Ladislav Kohout

Fluorescent castasterone reveals BRI1 signaling from the plasma membrane

Receptor-mediated endocytosis is an integral part of signal transduction as it mediates signal attenuation and provides spatial and temporal dimensions to signaling events. One of the best-studied leucine-rich repeat receptor-like kinases in plants, BRASSINOSTEROID INSENSITIVE 1 (BRI1), perceives its ligand, the brassinosteroid (BR) hormone, at the cell surface and is constitutively endocytosed. However, the importance of endocytosis for BR signaling remains unclear. Here we developed a bioactive, fluorescent BR analog, Alexa Fluor 647-castasterone (AFCS), and visualized the endocytosis of BRI1-AFCS complexes in living Arabidopsis thaliana cells. Impairment of endocytosis dependent on clathrin and the guanine nucleotide exchange factor for ARF GTPases (ARF-GEF) GNOM enhanced BR signaling by retaining active BRI1-ligand complexes at the plasma membrane. Increasing the trans-Golgi network/early endosome pool of BRI1-BR complexes did not affect BR signaling. Our findings provide what is to our knowledge the first visualization of receptor-ligand complexes in plants and reveal clathrin- and ARF-GEF-dependent endocytic regulation of BR signaling from the plasma membrane.

New Techniques for the Estimation of Naturally Occurring Brassinosteroids

We have developed enzyme-linked immunosorbent assays (ELISAs) for measuring 24-epicastasterone and related brassinolide analogs, with detection ranges of 0.005 to 50 pmoles. Polyclonal antibodies used in these assays were raised against 24-epicastasterone carboxymethyloxime-bovine serum albumin conjugates and were found to have high specificity for 24-epibrassinosteroids. Natural brassinosteroids (BRs), such as brassinolide and 24-epibrassinolide, exhibited relatively high cross-reactivities with the generated antibodies, whereas other BR analogs with β-oriented hydroxyl groups at C-2, C-3, C-22, and C23 lacked immunoreactivity. Through the use of internal standardization, dilution assays, recovery of authentic [3H]24-epicastasterone, and immunohistograms, the ELISAs have been shown to be applicable for estimating 24-epibrassinosteroid levels in crude plant extracts. To analyze brassinosteroids in tissues from young bean (Phaseolus vulgaris L., cv. Pinto), Daucus carota ssp.sativus plants and Arabidopsis thaliana L. Heynh. seedlings, and rape (Brassica napus L.) pollen, the extracts were fractionated by high performance liquid chromatography (HPLC) and the resulting fractions were analyzed by the ELISA method. Immunohistogram ELISA analysis of HPLC fractions indicated that major peaks of immunoreactivity co-chromatographed with the labeled and unlabeled 24-epibrassinolide. A highly sensitive electrospray ionization mass spectrometry (MS) technique (LOD: 50 fmol) was also developed and the results obtained by the HPLC-ELISA and HPLC-MS approaches were compared.

Platinum(II) complexes with steroidal esters of l-methionine and l-histidine: Synthesis, characterization and cytotoxic activity

Twelve steroidal platinum(II) complexes were synthesized by reaction of potassium tetrachloroplatinate with steroidal esters of L-methionine and L-histidine. The steroidal esters coordinated as bidentate ligands via S and N donor atoms of L-methionine and via two N donor atoms of L-histidine. Cholesterol, cholestanol, diosgenine, pregnenolone, dehydroepiandrosterone, testosterone, estrone, and estradiol were used as the steroidal compounds. The esters and complexes prepared were characterized by infrared, mass, and (1)H NMR spectroscopy and elemental analysis. Platinum complexes were tested for in vitro cytotoxicity against several cancer cell lines: T-lymphoblastic leukemia CEM, breast carcinoma MCF-7, lung carcinoma A-549, multiple myeloma RPMI 8226, and one normal cell line human fibroblast BJ.

Brassinosteroids: synthesis and activity of some fluoro analogues.

Three types of 5alpha-androstane and ergostane analogues of brassinolide, containing a fluorine atom in either the 3alpha or the 5alpha positions or in 3alpha and 5alpha positions, were prepared using standard operations (reaction of 3beta-alcohols with (diethylamino)sulfur trifluoride, cleavage of epoxide with HF in py or BF 3.Et 2O). The 5alpha-fluorine was found to affect chemical reactivity (e.g., electrophilic addition to the Delta (2)-double bond) as well as physical properties (e.g., NMR, chromatographic behavior) of the products. Cytotoxicity of the products was studied using human normal and cancer cell lines with 28-homocastasterone as positive control and their brassinolide type activity was established using the bean second-internode test with 24-epibrassinolide as standard. The equivalence of F and OH groups was observed in some of the active compounds. The anticancer and the brassinolide-type activity do not correlate with each other: ergostane derivatives were most active in the former test while androstane derivatives were best in the latter.

Synthesis and cytotoxic activities of estrone and estradiol cis-dichloroplatinum(II) complexes.

Sixteen platinum(II) complexes of estrone and estradiol were synthesized in this work to evaluate their cytotoxic activity against several cancer cell lines including estrogen dependent and independent ones. The synthesis of all the complexes was done in three steps. The reaction of steroids with dibromoalkanes was followed by a reaction of the bromoalkyl steroids with 2-(aminomethyl)pyridine or 2-(2-aminoethyl)pyridine. The last step was a reaction of steroidal diamino ligands with potassium tetrachloroplatinate to obtain the desired platinum(II) complexes. Cytotoxicity assays showed that most of the complexes prepared are active against the cancer cell lines used-CEM, U-2 OS, MCF7, MCF7 AL, MDA-MB-468, BT-474, BT-549, and BJ fibroblasts. The six-membered platinum complexes are more active than five-membered ones.

Affinity chromatography reveals RuBisCO as an ecdysteroid-binding protein

The aim of this work was to isolate plant ecdysteroid-binding proteins using affinity chromatography. Ecdysteroids as insect hormones have been investigated thoroughly but their function and the mechanism of action in plants and other organisms is still unknown although ecdysteroids occur in some plants in a relatively large amount. Therefore, 20-hydroxyecdysone was immobilized on a polymeric carrier as a ligand for affinity chromatography in order to isolate plant ecdysteroid-binding proteins from the cytosolic extract of New Zealand spinach (Tetragonia tetragonoides). Non-specifically bound proteins were eluted with a rising gradient of concentration of sodium chloride, and 3% (v/v) acetic acid was used for the elution of the specifically bound proteins. Using this method, ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) was isolated. The influence of ecdysteroids on RuBisCO was further studied. Our results show that ecdysteroids are able to increase the yield of RuBisCO-mediated reaction in which CO(2) is fixed into organic matter by more than 10%.

Steroids with modified ring A or B: Screening for potential antiandrogenic and synandrogenic activity

Abstract A series of 14 steroids with modified ring A or B (A-homosteroids, B-homosteroids and 3,6-cyclo-A-nor-3,5-seco-6β-androstanes) were tested for potential antiandrogenic activity by binding assays, using prostate cytosol receptor or sex hormone binding globulin as binder, and by the inhibition of 5α-reductase. The results of in vitro screening were compared with in vivo bioassay. The antiandrogenic or synandrogenic activity of these compounds seemed to have an integrated effect on steroid binding to the cytosol receptors, displacement of the endogenous androgens from the binding to sex hormone binding globulin and influence the transformation of testosterone into 5α-dihydrotestosterone.

The effect of exogenous 24-epibrassinolide on the ecdysteroid content in the leaves of Spinacia oleracea L.

The aim of this study was to show whether/how the application of exogenous 24-epibrassinolide can affect the content of ecdysteroids in spinach leaves. Brassinosteroids and ecdysteroids, structurally related phytosterols, show effect on a range of processes in plants. Brassinosteroids increase biomass yield in some species, photosynthesis and resistance to stress, and ecdysteroids show effect on proteins responsible for binding of CO2 or water cleavage. The mutual interaction of these sterols in plants is unclear. The UPLC-(+)ESI-MS/MS analyses of extracts of treated and untreated spinach (Spinacia oleracea L.) leaves show that the application of exogenous 24-epibrassinolide does influence the ecdysteroid content in plant tissues. The response differs for the major ecdysteroids and also differs from that for the minor ones and is dependent on the developmental stage of the leaves within the same plant or the 24-epibrassinolide concentration applied.

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.