detection (indole-3-acetic acid (IAA) and indole-3-pyruvic acid (IPA)), high-performance liquid chromatography system. The separation was carried out on Zorbax Eclipse XDB C8 column (150  ×  4.6  mm I.D) with a mobile phase composed of methanol and 1% acetic acid (60 : 40 v/v) in isocratic mode at a flow rate of 1 ml min−1. The detection was monitored at 270 nm (ABA) and at 282 nm (Ex) and 360 nm (Em) (IAA, IPA). The developed method was validated in terms of accuracy, precision, linearity, limit of detection, limit of quantification, and robustness. The determined validation parameters are in the commonly acceptable ranges for that kind of analysis.

Differential disease resistance response in the barley necrotic mutant nec1

BackgroundAlthough ion fluxes are considered to be an integral part of signal transduction during responses to pathogens, only a few ion channels are known to participate in the plant response to infection. CNGC4 is a disease resistance-related cyclic nucleotide-gated ion channel. Arabidopsis thaliana CNGC4 mutants hlm1 and dnd2 display an impaired hypersensitive response (HR), retarded growth, a constitutively active salicylic acid (SA)-mediated pathogenesis-related response and elevated resistance against bacterial pathogens. Barley CNGC4 shares 67% aa identity with AtCNGC4. The barley mutant nec1 comprising of a frame-shift mutation of CNGC4 displays a necrotic phenotype and constitutively over-expresses PR-1, yet it is not known what effect the nec1 mutation has on barley resistance against different types of pathogens.Resultsnec1 mutant accumulated high amount of SA and hydrogen peroxide compared to parental cv. Parkland. Experiments investigating nec1 disease resistance demonstrated positive effect of nec1 mutation on non-host resistance against Pseudomonas syringae pv. tomato (Pst) at high inoculum density, whereas at normal Pst inoculum concentration nec1 resistance did not differ from wt. In contrast to augmented P. syringae resistance, penetration resistance against biotrophic fungus Blumeria graminis f. sp. hordei (Bgh), the causal agent of powdery mildew, was not altered in nec1. The nec1 mutant significantly over-expressed race non-specific Bgh resistance-related genes BI-1 and MLO. Induction of BI-1 and MLO suggested putative involvement of nec1 in race non-specific Bgh resistance, therefore the effect of nec1on mlo-5-mediated Bgh resistance was assessed. The nec1/mlo-5 double mutant was as resistant to Bgh as Nec1/mlo-5 plants, suggesting that nec1 did not impair mlo-5 race non-specific Bgh resistance.ConclusionsTogether, the results suggest that nec1 mutation alters activation of systemic acquired resistance-related physiological markers and non-host resistance in barley, while not changing rapid localized response during compatible interaction with host pathogen. Increased resistance of nec1 against non-host pathogen Pst suggests that nec1 mutation may affect certain aspects of barley disease resistance, while it remains to be determined, if the effect on disease resistance is a direct response to changes in SA signaling.

Chemical Composition Analysis, Antimicrobial Activity and Cytotoxicity Screening of Moss Extracts (Moss Phytochemistry)

Mosses have been neglected as a study subject for a long time. Recent research shows that mosses contain remarkable and unique substances with high biological activity. The aim of this study, accordingly, was to analyze the composition of mosses and to screen their antimicrobial and anticancer activity. The total concentration of polyphenols and carbohydrates, the amount of dry residue and the radical scavenging activity were determined for a preliminary evaluation of the chemical composition of moss extracts. In order to analyze and identify the substances present in mosses, two types of extrahents (chloroform, ethanol) and the GC/MS and LC-TOF-MS methods were used. The antimicrobial activity was tested on four bacteria strains, and the anticancer activity on six cancer cell lines. The obtained results show the presence of a high number of primary (fatty acids and amino acids), but mainly secondary metabolites in moss extracts—including, sterols, terpenoids, polyphenols and others—and a high activity with respect to the studied test organisms.

Collision-induced dissociation of imidazolium-based zwitterionic liquids

Fragmentation pathways of some imidazolium based zwitterionic liquids—3-(3-alkyl-1-imidazolio)-propane sulfonates and 3-(2-methyl-3-alkyl-1-imidazolio)-propane sulfonates—have been studied by tandem electrospray mass spectrometry and collision-induced dissociation. The relative abundances of the lowest energy fragment ions depend on the length of the alkyl chain at the IIN of the imidazolium ring and the cone voltage. The first fragment ions originate from the scission of Cnon aromatic–N bond of compounds investigated, but with increasing collision energy, scission of C–C bonds occurs. Aggregates of the general formula [(M + H) x + (M) y ]+ (x;y = 1–2) formed. Methyl substituted zwitterionic liquids show higher molecular stability than 3-(3-alkyl-1-imidazolio)-propane sulfonates.

Structure Effect of Imidazolium-Based Dicationic Ionic Liquids on Claisen Rearrangement

Abstract A large group of imidazolium-based dicationic ionic liquids (DILs) has been prepared in good yields. Thermal stability of all DILs has been determined. The effects of the reaction time, cation, imidazolium C2–H acidity, and anion on the Claisen rearrangement of allyl phenyl ether have been investigated. Type of anion and the presence of the acidic C2–H bond in the imidazolium moieties have proven to be essential for this reaction. The simple procedure and the possibility of avoiding the use catalysts and volatile organic solvents make this synthetic method environmentally benign and adaptable for large-scale applications. GRAPHICAL ABSTRACT

Primary culture of avian embryonic heart forming region cells to study the regulation of vertebrate early heart morphogenesis by vitamin A

BackgroundImportant knowledge about the role of vitamin A in vertebrate heart development has been obtained using the vitamin A-deficient avian in ovo model which enables the in vivo examination of very early stages of vertebrate heart morphogenesis. These studies have revealed the critical role of the vitamin A-active form, retinoic acid (RA) in the regulation of several developmental genes, including the important growth regulatory factor, transforming growth factor-beta2 (TGFβ2), involved in early events of heart morphogenesis. However, this in ovo model is not readily available for elucidating details of molecular mechanisms determining RA activity, thus limiting further examination of RA-regulated early heart morphogenesis. In order to obtain insights into RA-regulated gene expression during these early events, a reliable in vitro model is needed. Here we describe a cell culture that closely reproduces the in ovo observed regulatory effects of RA on TGFβ2 and on several developmental genes linked to TGFβ signaling during heart morphogenesis.ResultsWe have developed an avian heart forming region (HFR) cell based in vitro model that displays the characteristics associated with vertebrate early heart morphogenesis, i.e. the expression of Nkx2.5 and GATA4, the cardiogenesis genes, of vascular endothelial growth factor (VEGF-A), the vasculogenesis gene and of fibronectin (FN1), an essential component in building the heart, and the expression of the multifunctional genes TGFβ2 and neogenin (NEO). Importantly, we established that the HFR cell culture is a valid model to study RA-regulated molecular events during heart morphogenesis and that the expression of TGFβ2 as well as the expression of several TGFβ2-linked developmental genes is regulated by RA.ConclusionsOur findings reported here offer a biologically relevant experimental in vitro system for the elucidation of RA-regulated expression of TGFβ2 and other genes involved in vertebrate early cardiovascular morphogenesis.

Multi-technique approach for qualitative and quantitative characterization of furazidin degradation kinetics under alkaline conditions

Degradation of drug furazidin was studied under different conditions of environmental pH (11-13) and temperature (30-60°C). The novel approach of hybrid hard- and soft-multivariate curve resolution-alternating least squares (HS-MCR-ALS) method was applied to UV-vis spectral data to determine a valid kinetic model and kinetic parameters of the degradation process. The system was found to be comprised of three main species and best characterized by two consecutive first-order reactions. Furazidin degradation rate was found to be highly dependent on the applied environmental conditions, showing more prominent differences between both degradation steps towards higher pH and temperature. Complimentary qualitative analysis of the degradation process was carried out using HPLC-DAD-TOF-MS. Based on the obtained chromatographic and mass spectrometric results, as well as additional computational analysis of the species (theoretical UV-vis spectra calculations utilizing TD-DFT methodology), the operating degradation mechanism was proposed to include formation of a 5-hydroxyfuran derivative, followed by complete hydrolysis of furazidin hydantoin ring.

Increased Auxin Content and Altered Auxin Response in Barley Necrotic Mutant nec1

The role of hormone crosstalk in plant immunity is lately emerging as significant topic of plant physiology. Although crosstalk between salicylic acid and auxin affects plant disease resistance, molecular mechanisms of this process have not yet been uncovered in details. Mutations disrupting cyclic nucleotide-gated ion channel 4 (CNGC4) affect SA-mediated disease resistance in barley Hordeum vulgare and in A. thaliana. Significantly, decreased stomatal apertures of barley CNGC4 mutant nec1 and dwarfed stature of A. thaliana CNGC4 mutant dnd2 suggest that nonfunctional CNGC4 might be affecting also auxin signaling. Excised coleoptile elongation, stomatal conductance, and cell size measurements assaying physiological effect of exogenous auxin treatment suggested altered auxin signaling in nec1 mutant. Real-time qPCR analysis identified significant change in mRNA abundance of four auxin-related genes – YUCCA1, VT2, HVP1, and TIR1. Analysis of endogenous auxin content of nec1 plants detected ca. fourfold increase in indole acetic acid (IAA) content in nec1 leaves and roots compared to wt plants, as measured by HPLC. These results suggest that apart from SA-related disease resistance, CNGC4 functions also in auxin signaling in barley; therefore, barley nec1 mutant could serve as model system revealing role of SA-auxin crosstalk in plant disease resistance.

Pharmacological research on natural substances in Latvia: Focus on lunasin, betulin, polyprenol and phlorizin

In this concise review the current research in plant bioactive compound studies in Latvia is described. The paper summarizes recent studies on substances from edible plants (e.g., cereals and apples) or their synthetic analogues, such as peptide lunasin, as well as substances isolated from inedible plants (e.g., birch and conifer), such as pentacyclic triterpenes (e.g., betulin, betulinic acid, and lupeol) and polyprenols. Latvian researchers have been first to demonstrate the presence of lunasin in triticale and oats. Additionally, the impact of genotype on the levels of lunasin in cereals was shown. Pharmacological studies have revealed effects of lunasin and synthetic triterpenes on the central nervous system in rodents. We were first to show that synthetic lunasin causes a marked neuroleptic/cataleptic effect and that betulin antagonizes bicuculline-induced seizures (a GABA A receptor antagonist). Studies on the mechanisms of action showed that lunasin binds to dopamine D1 receptors and betulin binds to melanocortin and gamma-aminobutyric acid A receptors therefore we suggest that these receptors play an essential role in lunasins and betulins central effects. Recent studies on conifer polyprenols demonstrated the ability of polyprenols to prevent statin-induced muscle weakness in a rat model. Another study on plant compounds has demonstrated the anti-hyperglycemic activity of phlorizin-containing unripe apple pomace in healthy volunteers. In summary, research into plant-derived compounds in Latvia has been focused on fractionating, isolating and characterizing of lunasin, triterpenes, polyprenols and phlorizin using in vitro, and in vivo assays, and human observational studies.

The influence of pH on the stability of antazoline: kinetic analysis

Degradation of the drug antazoline was studied in aqueous solutions by means of pH-rate profiling (pH 0–7.4). The novel approach of Runge–Kutta numerical integration in combination with multi-parameter optimisation was applied to UV-Vis spectral data to determine a valid kinetic model and kinetic parameters of the degradation process. The overall degradation mechanism was found to be dependent on the environmental pH. In the pH range of 3.0–7.4, the formation of the antazoline hydrolysis product (N-(2-aminoethyl)-2-(N-benzylanilino)acetamide) through three different pathways (acidic, non-catalysed, and semi-alkaline hydrolysis) was observed. In highly acidic media (pH 0–2), the degradation mechanism was found to be more complex. Although the same primary degradation product formed, a colourful (dark blue/violet) intermediate was also observed and further investigated by HPLC/TOF-MS.