The Oxime Ethers with Heterocyclic, Alicyclic and Aromatic Moiety as Potential Anti-Cancer Agents.

Chemotherapy is one of the most commonly used methods of cancer disease treatment. Due to the acquisition of drug resistance and the possibility of cancer recurrence, there is an urgent need to search for new molecules that would be more effective in destroying cancer cells. In this study, 1-(benzofuran-2-yl)ethan-1-one oxime and 26 oxime ethers containing heterocyclic, alicyclic or aromatic moiety were screened for their cytotoxicity against HeLa cancer cell line. The most promising derivatives with potential antitumor activity were 2-(cyclohexylideneaminoxy)acetic acid (18) and (E)-acetophenone O-2-morpholinoethyl oxime (22), which reduced the viability of HeLa cells below 20% of control at concentrations of 100-250 µg/mL. Some oxime ethers, namely thiazole and benzothiophene derivatives (24-27), also reduced HeLa cell viability at similar concentrations but with lower efficiency. Further cytotoxicity evaluation confirmed the specific toxicity of (E)-acetophenone O-2-morpholinoethyl oxime (22) against A-549, Caco-2, and HeLa cancer cells, with an EC50 around 7 µg/mL (30 µM). The most potent and specific compound was (E)-1-(benzothiophene-2-yl)ethanone O-4-methoxybenzyl oxime (27), which was selective for Caco-2 (with EC50 116 µg/mL) and HeLa (with EC50 28 µg/mL) cells. Considering the bioavailability parameters, the tested derivatives meet the criteria for good absorption and permeation. The presented results allow us to conclude that oxime ethers deserve more scientific attention and further research on their chemotherapeutic activity.

[Suppression of PAMP-triggered immunity (PTI) by effector proteins synthesized by phytopathogens and delivered into cells of infected plant]. / Supresja odpornosci podstawowej typu PTI przez syntetyzowane w fitopatogenach bialka efektorowe wprowadzane do wnetrza komórek infekowanej rosliny.

Plant immunity is constituted by multilayered system involving two intertwined lines of defence: a first level of immunity termed PAMP-triggered immunity (PTI) or basal resistance, and a second layer of plant defence, called effector-triggered immunity (ETI). The second line of defence depends on the ability of the plant to recognize phytopathogen-synthesized effector proteins delivered into host plant cells. The effector proteins employ common as well as pathogen-specific strategies to disturb plant immunity and to promote pathogen survival and favor their multiplication. They target pattern-recognition receptors (PRRs) and key components in the PTI signaling pathways, as well as, they interfere with many cellular processes including vesicle transport, cytoskeleton reorganization, proteasome-dependent protein degradation, phytohormone biosynthesis and signaling, and gene expression. This results in effector-triggered susceptibility (ETS). However, in some cases, pathogen effectors are recognized by plant intracellular immune receptors NB-LRR/NLR that identify effector proteins. Conformational changes in the NB-LRR/NLR immune receptors accompanying the recognition of the effector proteins activate intracellular signaling pathways initiating a whole range of defence responses that form the second line of local defence.

[Effector proteins of phytopathogens recognized by intracellular immune receptors NB-LRR/NLR that activate second line of defence of the plant immune system]. / Immunoreceptory wewnatrzkomórkowe NB-LRR/NLR rozpoznajace bialka efektorowe fitopatogenów i aktywujace druga linie obrony ukladu odpornosciowego roslin.

The effector proteins employ common as well as pathogen-specific strategies to disturb plant immunity and to promote pathogen survival and favor their multiplication. However, in some cases, pathogen effectors are recognized by plant intracellular immune receptors NB-LRR/NLR that identify effector proteins, either directly by physical interaction or indirectly through monitoring of host proteins modification. NB-LRR immune receptors are characterized by the central nucleotide binding domain NB-ARC, C-terminal Leucine-Rich Repeats (LRRs) domain, and N-terminal TIR, CC or CCR domain. Immune receptors localized to the nucleus appears to be more directly associated with transcriptional regulation of defence gene expression, while the detection of pathogen effectors by membrane- or cytoplasm-localized immune receptors NB-LRR activate calcium-mediated signaling, ion flow, mitogen-activated protein kinase cascades, oxidative burst, biosynthesis of plant defence hormones and antimicrobial compounds, as well, far-reaching transcriptional reprogramming. Effector-triggered immunity normally culminates in programmed cell death called hypersensitive response (HR).

[Membrane receptors recognizing MAMP/PAMP and DAMP molecules that activate first line of defence in plant immune system]. / Receptory blonowe wiazace czasteczki typu MAMP/PAMP i DAMP aktywujace pierwsza linie obrony lokalnej ukladu odpornosciowego roslin

Plants have evolved a multilevel immune system to protect them against infection by a diverse range of pathogens. The first line of plant defense consists of the integral plasma membrane receptors, known as pattern-recognition receptors (PRRs), which recognize MAMP/PAMP or DAMP molecules. PRRs activate downstream signaling cascades that culminate in generation of the innate immune response, called PAMP-triggered immunity (PTI). The activation of PTI leads to the induction of basal defense responses, which include influx of Ca2+ in the cytosol, production of reactive oxygen species (ROS) and nitric oxide, activation of mitogen-activated protein kinase cascades, callose deposition, stomatal closure, phytoalexins biosynthesis, defence hormone biosynthesis and transcriptional induction of a large suite of defence-related genes.

Gene Expression and Activity Profiling Reveal a Significant Contribution of Exo-Phosphotransferases to the Extracellular Nucleotides Metabolism in HUVEC Endothelial Cells.

Purinergic signaling maintains local tissue homeostasis in blood vessels via the regulation of vascular tone, blood platelet aggregation, cell proliferation, and differentiation as well as inflammatory responses. Extracellular purines are important signaling molecules in the vasculature, and both purine-hydrolysing as well as -phosphorylating enzymes are considered to selectively govern extracellular nucleotide/nucleoside metabolism. Recent studies have provided some evidence for the existence of these enzymes in a soluble form in human blood and their secretion into the extracellular space under physiological and pathological conditions. However, the comprehensive analysis of endothelium-derived enzymes involved in purine metabolic pathways has received no attention so far. In the presented study, in vitro cultured human umbilical vein endothelial cells (HUVEC) are shown to be an abundant source of exo-nucleotidases comprising 5'-nucleotidase (exo-5'-NT), and nucleoside triphosphate diphosphohydrolases (exo-NTPDase) as well as phosphotransferases, represented by nucleoside diphosphate kinase (exo-NDPK) and adenylate kinase (exo-AK). An attempt is also made to demonstrate that, in contrast to the metabolic pattern determined on the endothelial cell surface, exo-phosphorylating activities markedly predominate over exo-hydrolytic ones. We present for the first time the expression profiles of genes encoding isoenzymes belonging to distinct nucleotide kinase and nucleotidase families. The genes encoding NDPK1, NDPK2, AK1, and AK2 phosphotransferases have been shown to be expressed at the highest level in HUVEC cells. The data indicate the coexistence of secreted and cell-associated factors of endothelial origin mediating ATP-consuming and ATP-generating pathways with the predominance of exo-phosphotransferases activity. The described enzymes contribute to the regulation of purinergic signal duration and extent in the venous vasculature. J. Cell. Biochem. 118: 1341-1348, 2017. © 2016 Wiley Periodicals, Inc.

[Human adenylate kinases - classification, structure, physiological and pathological importance]. / Kinazy adenylanowe czlowieka-klasyfikacja, budowa oraz znaczenie w fizjologii i patologii.

Adenylate kinase (AK, EC 2.7.4.3) is a ubiquitous phosphotransferase which catalyzes the reversible transfer of high-energy ß - and γ-phosphate groups between nucleotides. All classified AKs show a similar structure: they contain a large central CORE region, nucleoside monophosphate and triphosphate binding domains (NMPbd and NTPbd) and the LID domain. Analysis of amino acid sequence similarity revealed the presence of as many as nine human AK isoenzymes, which demonstrate different organ-tissue and intercellular localization. Among these kinases, only two, AK1 and AK2, fulfill the structural and functional criterion by the highest affinity for adenine nucleotides and the utilization of only AMP or dAMP as phosphate acceptors. Human AK isoenzymes are involved in nucleotide homeostasis and monitor disturbances of cell energy charge. Participating in large regulatory protein complexes, AK supplies high energy substrates for controlling the functions of channels and transporters as well as ligands for extracellular P2 nucleotide receptors. In pathological conditions AK can take over the function of other kinases, such as creatine kinase in oxygen-depleted myocardium. Directed mutagenesis and genetic studies of diseases (such as aleukocytosis, hemolytic anemia, primary ciliary dyskinesia (PCD)) link the presence and activity of AK with etiology of these disturbances. Moreover, AK participates in regulation of differentiation and maturation of cells as well as in apoptosis and oncogenesis. Involvement of AK in a wide range of processes and the correlation between AK and etiology of diseases support the medical potential for the use of adenylate kinases in the diagnosis and treatment of certain diseases. This paper summarizes the current knowledge on the structure, properties and functions of human adenylate kinase.

Adenylate kinase immobilized on graphene oxide impairs progression of human lung carcinoma epithelial cells through adenosinergic pathway.

Purinergic signaling, the oldest evolutionary transmitter system, has been increasingly studied as a pivotal target for novel anti-cancer therapies. In the present work, the developed nanobiocatalytic system consisting of adenylate kinase immobilized on graphene oxide (AK-GO) was characterized in terms of its physicochemical and biochemical properties. We put special emphasis on the AK-GO influence on purinergic signaling components, that is, ecto-nucleotides concentration and ecto-enzymes expression and activity in human lung carcinoma epithelial (A549) cells. The immobilization-dependent modification of AK kinetic parameters allowed for the removal of ATP excess while maintaining low ATP concentrations, efficient decrease in adenosine concentration, and control of the nucleotide balance in carcinoma cells. The cyto- and hemocompatibility of developed AK-GO nanobiocatalytic system indicates that it can be successfully harnessed for biomedical applications. In A549 cells treated with AK-GO nanobiocatalytic system, the significantly decreased adenosinergic signaling results in reduction of the proliferation and migration capability of cancer cells. This finding is particularly relevant in regard to AK-GO prospective anti-cancer applications.

[Small, monomeric G proteins in plants]. / Male, monomeryczne bialka G roslin.

The superfamily of small, monomeric GTP-binding proteins, in Arabidopsis thaliana comprising 93 members, is classified into four families: Arf/Sar, Rab, Rop/Rac, and Ran families. All monomeric G proteins function as molecular switches that are activated by GTP and inactivated by the hydrolysis of GTP to GDP. GTP/GDP cycling is controlled by three classes of regulatory protein: guanine-nucleotide-exchange factors (GEFs), GTPase-activating proteins (GAPs), and guanine-nucleotide-dissociation inhibitors (GDIs). Proteins of Arf family are primarily involved in regulation of membrane traffic and organization of the cytoskeleton. Arf1/Sar1 proteins regulate the formation of vesicle coat at different steps in the exocytic and endocytic pathways. Rab GTPases are regulators of vesicular transport. They are involved in vesicle formation, recruitment of cytoskeletal motor proteins, and in vesicle tethering and fusion. Rop proteins serve as key regulators of cytoskeletal reorganization in response to extracellular signals. Several data have also shown that Rop proteins play additional roles in membrane trafficking and regulation of enzymes activity. Ran proteins are involved in nucleocytoplasmic transport.

Nucleoside diphosphate kinase isoforms regulated by phytochrome A isolated from oat coleoptiles.

Nucleoside diphosphate kinase (NDPK) (EC 2.7.4.6), the enzyme transferring the phosphate residue from ATP to nucleoside diphosphates, is localized mainly in the cytoplasm and mitochondria and in smaller amounts in cell nuclei and the microsomal fraction. Exposure of etiolated oat seedlings to red light causes an increase of the enzyme activity by about 42% in nuclear fraction, 7% in etioplastic and 14% in postetioplastic fraction. Endogenous phytochrome A, as visualized by an immunochemical method, translocates from the cytoplasm into the nucleus upon red, far-red or white light activation. Nuclei purified from oat seedlings contain two, and the postnuclear fraction four easily separated forms of NDPK. One of the nuclear isoforms (I(n)) and one isoform isolated from the postnuclear fraction (II(pn)) are activated by red light in the presence of phytochrome A purified from etiolated oat coleoptiles. Both phytochrome A-activated NDPKs purified to electrophoretic homogeneity have the same molecular mass (17-18 kDa) determined by SDS/PAGE. Both enzymes in the native form have similar molecular masses (71 and 63 kDa).

[G-protein-coupled receptors, heterotrimeric G-proteins and protein effectors in plants]. / Receptory sprzezone z bialkami G, heterotrimeryczne bialka G i bialka efektorowe roslin.

The superfamily of G-protein-coupled receptors (GPCRs) is one of the largest and most diverse family of proteins in mammals. Plants, in contrast to animals, have a greatly simplified repertoire of GPCRs. To date, only AtGCR1 and AtRGS1 have been shown to physically interacts with a plant G-protein. Also, the number of G-protein complex components in plants is dramatically less than in other Eukaryotes. Only one prototypical Ga subunit, one Gbeta and two Ggamma subunits have been identified in A. thaliana and rice. Similarly, there are few known downstream effectors that physicaly interact with either the plant Ga subunit or the Gbetagamma dimer. The best-characterized effector is phospholipase D (PLDalpha1). Besides, the potential Galpha effectors are: cupin-domain protein designated as Atpirin1, prephenate dehydratase--a cytosolic enzyme involved in regulation of the shikimate pathway, and THF1 protein located in plastid membranes. Indirect evidences suggest that Galpha may interact with ion channels and phospholipases A2 and C, whereas Gbetagamma dimer supposedly interacts with a Golgi-localized hexose transporter.

Novel biocatalytic systems for maintaining the nucleotide balance based on adenylate kinase immobilized on carbon nanostructures.

In this study graphene oxide (GO), carbon quantum dots (CQD) and carbon nanoonions (CNO) have been characterized and applied for the first time as a matrix for recombinant adenylate kinase (AK, EC 2.7.4.3) immobilization. AK is an enzyme fulfilling a key role in metabolic processes. This phosphotransferase catalyzes the interconversion of adenine nucleotides (ATP, ADP and AMP) and thereby participates in nucleotide homeostasis, monitors a cellular energy charge as well as acts as a component of purinergic signaling system. The AK activity in all obtained biocatalytic systems was higher as compared to the free enzyme. We have found that the immobilization on carbon nanostructures increased both activity and stability of AK. Moreover, the biocatalytic systems consisting of AK immobilized on carbon nanostructures can be easily and efficiently lyophilized without risk of desorption or decrease in the catalytic activity of the investigated enzyme. The positive action of AK-GO biocatalytic system in maintaining the nucleotide balance in in vitro cell culture was proved.

Indole-3-acetic acid UDP-glucosyltransferase from immature seeds of pea is involved in modification of glycoproteins.

The glycosylation of auxin is one of mechanisms contributing to hormonal homeostasis. The enzyme UDPG: indole-3-ylacetyl-ß-D-glucosyltransferase (IAA glucosyltransferase, IAGlc synthase) catalyzes the reversible reaction: IAA+UDPG↔1-O-IA-glucose+UDP, which is the first step in the biosynthesis of IAA-ester conjugates in monocotyledonous plants. In this study, we report IAA-glucosyltransferase isolated using a biochemical approach from immature seed of pea (Pisum sativum). The enzyme was purified by PEG fractionation, DEAE-Sephacel anion-exchange chromatography and preparative PAGE. LC-MS/MS analysis of tryptic peptides of the enzyme revealed the high identity with maize IAGlc synthase, but lack of homology with other IAA-glucosyltransferases from dicots. Biochemical characterization showed that of several acyl acceptors tested, the enzyme had the highest activity on IAA as the glucosyl acceptor (Km=0.52 mM, Vmax=161 nmol min(-1), kcat/Km=4.36 mM s(-1)) and lower activity on indole-3-propionic acid and 1-naphthalene acetic acid. Whereas indole-3-butyric acid and indole-3-propionic acid were competitive inhibitors of IAGlc synthase, D-gluconic acid lactone, an inhibitor of ß-glucosidase activity, potentiated the enzyme activity at the optimal concentration of 0.3mM. Moreover, we demonstrated that the 1-O-IA-glucose synthesized by IAGlc synthase is the substrate for IAA labeling of glycoproteins from pea seeds indicating a possible role of this enzyme in the covalent modification of a class of proteins by a plant hormone.