EFFECT OF THE ZINC PHTHALOCYANINE MEDIATED PHOTODYNAMIC THERAPY ON CYTOSKELETAL APPARATUS OF HELA CELLS

This study deals with the utilization of photosensitizer (λmax ~ 660 nm) from the group of the phthalocyanines, in photodynamic therapy. Effect of the zinc phthalocyanine photosensitizer mediated photodynamic therapy was evaluated in vitro on the tumor cell line – HeLa (cervical cancer cells) using mass spectrometry and atomic force and fluorescent microscopy techniques

Phosphorylation of human Argonaute proteins affects small RNA binding

Argonaute (Ago) proteins are highly conserved between species and constitute a direct-binding platform for small RNAs including short-interfering RNAs (siRNAs), microRNAs (miRNAs) and Piwi interacting RNAs (piRNAs). Small RNAs function as guides whereas Ago proteins are the actual mediators of gene silencing. Although the major steps in RNA-guided gene silencing have been elucidated, not much is known about Ago-protein regulation. Here we report a comprehensive analysis of Ago2 phosphorylation in human cells. We find that the highly conserved tyrosine Y529, located in the small RNA 5′-end-binding pocket of Ago proteins can be phosphorylated. By substituting Y529 with a negatively charged glutamate (E) mimicking a phosphorylated tyrosine, we show that small RNA binding is strongly reduced. Our data suggest that a negatively charged phospho-tyrosine generates a repulsive force that prevents efficient binding of the negatively charged 5′ phosphate of the small RNA

Intact cell MALDI-TOF mass spectrometric analysis of Chroococcidiopsis cyanobacteria for classification purposes and identification of possible marker proteins.

Cyanobacteria represent a bacterial phyllum characteristic by the ability to photosynthesize. They are potentially applicable for the production of useful compounds but may also cause poisoning or at least health problems as they can produce cyanotoxins. The introduction of a fast methodology is important not only for fundamental taxonomic purposes, but also for reliable identifications in biological studies. In this work, we have used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of intact cells to study Chroococcidiopsis strains. A library of the obtained reference mass spectra containing characteristic peptide/protein profiles was examined by software tools to characterize similarities and differences applicable for diagnostics and taxonomy. Both a similarity tree and heat map constructed from the mass spectrometric data proved consistent with 16S rRNA sequencing results. We show as novelty that a binary matrix combining ferulic and sinapinic acids performs well in acquiring reproducible mass spectra of cyanobacteria. Using the matrix solvent, a protein extraction from cells was done. After polyacrylamide gel electrophoresis, the separated protein fractions were in-gel digested and the resulting peptides analyzed by liquid chromatography coupled with tandem mass spectrometry. For the first time, photosystem protein components, phycobilisome proteins, electron transport proteins, nitrogen-metabolism and nucleic acids binding-proteins, cytochromes plus other enzymes and various uncharacterized proteins could be assigned to characteristic peaks in the mass spectrometric profiles and some of them suggested as markers in addition to 30S and 50S ribosomal proteins known from previous studies employing intact cell mass spectrometry of microorganisms

Purification of Maize Nucleotide Pyrophosphatase/Phosphodiesterase Casts Doubt on the Existence of Zeatin Cis–Trans Isomerase in Plants

Almost 25 years ago, an enzyme named zeatin cis–trans isomerase from common bean has been described by Bassil et al. (1993). The partially purified enzyme required an external addition of FAD and dithiothreitol for the conversion of cis-zeatin to its trans- isomer that occurred only under light. Although an existence of this important enzyme involved in the metabolism of plant hormones cytokinins was generally accepted by plant biologists, the corresponding protein and encoding gene have not been identified to date. Based on the original paper, we purified and identified an enzyme from maize, which shows the described zeatin cis–trans isomerase activity. The enzyme belongs to nucleotide pyrophosphatase/phosphodiesterase family, which is well characterized in mammals, but less known in plants. Further experiments with the recombinant maize enzyme obtained from yeast expression system showed that rather than the catalytic activity of the enzyme itself, a non-enzymatic flavin induced photoisomerization is responsible for the observed zeatin cis–trans interconversion in vitro. An overexpression of the maize nucleotide pyrophosphatase/phosphodiesterase gene led to decreased FAD and increased FMN and riboflavin contents in transgenic Arabidopsis plants. However, neither contents nor the ratio of zeatin isomers was altered suggesting that the enzyme is unlikely to catalyze the interconversion of zeatin isomers in vivo. Using enhanced expression of a homologous gene, functional nucleotide pyrophosphatase/phosphodiesterase was also identified in rice

The Gold(I) Complex with Plant Hormone Kinetin Shows Promising In Vitro Anticancer and PPARγ Properties

Motivated by the clinical success of gold(I) metallotherapeutic Auranofin in the effective treatment of both inflammatory and cancer diseases, we decided to prepare, characterize, and further study the [Au(kin)(PPh3)] complex (1), where Hkin = kinetin, 6-furfuryladenine, for its in vitro anti-cancer and anti-inflammatory activities. The results revealed that the complex (1) had significant in vitro cytotoxicity against human cancer cell lines (A2780, A2780R, PC-3, 22Rv1, and THP-1), with IC50 ≈ 1–5 μM, which was even significantly better than that for the conventional platinum-based drug Cisplatin while comparable with Auranofin. Although its ability to inhibit transcription factor NF-κB activity did not exceed the comparative drug Auranofin, it has been found that it is able to positively influence peroxisome-proliferator-activated receptor-gamma (PPARγ), and as a consequence of this to have the impact of moderating/reducing inflammation. The cellular effects of the complex (1) in A2780 cancer cells were also investigated by cell cycle analysis, induction of apoptosis, intracellular ROS production, activation of caspases 3/7 and disruption of mitochondrial membrane potential, and shotgun proteomic analysis. Proteomic analysis of R2780 cells treated with complex (1) and starting compounds revealed possible different places of the effect of the studied compounds. Moreover, the time-dependent cellular accumulation of copper was studied by means of the mass spectrometry study with the aim of exploring the possible mechanisms responsible for its biological effects

Rotaxanes Capped with Host Molecules: Supramolecular Behavior of Adamantylated Bisimidazolium Salts Containing a Biphenyl Centerpiece

Bisimidazolium salts with one central biphenyl binding site and two terminal adamantyl binding sites form water-soluble binary or ternary aggregates with cucurbit[7]uril (CB7) and β-cyclodextrin (β-CD) with rotaxane and pseudorotaxane architectures. The observed arrangements result from cooperation of the supramolecular stopper binding strength and steric barriers against free slippage of the CB7 and β-CD host molecules over the bisimidazolium guest axle. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.internal funding agency of Tomas Bata University in Zlin [IGA/FT/2015/005]; program "Projects of Large Infrastructure for Research, Development, and Innovations" [LM2010005

Does the Pollen Diet Influence the Production and Expression of Antimicrobial Peptides in Individual Honey Bees?

We investigated the importance of protein nutrition for honey bee immunity. Different protein diets (monofloral pollen of Helianthus spp., Sinapis spp., Asparagus spp., Castanea spp., a mixture of the four different pollen and the pollen substitute FeedbeeTM) were fed to honey bees in cages ad libitum. After 18 days of feeding, apidaecin 1 isoforms concentration in the thorax were measured using nanoflow liquid chromatography coupled with mass spectrometry. Expression levels of genes, coding for apidaecins and abaecin in the abdomen were determined using quantitative PCR. The results indicate that protein-containing nutrition in adult worker honey bees can trigger certain metabolic responses. Bees without dietary protein showed lower apidaecin 1 isoforms concentrations. The significantly lowest concentration of apidaecin 1 isoforms was found in the group that was fed no pollen diet when compared to Asparagus, Castanea, Helianthus, and Sinapis pollen or the pollen supplement FeedBeeTM. Expression levels of the respective genes were also affected by the protein diets and different expression levels of these two antimicrobial peptides were found. Positive correlation between concentration and gene expression of apidaecins was found. The significance of feeding bees with different protein diets, as well as the importance of pollen nutrition for honey bee immunity is demonstrated

Structural characterization of a plant photosystem I and NAD(P)H dehydrogenase supercomplex

Cyclic electron transport (CET) around photosystem I (PSI) plays an important role in balancing the ATP/NADPH ratio and the photoprotection of plants. The NAD(P)H dehydrogenase complex (NDH) has a key function in one of the CET pathways. Current knowledge indicates that, in order to fulfill its role in CET, the NDH complex needs to be associated with PSI; however, until now there has been no direct structural information about such a supercomplex. Here we present structural data obtained for a plant PSI–NDH supercomplex. Electron microscopy analysis revealed that in this supercomplex two copies of PSI are attached to one NDH complex. A constructed pseudo-atomic model indicates asymmetric binding of two PSI complexes to NDH and suggests that the low-abundant Lhca5 and Lhca6 subunits mediate the binding of one of the PSI complexes to NDH. On the basis of our structural data, we propose a model of electron transport in the PSI–NDH supercomplex in which the association of PSI to NDH seems to be important for efficient trapping of reduced ferredoxin by NDH.

The Gold(I) Complex with Plant Hormone Kinetin Shows Promising In Vitro Anticancer and PPARγ Properties

Motivated by the clinical success of gold(I) metallotherapeutic Auranofin in the effective treatment of both inflammatory and cancer diseases, we decided to prepare, characterize, and further study the [Au(kin)(PPh3)] complex (1), where Hkin = kinetin, 6-furfuryladenine, for its in vitro anti-cancer and anti-inflammatory activities. The results revealed that the complex (1) had significant in vitro cytotoxicity against human cancer cell lines (A2780, A2780R, PC-3, 22Rv1, and THP-1), with IC50 ≈ 1–5 μM, which was even significantly better than that for the conventional platinum-based drug Cisplatin while comparable with Auranofin. Although its ability to inhibit transcription factor NF-κB activity did not exceed the comparative drug Auranofin, it has been found that it is able to positively influence peroxisome-proliferator-activated receptor-gamma (PPARγ), and as a consequence of this to have the impact of moderating/reducing inflammation. The cellular effects of the complex (1) in A2780 cancer cells were also investigated by cell cycle analysis, induction of apoptosis, intracellular ROS production, activation of caspases 3/7 and disruption of mitochondrial membrane potential, and shotgun proteomic analysis. Proteomic analysis of R2780 cells treated with complex (1) and starting compounds revealed possible different places of the effect of the studied compounds. Moreover, the time-dependent cellular accumulation of copper was studied by means of the mass spectrometry study with the aim of exploring the possible mechanisms responsible for its biological effects