Design and synthesis of new substrates of HtrA2 protease

HtrA2 belongs to the HtrA (high temperature requirement A) family of ATP-independent serine proteases. The primary function of HtrA2 includes maintaining the mitochondria homeostasis, cell death (by apoptosis, necrosis, or anoikis), and contribution to the cell signaling. Several recent reports have shown involvement of HtrA2 in development of cancer and neurodegenerative disorders. Here, we describe the profiling of HtrA2 protease substrate specificity via the combinatorial chemistry approach that led to the selection of novel intramolecularly quenched substrates. For all synthesized compounds, the highest HtrA2-mediated hydrolysis efficiency and selectivity among tested HtrA family members was observed for ABZ-Ile-Met-Thr-Abu-Tyr-Met-Phe-Tyr(3-NO₂)-NH₂, which displayed a specificity constant k_cat/K_M value of 14,535 M⁻¹ s⁻¹.     

Centipede (Chilopoda) richness and diversity in the Bug River valley (Eastern Poland)

The main aim of the survey was to describe the diversity and richness of Chilopoda in the selected area of the Bug River valley. The study sites were located in two regions differing in the shape of the valley, the presence of thermophilous habitats and the size of riparian forests. Pitfall traps were used as a sampling method. As a result, 444 specimens belonging to 12 centipede species of two orders – Geophilomorpha (four species) and Lithobiomorpha (eight species) were caught. Lithobius (Monotarsobius) curtipes C.L.Koch, 1847, Pachymerium ferrugineum (C.L.Koch, 1835), Lamyctes (Lamyctes) emarginatus (Newport, 1844) and Lithobius (Monotarsobius) dudichi Loksa, 1947 were the most common and the most numerous species. Of particular note is Lithobius dudichi found in Poland for the first time and previously known based on a single specimen. Two to 10 Chilopoda species were found in each habitat under investigation. The greatest species richness was found in thermophilous thickets (10 species), sandy grasslands (eight), xerothermic grasslands (eight) and mesic meadows (six). The fewest number of species (two) was found in rushes at oxbows and in wet meadows. We found differences in the species composition and the number of Chilopoda between the lower (102 specimens, six species) and the middle (324 specimens, 11 species) section of the river valley. Our results confirm the need to protect xerothermic habitats, unique almost throughout entire Central Europe, which due to their distribution and their small area covered are fairly easily subject to the process of destruction.     

Metalloporphyrin intercalation in liposome membranes: ESR study

Liposomes characterized by membranes featuring diverse fluidity (liquid-crystalline and/or gel phase), prepared from egg yolk lecithin (EYL) and dipalmitoylphosphatidylcholine (DPPC), were doped with selected metalloporphyrins and the time-related structural and dynamic changes within the lipid double layer were investigated. Porphyrin complexes of Mg(II), Mn(III), Fe(III), Co(II), Ni(II), Cu(II), Zn(II), and the metal-free base were embedded into the particular liposome systems and tested for 350 h at 24°C using the electron spin resonance (ESR) spin probe technique. 5-DOXYL, 12-DOXYL, and 16-DOXYL stearic acid methyl ester spin labels were applied to explore the interior of the lipid bilayer. Only the 16-DOXYL spin probe detected evident structural changes inside the lipid system due to porphyrin intercalation. Fluidity of the lipid system and the type of the porphyrin complex introduced significantly affected the intermolecular interactions, which in certain cases may result in self-assembly of metalloporphyrin molecules within the liposome membrane, reflected in the presence of new lines in the relevant ESR spectra. The most pronounced time-related effects were demonstrated by the EYL liposomes (liquid-crystalline phase) when doped with Mg and Co porphyrins, whereas practically no spectral changes were revealed for the metal-free base and both the Ni and Zn dopants. ESR spectra of the porphyrin-doped gel phase of DPPC liposomes did not show any extra lines; however, they indicated the formation of a more rigid lipid medium. Electronic configuration of the porphyrin’s metal center appeared crucial to the degree of molecular reorganization within the phospholipid bilayer system.     

Characterization of N-cadherin unbinding properties in non-malignant (HCV29) and malignant (T24) bladder cells

The expression of N‐cadherin, characteristic of various cancers, very often leads to changes in the cells' adhesive properties. Thus, we sought to find out if N‐cadherin expressed in various, but cancer‐related cells, differs in its functional properties that could contribute to variations in cells' phenotypes. In our work, measurements of an unbinding force of a single N‐cadherin molecule, probed with the same antibody both on a surface of living non‐malignant (HCV29) and malignant cells (T24) of bladder cancer, were carried out with the use of an atomic force microscopy. The results show the enhanced N‐cadherin level in T24 malignant cells (8.7% vs. 3.6% obtained for non‐malignant one), confirmed by the Western blot and the immunohistochemical staining. The effect was accompanied by changes in unbinding properties of an individual N‐cadherin molecule. Lower unbinding force values (26.1 ± 7.1 pN) in non‐malignant cells reveal less stable N‐cadherin complexes, as compared to malignant cells (61.7 ± 14.6 pN). This suggests the cancer‐related changes in a structure of the binding site of the antibody, located at the extracellular domain of N‐cadherin. Copyright © 2011 John Wiley & Sons, Ltd.     

Synthesis and in vitro study of antiviral and virucidal activity of novel 2-[(4-methyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetamide derivatives

2-[(4-Methyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetamide derivatives were synthesized and their structures were confirmed by 1H NMR, IR, and elemental analysis. Cytotoxicity of the compounds towards HEK-293 and GMK cells was evaluated. Moreover, the antiviral and virucidal activities of these compounds against human adenovirus type 5 and ECHO-9 virus were assessed. Some of the newly synthesized derivatives have the potential to reduce the viral replication of both tested viruses.     

Alterations in the expression of the Atp7a gene in the early postnatal development of the mosaic mutant mice (Atp7amo-ms) – An animal model for Menkes disease

Copper is a trace element that is essential for the normal growth and development of all living organisms. In mammals, the ATP7A Cu-transporting ATPase is a key protein that is required for the maintenance of copper homeostasis. In both humans and mice, the ATP7A protein is coded by the X-linked ATP7A/Atp7a gene. Disturbances in copper metabolism caused by mutations in the ATP7A/Atp7a gene lead to severe metabolic syndromes Menkes disease in humans and the lethal mottled phenotype in mice. Mosaic is one of numerous mottled mutations and may serve as a model for a severe Menkes disease variant. In Menkes patients, mutations in the ATP7A gene often result in a decreased level of the normal ATP7A protein. The aim of this study was to analyse the expression of the Atp7a gene in mosaic mutants in early postnatal development, a critical period for starting copper supplementation therapy in both Menkes patients and mutant mice. Using real-time quantitative RT-PCR, we analysed the expression of the Atp7a gene in the brain, kidney and liver of newborn (P0.5) and suckling (P14) mice. Our results indicate that in mosaic P0.5 mutants, the Atp7a mRNA level is decreased in all analysed organs in comparison with wild-type animals. In two week-old mutants, a significant decrease was observed only in the kidney. In contrast, their hepatic level of Atp7a tended to be higher than in wild-type mice. We speculate that disturbance in the expression of the Atp7a gene and, consequently, change in the copper concentration of the organs, may contribute to the early fatal outcome of mosaic males.     

Quantum-chemical study of interactions of <Emphasis Type="Italic">trans-</Emphasis>resveratrol with guanine-thymine dinucleotide and DNA-nucleobases

Trans-resveratrol, a natural phytoalexin present in red wine and grapes, has gained considerable attention because of its antiproliferative, chemopreventive and proapoptotic activity against human cancer cells. The accurate quantum-chemical computations based on the density functional theory (DFT) and ab initio second-order Møller-Plesset perturbation method (MP2) have been performed for the first time to study interactions of trans-resveratrol with guanine-thymine dinucleotide and DNA-derived nitrogenous bases: adenine, guanine, cytosine and thymine in vacuum and water medium. This compound is found to show high affinity to nitrogenous bases and guanine-thymine dinucleotide. The electrostatic interactions from intermolecular hydrogen bonding increase the stability of complexes studied. In particular, significantly strong hydrogen bonds between 4′-H atom of trans-resveratrol and imidazole nitrogen as well as carbonyl oxygen atoms of nucleobases studied stabilize these systems. The stabilization energies computed reveal that the negatively charged trans-resveratrol-dinucleotide complex is more energetically stable in water medium than in vacuum. MP2 method gives more reliable and significantly high values of stabilization energy of trans-resveratrol-dinucleotide, trans-resveratrol-guanine and trans-resveratrol-thymine complexes than B3LYP exchange-correlation functional because it takes into account London dispersion energy. According to the results, in the presence of trans-resveratrol the 3′-5′ phosphodiester bond in dinucleotide can be cleaved and the proton from 4′-OH group of trans-resveratrol migrates to the 3′-O atom of dinucleotide. It is concluded that trans-resveratrol is able to break the DNA strand. Hence, the findings obtained help understand antiproliferative and anticancer properties of this polyphenol.