Description of the immature stages of two Mononychus species (Coleoptera: Curculionidae: Ceutorhynchinae) and a study of the host preferences of M. punctumalbum for Iris species in central Europe

Last larval instars and pupae of Mononychus punctumalbum (Herbst, 1784) and Mononychus schoenherri Kolenati, 1859 (Curculionidae, Ceutorhynchinae) are described in detail for the first time. No immatures of other species of this genus had previously been described. The immatures of Mononychus were compared with available data on immatures of all genera in the subfamily Ceutorhynchinae. Both the larvae and pupae of the two Mononychus species are very distinctive, having distinct characters from other genera in the subfamily Ceutorhynchinae. New bionomic data for M. punctumalbum concerning its host preferences for Iris species in central Europe can be used to verify literature data indicating the important role of the iris weevil in the dramatic decline in the plant's abundance in natural habitats as well as in gardens and other anthropogenic associations.     

Exogenous melatonin expediently modifies proteome of maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) embryo during seed germination

Melatonin (N-acetyl-5-methoxytryptamine) has a great potential for plant biostimulation. Its role in plant physiology is intensively explored, and its important function in plant stress defence frequently underlined. Melatonin is particularly effective when applied as an additional factor of seed priming. In the presented research, hydroconditioning was chosen experimentally as optimal for maize (Zea mays L.) seeds. The following seed variants were compared: controlled non-treated, hydroprimed with water and hydroprimed with melatonin solutions 50 and 500 μM. To identify modifications in proteome of maize seeds caused by the applied hydroconditioning techniques, protein extracts of germinated seed embryos (24 h, 25 °C) were separated by 2D-PAGE. Next, obtained maps of proteomes were compared (statistically and graphically) using PDQuest software, and characteristic spots of proteins were analysed qualitatively by mass-spectrometric techniques and identified in the Mascot protein databases. Research helped to identify hydropriming-associated proteins, and for the first time those which were expressed only in the presence of melatonin. Study confirmed that suitably selected pre-sowing treatment with melatonin, by embryo proteome modification, effectively prepares plants to adverse environmental conditions. In melatonin treated seeds during the initial state of embryos growth, even under optimal conditions, additional antioxidative, detoxifying, anti-stresses proteins were synthesized. Moreover, the supply of energy from seed storage substances was pretty intensified. The presented results partially explain how melatonin acts in plant stress defence, and why plants with higher melatonin content have exhibited increased capacity for stress tolerance.     

Novel 5-HT7R antagonists,arylsulfonamide derivatives of (aryloxy)propyl piperidines: Add-on effect to the antidepressant activity of SSRI and DRI,and pro-cognitive profile

A novel series of arylsulfonamide derivatives of (aryloxy)propyl piperidines was designed to obtain potent 5-HT₇R antagonists. Among the compounds evaluated herein, 3-chloro-N-{1-[3-(1,1-biphenyl-2-yloxy)2-hydroxypropyl]piperidin-4-yl}benzenesulfonamide (25) exhibited antagonistic properties at 5-HT₇R and showed selectivity over selected serotoninergic and dopaminergic receptors, as well as over serotonin, noradrenaline and dopamine transporters. Compound 25 demonstrated significant antidepressant-like activity in the forced swim test (0.625–2.5 mg/kg, i.p.) and in the tail suspension test (1.25 mg/kg, i.p.), augmented the antidepressant effect of inactive doses of escitalopram (selective serotonin reuptake inhibitor) and bupropion (dopamine reuptake inhibitor) in the FST in mice, and similarly to SB-269970, exerted pro-cognitive properties in the novel object recognition task in cognitively unimpaired conditions in rats (0.3 mg/kg, i.p.). Such an extended pharmacological profile, especially the augmentation effect of the identified 5-HT₇R antagonist on SSRI activity, seems promising regarding the complexity of affective disorders and potentially improved outcomes, including mnemonic performance.     

Spatial interactions between grey wolves and Eurasian lynx in Białowieża Primeval Forest, Poland

Various species of large predators are reported to influence each other through interference or exploitation competition that may affect demography and survival of the subordinate species. We analyzed spatial relationships between grey wolf (Canis lupus) and Eurasian lynx (Lynx lynx) in Białowieża Primeval Forest (BPF, eastern Poland) to determine how they partitioned the space. The wolves (n = 8) and lynx (n = 14) were radio-tracked in 1991–1999. Three wolves and seven lynx were radio-tracked simultaneously in 1994–1996. Territories of wolf packs and home ranges of lynx overlapped considerably (76% of wolf territories and 50% of lynx home ranges, on average). In three cases, their core areas were also overlapping. Wolf-lynx dyads with overlapping home ranges were simultaneously located at distances from 0 to 28 km from each other. We found neither avoidance nor attraction between wolves and lynx occupying the same areas. We concluded that in BPF, the two large predators coexist due to specialization on different preferred prey and heterogeneous habitat.     

DNA methylation changes in triticale due to in vitro culture plant regeneration and consecutive reproduction

Doubled haploids of triticale are of interest for plant breeders due to hybrid breeding programs based on cytoplasmic male sterility Tt phenomenon. However, (epi)mutations appearing during in vitro culture regeneration may lead to a phenotypic variation that makes the uniformity of plant materials questionable. Using RP-HPLC genomic DNA methylation of donor doubled haploid plants utilized as a source of tissues for the in vitro regeneration (via androgenesis and somatic embryogenesis) of triticale cv. Bogo and their consecutive generative progeny was evaluated. It was demonstrated that in vitro cultures induced a decrease of the DNA methylation of the regenerants independently of the approach used for plant regeneration. The decrease in DNA methylation of genomic DNA proceeded up to the first/second successive generations followed by the beginning of its reestablishment. Moreover, somatic embryogenesis resulted in a higher level of genomic DNA demethylation in regenerants than androgenesis and the process of methylation seems to be affected by donor plant. It is being speculated that long term changes in genomic DNA methylation may be a source of off-type individuals that may spontaneously arise during plant breeding.     

Light-induced isomerization of the LHCII-bound xanthophyll neoxanthin: possible implications for photoprotection in plants

Light-harvesting pigment-protein complex of Photosystem II (LHCII) is the largest photosynthetic antenna complex of plants and the most abundant membrane protein in the biosphere. Plant fitness and productivity depend directly on a balance between excitations in the photosynthetic apparatus, generated by captured light quanta, and the rate of photochemical processes. Excess excitation energy leads to oxidative damage of the photosynthetic apparatus and entire organism and therefore the balance between the excitation density and photosynthesis requires precise and efficient regulation, operating also at the level of antenna complexes. We show that illumination of the isolated LHCII leads to isomerization of the protein-bound neoxanthin from conformation 9'-cis to 9',13- and 9',13'-dicis forms. At the same time light-driven excitation quenching is observed, manifested by a decrease in chlorophyll a fluorescence intensity and shortened fluorescence lifetimes. Both processes, the neoxanthin isomerization and the chlorophyll excitation quenching, are reversible in dim light. The results of the 77K florescence measurements of LHCII show that illumination is associated with appearance of the low-energy states, which can serve as energy traps in the pigment-protein complex subjected to excess excitation. Possible sequence of the molecular events is proposed, leading to a protective excess excitation energy quenching: neoxanthin photo-isomerization→formation of LHCII supramolecular structures which potentiate creation of energy traps→excitation quenching.     

Induction of monocyte antitumor response by human cancer cells transduced with TNF-GFP fusion gene: possible implications for immunotherapy of cancer

This study was undertaken to determine how human pancreatic cancer (HPC-4) cells transduced with the TNF-GFP fusion gene (TLG) alter the antitumor response of human monocytes in vitro and whether they could act as an antitumor vaccine. In our model, HPC-4 cells were transduced with retroviral vector harboring TLG gene and designated as HPC-4(TLG). The TLG protein expression was confirmed by Western blot and flow cytometry analysis. Monocytes were co-cultured with transduced and control HPC-4 cells. The secretion of TNF, IL-10 and IL-12 was measured by ELISA. The cytotoxicity of monocytes against HPC-4 cells was determined by MTT test. The results show that the HPC-4(TLG) cells expressed membrane-bound, intracellular and secretory TLG protein. When cultured with HPC-4(TLG) cells, monocytes released a higher amount of TNF, but IL-10 and IL-12 secretion was inhibited. The pre-exposure of monocytes to HPC-4(TLG), but not to HPC-4, cells did not decrease TNF nor increase IL-10 production, thus not leading to monocyte deactivation. Also, the antitumor cytotoxicity of monocytes stimulated with HPC-4(TLG) was not downregulated, which occurred when non-transduced HPC-4 cells were used. In conclusion, compared to parental HPC-4 cells, TLG gene transduced HPC-4 cells induced stronger antitumor response of monocytes in vitro and prevented deactivation of monocytes.     

MicroRNA in lung cancer diagnostics and treatment

The measurement of serine139-phosphorylated histone H2AX (γH2AX) provides a biomarker of DNA double-strand breaks (DSBs) and may identify potential genotoxic activity. In order to evaluate a flow cytometry assay for γH2AX detection (hereafter termed the γH2AX by flow assay), 6 prototypical (3 pro- and 3 proximate) genotoxins, i.e. dimethylbenz[a]anthracene (DMBA), 2-acetylaminofluorene (2-AAF), benzo[a]pyrene (B[a]P), methyl methane sulphonate (MMS), methyl nitrosourea (MNU) and 4-nitroquinoline oxide (4NQO), were selected to define assay evaluation criteria. In addition, 3 non-genotoxic cytotoxins (phthalic anhydride, n-butyl chloride and hexachloroethane) were included to investigate the influence of cytotoxicity on assay performance. At similar cytotoxicity levels (relative cell counts; RCC 75-40%) all prototypical genotoxins induced marked concentration-dependent increases in γH2AX compared with the non-genotoxins. As a result, assay evaluation criteria for a positive effect were defined as >1.5-fold γH2AX @ RCC >25%. Twenty five additional chemicals with diverse structures and genotoxic activity were selected to evaluate the γH2AX by flow assay. Results were compared with Ames bacterial and in vitro mammalian genotoxicity tests (mouse lymphoma assay and/or chromosome aberration assay). γH2AX by flow assay results were highly predictive of Ames (sensitivity 100%; specificity 67%; concordance 82%) and in vitro mammalian genotoxicity tests (sensitivity 91%; specificity 89%; concordance 91%) and provide additional evidence that γH2AX is a biomarker of potential genotoxic activity, underpinned mechanistically by the cellular response to DSBs. Discordant findings were predominately attributed to differences in specificity for some mammalian cell genotoxins that are Ames non-mutagens or for "biologically-irrelevant" positives in the mammalian tests. Simple anilines were classified as genotoxic following rat liver S9-mediated bioactivation, however, effects on γH2AX were atypical and limited to a small sub-population of S-phase nuclei. Nevertheless, the γH2AX by flow assay represents a novel genotoxicity assay with the potential to flag both pro- and proximate genotoxins.     

Haemolysis and Perturbations in the Systemic Iron Metabolism of Suckling,Copper-Deficient Mosaic Mutant Mice – An Animal Model of Menkes Disease

The biological interaction between copper and iron is best exemplified by the decreased activity of multicopper ferroxidases under conditions of copper deficiency that limits the availability of iron for erythropoiesis. However, little is known about how copper deficiency affects iron homeostasis through alteration of the activity of other copper-containing proteins, not directly connected with iron metabolism, such as superoxide dismutase 1 (SOD1). This antioxidant enzyme scavenges the superoxide anion, a reactive oxygen species contributing to the toxicity of iron via the Fenton reaction. Here, we analyzed changes in the systemic iron metabolism using an animal model of Menkes disease: copper-deficient mosaic mutant mice with dysfunction of the ATP7A copper transporter. We found that the erythrocytes of these mutants are copper-deficient, display decreased SOD1 activity/expression and have cell membrane abnormalities. In consequence, the mosaic mice show evidence of haemolysis accompanied by haptoglobin-dependent elimination of haemoglobin (Hb) from the circulation, as well as the induction of haem oxygenase 1 (HO1) in the liver and kidney. Moreover, the hepcidin-ferroportin regulatory axis is strongly affected in mosaic mice. These findings indicate that haemolysis is an additional pathogenic factor in a mouse model of Menkes diseases and provides evidence of a new indirect connection between copper deficiency and iron metabolism.