Diazenedicarboxamides as inhibitors of D-alanine-D-alanine ligase (Ddl)

D-Alanine-D-alanine ligase (Ddl) catalyzes the biosynthesis of an essential bacterial peptidoglycan precursor D-alanyl-D-alanine and it represents an important target for development of new antibacterial drugs. A series of semicarbazides, aminocarbonyldiazenecarboxylates, diazenedicarboxamides, and hydrazinedicarboxamides was synthesized and screened for inhibition of DdlB from Escherichia coli. Compounds with good inhibitory activity were identified, enabling us to deduce initial structure-activity relationships. Thirteen diazenedicarboxamides were better inhibitors than D-cycloserine and some of them also possess antibacterial activity, which makes them a promising starting point for further development.     

Characterization of proliferating cell nuclear antigen (PCNA) isoforms in normal and cancer cells: there is no cancer-associated form of PCNA

In order to clarify the status of PCNA in normal and transformed cells, we performed analysis of this protein by 2D-PAGE, Western blot and mass spectrometry. All the cell lines examined contained the major PCNA form (pI 4.57/30kDa), that is not post-translationally modified. In addition to the major form, two minor isoforms (pI 4.52/30kDa and pI 4.62/30kDa) were also detected in all the cell lines examined. However, the level of PCNA in cancer cells is 5-6 folds higher than those in primary and most of the immortalized cells. Taken together, the significant difference in PCNA status between cancer and normal cells is not at the post-translational modifications but in the overall levels of PCNA.     

Insects did it first: a micropatterned adhesive tape for robotic applications

Based on the structural and experimental studies of more than 300 insect species from different lineages, we have developed and characterized a bioinspired polymer material with the ability of multiple glue-free bonding and debonding. The material surface is covered with a pattern of microstructures, which resembles the geometry of tenent hairs previously described from the feet of flies, beetles, earwigs and other insects. The tape with such a microstructure pattern demonstrates at least two times higher pull-off force per unit apparent contact area compared to the flat polymer. Additionally, the tape is less sensitive to contamination by dust particles than a commercially available pressure-sensitive adhesive tape. Even if the 'insect tape' is contaminated, it can be washed with a soap solution in water, in order to completely recover its adhesive properties. We have successfully applied the tape to the 120 g wall-climbing robot Mini-Whegs. Furthermore, the tape can be used for multiple adhering of objects to glass surfaces or as a protective tape for sensitive glass surfaces of optical quality. Another area of potential applications is gripping and manipulation of objects with smooth surfaces.     

Species‐specific habitat preferences do not shape the structure of a crested newt hybrid zone (Triturus cristatus x T. carnifex)

Reproductive isolation barriers maintain the integrity of species by preventing interspecific gene flow. They involve temporal, habitat or behavioral isolation acting before fertilization, and postzygotic isolation manifested as hybrid mortality or sterility. One of the approaches of how to study reproductive isolation barriers is through the analysis of hybrid zones. In this paper, we describe the structure of a hybrid zone between two crested newt species (Triturus cristatus and T. carnifex) in the southern part of the Czech Republic using morphological, microsatellite, and mitochondrial (mtDNA) markers. Specifically, we tested the hypothesis that the structure of the hybrid zone is maintained by species‐specific habitat preferences. Comparing the genetic structure of populations with geographical and ecological parameters, we found that the hybrid zone was structured primarily geographically, with T. cristatus‐like populations occurring in the northeast and T. carnifex‐like populations in the southwest. Despite T. cristatus tending to occur in deeper ponds and T. carnifex on localities with more shading, the effect of both ecological parameters on the structure of the zone was minimal. Next, we corroborated that T. carnifex individuals and some hybrids possess mtDNA of T. dobrogicus, whose nuclear background was not detected in the studied hybrid zone. Hybridization between T. carnifex and T. dobrogicus (resulting in unidirectional mtDNA introgression) had to predate subsequent formation of the hybrid zone between T. cristatus and T. carnifex. Populations of crested newts in the southern part of the Czech Republic thus represent a genetic mosaic of nuclear and mitochondrial genomes of three species.     

Novel inhibitors of beta-ketoacyl-ACP reductase from Escherichia coli

Bacterial beta-ketoacyl-[acyl carrier protein] (beta-ketoacyl-ACP) reductase (FabG) is a highly conserved and ubiquitously expressed enzyme of the fatty-acid biosynthetic pathway of prokaryotic organisms that catalyzes NADPH-dependent reduction of beta-ketoacyl-ACP intermediates. Therefore, FabG represents an appealing target for the development of new antimicrobial agents. A number of trans-cinnamic acid derivatives were designed and screened for inhibitory activities against FabG from Escherichia coli. These inhibited FabG enzymatic activity with IC(50) values in the microM range, and were used as templates for the subsequent diversification of the chemotype. Introduction of an electron-withdrawing 4-cyano group to the phenol substituent showed improved inhibition over the non-substituted compound. The benzo-[1,3]-dioxol moiety also appeared to be essential for inhibitory activity of trans-cinnamic acid derivatives against FabG from E. coli. To explain the possible binding position, the best inhibitor from the present study was docked in the active site of FabG. The results for the best scoring conformers chosen by the docking programme revealed that cinnamic acid derivatives can be accommodated in the substrate-binding region of the active site, above the nicotinamide moiety of the NADPH cofactor. Additionally, a phage-displayed library of random linear 15-mer peptides was screened against FabG, to identify ligands with the common PPLTXY motif.     

TMEM70 protein — A novel ancillary factor of mammalian ATP synthase

An increasing number of patients with nuclear genetic defects of mitochondrial ATP synthase have been identified in recent years. They are characterized by early onset, lactic acidosis, 3-methylglutaconic aciduria, hypertrophic cardiomyopathy and encephalopathy and most cases have a fatal outcome. Patient tissues show isolated defect of the ATP synthase complex and its content decreases to ≥ 30% of normal due to altered enzyme biosynthesis and assembly. Gene mapping and complementation studies have identified mutations in TMEM70 gene encoding a 30kD mitochondrial protein of unknown function as the cause of the disease. An altered synthesis of this new ancillary factor in ATP synthase biogenesis was found in most of the known patients with decreased ATP synthase content. As revealed by phylogenetic analysis, TMEM70 is specific for higher eukaryotes.     

Basic morphological data of native Czech Emys orbicularis revealed by subfossil finds

Holocene Czech Emys orbicularis are rare in museum collections because the species disappeared from this region centuries ago. Fifteen reconstructed shells of subfossil Czech specimens of E. orbicularis (ranging in age from 3800–3500 BC to 1300 AD) from archaeological sites were used to assess basic morphological data. Our results provide evidence that the extinct Czech turtles fall into the size range of extant Central European E. orbicularis populations from the Hungarian Lowland. Maximum male and female straight-line carapacial lengths of the Czech specimens reached at least 160 and 175 mm, respectively.     

Synthesis and biological evaluation of new glutamic acid-based inhibitors of MurD ligase

Mur ligases catalyze the biosynthesis of the UDP-MurNAc-pentapeptide precursor of peptidoglycan, an essential polymer of bacterial cell-wall. They constitute attractive targets for the development of novel antibacterial agents. Here we report on the synthesis of a series of 2,4-diaminoquinazolines, quinazoline-2,4(1H,3H)-diones, 5-benzylidenerhodanines and 5-benzylidenethiazolidine-2,4-diones and their inhibitory activities against MurD from Escherichia coli. Compounds (R)-27 and (S)-27 showed inhibitory activity against MurD with IC₅₀ values of 174 and 206 μM, respectively, which makes them promising starting points for optimization.     

Overlapping expression patterns and differential transcript levels of phosphate transporter genes in arbuscular mycorrhizal,P<Subscript>i</Subscript>-fertilised and phytohormone-treated <Emphasis Type="Italic">Medicago truncatula</Emphasis> roots

A microarray carrying 5,648 probes of Medicago truncatula root-expressed genes was screened in order to identify those that are specifically regulated by the arbuscular mycorrhizal (AM) fungus Gigaspora rosea, by P_i fertilisation or by the phytohormones abscisic acid and jasmonic acid. Amongst the identified genes, 21% showed a common induction and 31% a common repression between roots fertilised with P_i or inoculated with the AM fungus G. rosea, while there was no obvious overlap in the expression patterns between mycorrhizal and phytohormone-treated roots. Expression patterns were further studied by comparing the results with published data obtained from roots colonised by the AM fungi Glomus mosseae and Glomus intraradices, but only very few genes were identified as being commonly regulated by all three AM fungi. Analysis of P_i concentrations in plants colonised by either of the three AM fungi revealed that this could be due to the higher P_i levels in plants inoculated by G. rosea compared with the other two fungi, explaining that numerous genes are commonly regulated by the interaction with G. rosea and by phosphate. Differential gene expression in roots inoculated with the three AM fungi was further studied by expression analyses of six genes from the phosphate transporter gene family in M. truncatula. While MtPT4 was induced by all three fungi, the other five genes showed different degrees of repression mirroring the functional differences in phosphate nutrition by G. rosea, G. mosseae and G. intraradices. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The influence of simvastatin, atorvastatin and high-cholesterol diet on acetylcholinesterase activity, amyloid beta and cholesterol synthesis in rat brain

OBJECTIVE: There is evidence to suppose that cholesterol-lowering medicine might confer protection against dementia, probably via modulation of cholesterol synthesis in the brain. The aim of the present study was to investigate the potential influence of statins and cholesterol diet on selected parameters relevant to Alzheimer's disease pathophysiology. METHODS: For 15 days, rats were orally administered simvastatin (10 or 20mg/kg b.wt.), atorvastatin (10 or 20mg/kg b.wt.), or aqua (control group); and one group was fed high-cholesterol (2%) diet. At the end of experiments brain (and plasma) cholesterol, lathosterol, hydroxymethylglutaryl-coenzyme A reductase protein, acetylcholinesterase activity, amyloid beta (40 and 42) and cholesterol synthesis rate (using the incorporation of deuterium from deuterated water) were determined and statistically compared to those of aqua. RESULTS: Both statins were able to lower cholesterol in the plasma, but none elicited an effect on total brain cholesterol. Significant reductions of brain lathosterol and cholesterol synthesis rate were observed after simvastatin and atorvastatin treatment. Acetylcholinesterase activity, amyloid beta and hydroxymethylglutaryl-coenzyme A reductase levels remained unaffected by the two drugs. CONCLUSIONS: This study brings additional evidence of a role for statins in cholesterol synthesis in the brain. Our data question the relationship between amyloid beta, acetylcholinesterase activity and cholesterol synthesis in the rat brain as well as the assumption about no exchange between peripheral and brain cholesterol pools.