Karel Královec

Albumin‐coated monodisperse magnetic poly(glycidyl methacrylate) microspheres with immobilized antibodies: Application to the capture of epithelial cancer cells

Monodisperse (4 μm) macroporous crosslinked poly(glycidyl methacrylate) (PGMA) microspheres for use in microfluidic immunomagnetic cell sorting, with a specific application to the capture of circulating tumor cells (CTCs), were prepared by multistep swelling polymerization in the presence of cyclohexyl acetate porogen and hydrolyzed and ammonolyzed. Iron oxide was then precipitated in the microspheres to render them magnetic. Repeated precipitation made possible to raise the iron oxide content to more than 30 wt %. To minimize nonspecific adsorption of the microspheres in a microchannel and of cells on the microspheres, they were coated with albumin crosslinked with glutaraldehyde. Antibodies of epithelial cell adhesion molecule (anti-EpCAM) were then immobilized on the albumin-coated magnetic microspheres using the carbodiimide method. Capture of breast cancer MCF7 cells as a model of CTCs by the microspheres with immobilized anti-EpCAM IgG was performed in a batch experiment. Finally, MCF7 cells were captured by the anti-EpCAM-immobilized albumin-coated magnetic microspheres in an Ephesia chip. A very good rejection of lymphocytes was achieved. Thus, albumin-coated monodisperse magnetic PGMA microspheres with immobilized anti-EpCAM seem to be promising for capture of CTCs in a microfluidic device.

The effect of Amaryllidaceae alkaloids haemanthamine and haemanthidine on cell cycle progression and apoptosis in p53-negative human leukemic Jurkat cells

Plants from the Amaryllidaceae family have been shown to be a promising source of biologically active natural compounds of which some selected are currently in pre-clinical development. Regardless of interesting pioneer works, little is known about Amaryllidaceae alkaloids that have shown promising anti-cancer activities. The crinane group of the Amaryllidaceae, including haemanthamine and haemanthidine, was amongst the first of these compounds to exhibit an interesting cytotoxic potential against cancer cell lines. However, the mechanism of cytotoxic and anti-proliferative activity is not yet entirely clear. The primary objectives of the current study were to investigate the effects of haemanthamine and haemanthidine on the induction of apoptosis and the cell cycle regulatory pathway in p53-null Jurkat cells. Results indicate that haemanthamine and haemanthidine treatment decreases cell viability and mitochondrial membrane potential, leads to a decline in the percentage of cells in the S phase of the cell cycle, induces apoptosis detected by Annexin V staining and increases caspase activity. Dose dependent apoptosis was cross verified by fluorescence and bright field microscopy through Annexin V/propidium iodine staining and morphological changes which characteristically attend programmed cell death. The apoptotic effect of haemanthamine and haemanthidine on leukemia cells is more pronounced than that of gamma radiation. Contrary to gamma radiation, Jurkat cells do not completely halt the cell cycle 24h upon haemanthamine and haemanthidine exposure. Both Amaryllidaceae alkaloids accumulate cells preferentially at G1 and G2 stages of the cell cycle with increased p16 expression and Chk1 Ser345 phosphorylation. Concerning the pro-apoptotic effect, haemanthidine was more active than haemanthamine in the Jurkat leukemia cell line.

Synthesis and in vitro evaluation of new derivatives of 2-substituted-6-fluorobenzo[d]thiazoles as cholinesterase inhibitors

A series of novel cholinesterase inhibitors based on 2-substituted 6-fluorobenzo[d]thiazole were synthesised and characterised by IR, (1)H, (13)C and (19)F NMR spectroscopy and HRMS. Purity was checked by elemental analyses. The novel carbamates were tested for their ability to inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The toxicity of the most active compounds was investigated using a standard in vitro test with HepG2 cells, and the ratio between biological activity and toxicity was determined. In addition, the toxicity of the most active compounds was evaluated against MCF7 cells using the xCELLigence system. Structure-activity relationships reflecting the dependence of cholinesterase inhibitors on the lipophilicity of the compounds as well as on the Taft polar and steric substituent constants are discussed. The specific orientation of the inhibitors in the binding site of acetylcholinesterase was determined using molecular docking of the most active compound.

Inverse Correlation Between Plasma Beta-Carotene and Interleukin-6 in Patients with Advanced Coronary Artery Disease

The interrelationships between plasma beta-carotene, alpha-tocopherol, and the level of systemic inflammation and oxidative stress were investigated in patients with advanced coronary artery disease (CAD). Plasma beta-carotene, alpha-tocopherol, malondialdehyde, free radicals, interleukin-6, high sensitive C-reactive protein levels, and other risk factors of CAD were determined in a group of patients with advanced CAD [significant stenosis according to coronarographic examination (n=91) and a control group of examined patients with coronary arteries with no stenosis (n=49)]. Between-group differences in continuous variables were analyzed with the Hotelling T2-test (software NCSS2000), analyses of correlation matrix with the software STATISTICA. Advanced CAD coincided with significantly lower plasma concentrations of high-density lipoprotein (HDL)-cholesterol and beta-carotene as well as with elevated levels of all inflammatory markers, but only with mild increase of oxidative stress. Beta-carotene significantly inversely correlated with interleukin-6. This inverse correlation could suggest potential protective effect of beta-carotene on atherosclerosis due to the inhibition of inflammatory processes.

Synthesis, structural characterization, docking, lipophilicity and cytotoxicity of 1-[(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]-3-alkyl carbamates, novel acetylcholinesterase and butyrylcholinesterase pseudo-irreversible inhibitors

In the current study, sixteen novel derivatives of (R)-1-(6-fluorobenzo[d]thiazol-2-yl)ethanamine were synthesized as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors. Chemical structures together with purity of the synthesized compounds were substantiated by IR, (1)H, (13)C, (19)F NMR, high resolution mass spectrometry and elemental analysis. The optical activities were confirmed by optical rotation measurements. The synthesized compounds were evaluated for their AChE and BChE inhibitory activities. In addition, the cytotoxicity of the most active compounds was investigated against human cell lines employing XTT tetrazolium salt reduction assay and xCELLigence system allowing a label-free assessment of the cells proliferation. Our results demonstrated that the inhibitory mechanism was confirmed to be pseudo-irreversible, in line with previous studies on carbamates. Compounds indicated as 3b, 3d, 3l and 3n showed the best AChE inhibitory activity of all the evaluated compounds and were up to tenfold more potent than standard drug rivastigmine. The binding mode was determined using state-of-the-art covalent docking and scoring methodology. The obtained data clearly demonstrated that 3b, 3d, 3l and 3n benzothiazole carbamates possess high inhibitory activity against AChE and BChE and concurrently negligible cytotoxicity. In conclusion, our results indicate, that these derivatives could be promising in an effective therapeutic intervention for Alzheimers disease.

Silica-coated manganite and Mn-based ferrite nanoparticles: a comparative study focused on cytotoxicity

Magnetic oxide nanoparticles provide a fascinating tool for biological research and medicine, serving as contrast agents, magnetic carriers, and core materials of theranostic systems. Although the applications rely mostly on iron oxides, more complex oxides such as perovskite manganites may provide a much better magnetic performance. To assess the risk of their potential use, in vitro toxicity of manganite nanoparticles was thoroughly analysed and compared with another prospective system of Mn–Zn ferrite nanoparticles. Magnetic nanoparticles of La0.63Sr0.37MnO3 manganite were prepared by two distinct methods, namely the molten salt synthesis and the traditional sol–gel route, whereas nanoparticles of Mn0.61Zn0.42Fe1.97O4 ferrite, selected as a comparative material, were synthesized by a new procedure under hydrothermal conditions. Magnetic cores were coated with silica and, moreover, several samples of manganite nanoparticles with different thicknesses of silica shell were prepared. The size-fractionated and purified products were analysed using transmission electron microscopy, dynamic light scattering, measurement of the zeta-potential dependence on pH, IR spectroscopy, and SQUID magnetometry. The silica-coated products with accurately determined concentration by atomic absorption spectroscopy were subjected to a robust evaluation of their cytotoxicity by four different methods, including detailed analysis of the concentration dependence of toxicity, analysis of apoptosis, and experiments on three different cell lines. The results, comparing two manganese-containing systems, clearly indicated superior properties of the Mn–Zn ferrite, whose silica-coated nanoparticles show very limited toxic effects and thus constitute a promising material for bioapplications.

Synthesis and characterization of a pH-sensitive conjugate of isoniazid with Fe3O4@SiO2 magnetic nanoparticles

The Letter describes the preparation and characterization of a conjugate of isoniazid (INH) with magnetic nanoparticles Fe3O4@SiO2 115±60 nm in size. The INH molecules were attached to the surface of nanoparticles by a covalent pH-sensitive amidine bond. The conjugate was characterized by X-ray diffraction, SEM, dynamic light scattering, IR spectroscopy and microanalysis. The conjugate released isoniazid under in vitro conditions (pH=4; 37 °C; t1/2≈115 s). In addition, the cytotoxicity of the Fe3O4@SiO2-INH conjugate was evaluated in SK-BR-3 cells using the xCELLigence system.

Specific inhibition of Wee1 kinase and Rad51 recombinase: A strategy to enhance the sensitivity of leukemic T-cells to ionizing radiation-induced DNA double-strand breaks

Present-day oncology sees at least two-thirds of cancer patients receiving radiation therapy as a part of their anticancer treatment. The objectives of the current study were to investigate the effects of the small molecule inhibitors of Wee1 kinase II (681641) and Rad51 (RI-1) on cell cycle progression, DNA double-strand breaks repair and apoptosis following ionizing radiation exposure in human leukemic T-cells Jurkat and MOLT-4. Pre-treatment with the Wee1 681641 or Rad51 RI-1 inhibitor alone increased the sensitivity of Jurkat cells to irradiation, however combining both inhibitors together resulted in a further enhancement of apoptosis. Jurkat cells pre-treated with inhibitors were positive for γH2AX foci 24h upon irradiation. MOLT-4 cells were less affected by inhibitors application prior to ionizing radiation exposure. Pre-treatment with Rad51 RI-1 had no effect on apoptosis induction; however Wee1 681641 increased ionizing radiation-induced cell death in MOLT-4 cells.

Development of the olfactory and vomeronasal organs in Discoglossus pictus (Discoglossidae, Anura)

Using histological techniques and computer‐aided three‐dimensional reconstructions of histological serial sections, we studied the development of the olfactory and vomeronasal organs in the discoglossid frog Discoglossus pictus. The olfactory epithelium in larval D. pictus represents one continuous unit of tissue not divided into two separate portions. However, a small pouch of olfactory epithelium (the “ventromedial diverticulum”) is embedded into the roof of the buccal cavity, anteromedial to the internal naris. The lateral appendix is present in D. pictus through the entire larval period and disappears during the onset of metamorphosis. The disappearance of the lateral appendix at this time suggests that it is a typical larval organ related to aquatic life. The vomeronasal organ develops during hindlimb development, which is comparatively late for anurans. The development of the vomeronasal organ in D. pictus follows the same general developmental pattern recognized for neobatrachians. As with most anurans, the vomeronasal glands appear later than the vomeronasal organ. After metamorphosis, the olfactory organ of adult D. pictus is composed of a series of three interconnected chambers: the cavum principale, cavum medium, and cavum inferius. We suggest that the ventromedial diverticulum at the anterior border of the internal naris of larval D. pictus might be homologous with the ventral olfactory epithelium of bufonids and with the similar diverticulum of Alytes. J. Morphol. 2013.

Synthesis, characterization and in vitro evaluation of substituted N-(2-phenylcyclopropyl)carbamates as acetyl- and butyrylcholinesterase inhibitors.

Abstract A serie of O-substituted N-2-phenylcyclopropylcarbamates was prepared and characterized. These carbamates were tested as inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). It was found, that these compounds exhibit moderate inhibition activity with values of IC50 in the range of 54.8–94.4 μM (for AChE) and up to 5.8 μM (for BChE). The AChE/BChE selectivity for each carbamate was calculated. These values varied from 0.50 to 9.46, two carbamate derivatives inhibited only AChE selectively. The most promising derivative was prepared in all optically pure forms (four isomers). It was found that individual stereoisomers differed only slightly in the inhibition ability. The cytotoxicity of all carbamates was evaluated using the standard in vitro test with Jurkat cells. With regard to their inhibition activity and cytotoxicity as well as easy preparation, O-substituted N-2-phenylcyclopropylcarbamates can be considered as promising compounds for potential medicinal applications.