Miroslav Kvasnica

The Exogenous Application of Brassinosteroids to Zea mays (L.) Stressed by Long-Term Chilling Does Not Affect the Activities of Photosystem 1 or 2

The effect of various concentrations of exogenously applied 24-epibrassinolide (E) and 2α,3α,17β-trihydroxy-5α-androstan-6-one (A) on the activities of Photosystem 1 and the Hill reaction, the contents of photosynthetic pigments, and the growth of plants was examined in young maize (Zea mays L.) plants subjected to long-term chilling stress or grown in normal-temperature conditions. Neither the activity of Photosystem 1 nor the Hill reaction activity of plants was in any way affected by the treatment with brassinosteroids (BRs), which suggests that the photosynthetic complexes of thylakoid membranes are not the primary site of the influence of BRs on photosynthesis. An extremely low (10−14 M) concentration of A applied to the nonstressed plants significantly increased the length of their 4th to the 7th leaves and their height, as well as the contents of chlorophylls a and b and total carotenoids. However, under chilling conditions, this positive effect was significant for the chlorophyll content only and higher concentrations of BRs (10−12, 10−10, 10−8 M) usually had no effect at all.

The effect of brassinosteroids on the morphology, development and yield of field-grown maize.

The response of two field-grown inbred lines of maize (Zea mays L.) and their F1 hybrid to the application of 10−8–10−14 M solutions of 24-epibrassinolide or synthetic androstane analogue of castasterone in V3/4 and V6/7 developmental stages was followed during the vegetative and early reproductive phases of plant development. Brassinosteroids (BRs) significantly affected (either positively or negatively, depending on the genotype and the developmental stage they were applied) the height of plants during the early weeks after their application, but not the final plant height nor the number of leaves. Spraying of plants with BRs in V3/4 developmental stage usually also increased the length of the 7th to 10th leaf, whereas the application in V6/7 developmental stage had the opposite effect. The beginning of the reproductive phase of plant development and the course of flowering was strongly influenced by the application of BRs. Treatment of plants in V3/4 stage delayed and treatment of plants in V6/7 stage advanced the dates of anthesis and silking, regardless of the type of BR used, its concentration or plant genotype. The influence of BRs on the development of the secondary ear was the least pronounced in the F1 hybrid; in both inbred lines it strongly depended on the concentrations of BRs used. Various yield parameters were also affected by treatment of plants with BRs, but this effect depended on the developmental stage during which the application of BRs occured, the plant genotype, the type of BR and its concentration.

The effects of brassinosteroids on photosynthetic parameters in leaves of two field-grown maize inbred lines and their F1 hybrid

The effect of foliar spray with 10−12 M aqueous solutions of 24-epibrassinolide or a synthetic androstane analogue of castasterone on the activity of photosystem (PS) 1, the Hill reaction activity, the content of photosynthetic pigments and the specific leaf mass was examined for three different leaves developed after brassinosteroid (BR) treatment in two inbred lines of field-grown maize and their F1 hybrid. The brassinosteroids significantly affected neither the efficiency of photosynthetic electron transport, nor the content of chlorophylls or carotenoids.

Biological activities of new monohydroxylated brassinosteroid analogues with a carboxylic group in the side chain

Thirteen monohydroxylated brassinosteroids analogues were synthesized and tested for their biological activity in plant and animal systems. The cytotoxic activity of the products was studied using human normal and cancer cell lines with 28-homocastasterone as positive control, their brassinolide type activity was established using the bean second-internode test with 24-epibrassinolide as standard.

15N-labelled pyrazines of triterpenic acids

Triterpenoid pyrazines from our research group were found selectively cytotoxic on several cancer cell lines with IC50 in low micromolar range. This sparked our interest in preparing their labeled analogs for metabolic studies. In this work, we prepared a set of non-labeled pyrazines from seven triterpenoid skeletal types along with their 15N labelled analogs. In this work, we present the synthesis and characterization of the target 15N labelled pyrazines. Currently, these compounds are being studied in complex metabolic studies.

Synthesis and antiproliferative properties of new hydrophilic esters of triterpenic acids

To improve the properties of cytotoxic triterpenoid acids 1-5, a large set of hydrophilic esters was synthesized. We choose betulinic acid (1), dihydrobetulinic acid (2), 21-oxoacid 3 along with highly active des-E lupane acids 4 and 5 as a model set of compounds for esterification of which the properties needed to be improved. As ester moieties were used - methoxyethanol and 2-(2-methoxyethoxy)ethanol and glycolic unit (type a-d), pyrrolidinoethanol, piperidinoethanol and morpholinoethanol (type f-h), and monosaccharide groups (type i-l). As a result, 56 triterpenic esters (49 new compounds) were obtained and their cytotoxicity on four cancer cell lines and normal human fibroblasts was tested. All new compounds were fully soluble at all tested concentrations, which used to be a problem of the parent compounds 1 and 2. 16 compounds had IC50xa0<xa010xa0μM on at least one cancer cell line, 12 compounds had cytotoxicity of <10xa0μM against at least three of four tested cancer cell lines. The highest activity was found for compound 3c (1.8xa0μM on MCF7, 2.8xa0μM on HeLa, and 1.6xa0μM on G-361xa0cells) which also had no toxicity on non-cancerous BJ fibroblasts at the highest tested concentration (50xa0μM). High selective cytotoxicity was also found in compounds 1k, 2k, 3c, and 3i that are ideal candidates for drug development. Therefore, more studies to identify the mechanism of action were performed in case of 1k, 3c, and 3g such as effects on cell cycle and apoptosis. It was found that compounds 3c and 3g can induce apoptosis via caspase-3 activation and modulation of protein Bcl-2 in G-361xa0cells. In conclusion, compounds 1k, 3c, and 3g show high and selective cytotoxicity, therefore they are significantly better candidates for anti-cancer drug development than the parent acids 1-5.

Synthesis of 3α-deuterated 7α-hydroxy-DHEA and 7-oxo-DHEA and application in LC-MS/MS plasma analysis.

7-Oxygenated metabolites of dehydroepiandrosterone (DHEA) are known for their neuroprotective and immunomodulatory properties. These neuroactive steroids are currently predominately analysed by mass spectrometry, for which the use of internal deuterated standards is necessary. The aim of this study was to synthesize the deuterated derivatives of 7α-hydroxy-DHEA and 7-oxo-DHEA and test them in liquid chromatography-tandem mass spectrometry (LC-MS/MS) in order to enhance the performance characteristics of this method. Here we report the synthesis of 3α deuterium-labelled 7α-hydroxy-DHEA and 7-oxo-DHEA. Deuterium was introduced into the 3α position by reduction of the corresponding 3-ketone with a protected 17-carbonyl group using NaBD4. Our new procedure allows the easier synthesis of deuterated steroid labelled compounds. The use of these deuterated steroids enabled us to improve the human plasma LC-MS/MS analysis of 7α-hydroxy-DHEA and 7-oxo-DHEA in terms of sensitivity, precision and recovery.

Synthesis of novel aryl brassinosteroids through alkene cross-metathesis and preliminary biological study

HIGHLIGHTSA series of phenyl analogues of brassinosteroids was prepared via cross‐metathesis.All new brassinosteroid analogues were docked into BRI1 receptor kinase.The activity was measured by the pea inhibition biotest and Arabidopsis root assay.Differences in the production of plant hormone ethylene were also studied. ABSTRACT A series of phenyl analogues of brassinosteroids was prepared via alkene cross‐metathesis using commercially available styrenes and 24‐nor‐5&agr;‐chola‐2,22‐dien‐6‐one. All derivatives were successfully docked into the active site of BRI1 using AutoDock Vina. Plant growth promoting activity was measured using the pea inhibition biotest and Arabidopsis root sensitivity assay and then was compared with naturally occuring brassinosteroids. Differences in the production of plant hormone ethylene were also observed in etiolated pea seedlings after treatment with the new and also five known brassinosteroid phenyl analogues. Antiproliferative activity was also studied using normal human fibroblast and human cancer cell lines.

Regulation of the membrane structure by brassinosteroids and progesterone in winter wheat seedlings exposed to low temperature

Graphical abstract Figure. No Caption available. HighlightsBrassinosteroids and progesterone alter structural properties of wheat membranes.Interaction of polar groups of steroids and lipids changes membrane flexibility.Steroid affinity to membranes depends on wheat cultivar/growth conditions.This is the first report describing brassinosteroid interaction with the lipid monolayer. Abstract Steroids constitute one of the most important groups of compounds of regulatory properties both in the animal and plant kingdom. In plants, steroids such as brassinosteroids or progesterone, by binding to protein receptors in cell membranes, regulate growth and initiate processes leading to increased tolerance to stress conditions. Due to their structural similarities to sterols, these steroids may also directly interact with cellular membranes. Our aim was to determine the changes of the structural parameters of lipid membranes under the influence of hydrophobic steroid compounds, i.e., 24‐epibrassinolide (EBR) and its precursor—24‐epicastasterone (ECS) and progesterone (PRO). Lipids were isolated from wheat seedlings with different tolerances to frost, grown at low temperatures (5 °C) for 1.5 and 3 weeks (acclimation process). Control plants were cultured continuously at 20 °C. From galactolipids and phospholipids, the main polar lipid fractions, the monolayers were formed, using a technique of Langmuir trough. EBR and ECS were introduced into monolayers, together with lipids, whereas the PRO was dissolved in the aqueous sub‐phase upon which the monolayers were spread. Measurements performed at 25 °C and 10 °C showed a significant action of the tested compounds on the physicochemical properties of the monolayers. EBR and PRO increased the area per lipid molecule in monolayers, resulting in formation of more flexible surface structures while the presence of the ECS induced the opposite effect. The influence of the polarity of lipids and steroids on the interactions in the monolayer was discussed. Lipids extracted from the membranes of wheat with the most tolerance to frost were characterized by the highest fatty acid unsaturation and steroids had a relatively weak effect on the parameters of the structure of their monolayers.

The novel brassinosteroid analog BR4848 inhibits angiogenesis in human endothelial cells and induces apoptosis in human cancer cells in vitro

We report the synthesis and detailed biological study of the synthetic brassinosteroid analog 2α,3α-dihydroxy-6-oxo-5α-androstan-17β-yl N-(tert-butoxycarbonyl)-D,L-valinate (BR4848). The panel of cancer cell lines was used for characterization of its antiproliferative activity, yet had no adverse effects in normal human fibroblasts. In HeLa cells, BR4848-induced apoptosis was accompanied by increase of apoptotic subG1 cells, PARP-1 and caspase-7 fragmentation, downregulation of Bcl-2 and Mcl-1, an increase in caspase activity and G2/M phase cell cycle arrest. Antiproliferative properties of BR4848 were exhibited by inhibition of phosphorylation of Akt, Erk1/2 and FAK. Furthermore, the developed analog exhibited in vitro antiangiogenic activity in human umbilical vein endothelial cells (HUVECs). BR4848-induced apoptosis accompanied with G2/M arrest was detected in endothelial cells. BR4848 also inhibited adhesion, tube formation and migration of endothelial cells by inhibition of FAK, Erk 1/2, CDK5, VEGFR2, TNFα-stimulated production of IL-6, angiopoietin-2 and Jagged1. Finally, BR4848 did not modulate the activity nor nuclear translocation of any of the steroid receptors (ERα, ERβ, AR, MR and PR) included in reporter cell-based assays, which excludes the genomic activity of steroid receptors as a contributing factor to the observed biological activities of BR4848.