Downregulation of PGY1/MDR1 mRNA level in human KB cells by antisense oligonucleotide conjugates. RNA accessibility in vitro and intracellular antisense activity

Inhibition of PGY1/MDR1 (multidrug resistance gene 1) mRNA expression in multidrug resistant KB-8-5 cells by 5'-bis-pyrenyl-3'-aminohexyl oligodeoxyribonucleotide conjugates targeted to four sites of this mRNA has been investigated. Three of the tested oligonucleotide conjugates specifically inhibited the expression of PGY1/MDR1 mRNA as monitored by the RT-PCR assay. The oligonucleotide conjugate targeted to the region (+178; +194) of the PGY1/MDR1 mRNA decreased level of this mRNA to 10% compared to the control. Nuclease-resistant analogs of oligonucleotide, complementary to this MDR1 mRNA region therefore, might be considered as a prototype compounds for development of gene-targeted therapeutic agents for overcoming the MDR phenotype caused by the overexpression of the PGY1/MDR1 gene.     

Parameters of lens protein fluorescence quenching in early stages of hereditary cataract

Several physico-chemical parameters of lenses were investigated in IOR/Hab mice with hereditary cataract aged 4, 6, 8 weeks, i.e. at the stage preceding the development of cataract. The level of restored glutathione in the IOR/Hab lenses /2.0 mM/ was the lowest as compared to other lines but almost the same as in CBA/J /2.3 mM/. In the process of studying protein fluorescence quenching in cortex homogenates by nitrate anions a significant increase of the quenching constant was discovered in IOR/Hab mice: 10.4 M-1 in four-week animals and up to 32.3 M-1 in eight-week animals. The data obtained suggest that a progressing growth of the quenching constant in line IOR/Hab lenses is related to a decreased density of the protein negative charge resulted from their decreased phosphorylation.     

Plant diversity and identity effects on predatory nematodes and their prey

There is considerable evidence that both plant diversity and plant identity can influence the level of predation and predator abundance aboveground. However, how the level of predation in the soil and the abundance of predatory soil fauna are related to plant diversity and identity remains largely unknown. In a biodiversity field experiment, we examined the effects of plant diversity and identity on the infectivity of entomopathogenic nematodes (EPNs, Heterorhabditis and Steinernema spp.), which prey on soil arthropods, and abundance of carnivorous non‐EPNs, which are predators of other nematode groups. To obtain a comprehensive view of the potential prey/food availability, we also quantified the abundance of soil insects and nonpredatory nematodes and the root biomass in the experimental plots. We used structural equation modeling (SEM) to investigate possible pathways by which plant diversity and identity may affect EPN infectivity and the abundance of carnivorous non‐EPNs. Heterorhabditis spp. infectivity and the abundance of carnivorous non‐EPNs were not directly related to plant diversity or the proportion of legumes, grasses and forbs in the plant community. However, Steinernema spp. infectivity was higher in monocultures of Festuca rubra and Trifolium pratense than in monocultures of the other six plant species. SEM revealed that legumes positively affected Steinernema infectivity, whereas plant diversity indirectly affected the infectivity of Heterorhabditis EPNs via effects on the abundance of soil insects. The abundance of prey (soil insects and root‐feeding, bacterivorous, and fungivorous nematodes) increased with higher plant diversity. The abundance of prey nematodes was also positively affected by legumes. These plant community effects could not be explained by changes in root biomass. Our results show that plant diversity and identity effects on belowground biota (particularly soil nematode community) can differ between organisms that belong to the same feeding guild and that generalizations about plant diversity effects on soil organisms should be made with great caution.     

Physiological roles of mitogen-activated-protein-kinase-activated p38-regulated/activated protein kinase

Mitogen-activated protein kinases (MAPKs) are a family of proteins that constitute signaling pathways involved in processes that control gene expression, cell division, cell survival, apoptosis, metabolism, differentiation and motility. The MAPK pathways can be divided into conventional and atypical MAPK pathways. The first group converts a signal into a cellular response through a relay of three consecutive phosphorylation events exerted by MAPK kinase kinases, MAPK kinase, and MAPK. Atypical MAPK pathways are not organized into this three-tiered cascade. MAPK that belongs to both conventional and atypical MAPK pathways can phosphorylate both non-protein kinase substrates and other protein kinases. The latter are referred to as MAPK-activated protein kinases. This review focuses on one such MAPK-activated protein kinase, MAPK-activated protein kinase 5 (MK5) or p38-regulated/activated protein kinase (PRAK). This protein is highly conserved throughout the animal kingdom and seems to be the target of both conventional and atypical MAPK pathways. Recent findings on the regulation of the activity and subcellular localization, bona fide interaction partners and physiological roles of MK5/PRAK are discussed.     

Effect of local weather on butterfly flight behaviour,movement, and colonization: significance for dispersal under climate change

Recent climate change is recognized as a main cause of shifts in geographical distributions of species. The impacts of climate change may be aggravated by habitat fragmentation, causing regional or large scale extinctions. However, we propose that climate change also may diminish the effects of fragmentation by enhancing flight behaviour and dispersal of ectothermic species like butterflies. We show that under weather conditions associated with anticipated climate change, behavioural components of dispersal of butterflies are enhanced, and colonization frequencies increase. In a field study, we recorded flight behaviour and mobility of four butterfly species: two habitat generalists (Coenonympha pamphilus; Maniola jurtina) and two specialists (Melitaea athalia; Plebejus argus), under different weather conditions. Flying bout duration generally increased with temperature and decreased with cloudiness. Proportion of time spent flying decreased with cloudiness. Net displacement generally increased with temperature. When butterflies fly longer, start flying more readily and fly over longer distances, we expect dispersal propensity to increase. Monitoring data showed that colonization frequencies moreover increased with temperature and radiation and decreased with cloudiness. Increased dispersal propensity at local scale might therefore lower the impact of habitat fragmentation on the distribution at a regional scale. Synergetic effects of climate change and habitat fragmentation on population dynamics and species distributions might therefore appear to be more complex than previously assumed.     

Modulation of F-actin rearrangement by the cyclic AMP/cAMP-dependent protein kinase (PKA) pathway is mediated by MAPK-activated protein kinase 5 and requires PKA-induced nuclear export of MK5

The MAPK-activated protein kinases belong to the Ca2+/calmodulin-dependent protein kinases. Within this group, MK2, MK3, and MK5 constitute three structurally related enzymes with distinct functions. Few genuine substrates for MK5 have been identified, and the only known biological role is in ras-induced senescence and in tumor suppression. Here we demonstrate that activation of cAMP-dependent protein kinase (PKA) or ectopic expression of the catalytic subunit Calpha in PC12 cells results in transient nuclear export of MK5, which requires the kinase activity of both Calpha and MK5 and the ability of Calpha to enter the nucleus. Calpha and MK5, but not MK2, interact in vivo, and Calpha increases the kinase activity of MK5. Moreover, Calpha augments MK5 phosphorylation, but not MK2, whereas MK5 does not seem to phosphorylate Calpha. Activation of PKA can induce actin filament accumulation at the plasma membrane and formation of actin-based filopodia. We demonstrate that small interfering RNA-triggered depletion of MK5 interferes with PKA-induced F-actin rearrangement. Moreover, cytoplasmic expression of an activated MK5 variant is sufficient to mimic PKA-provoked F-actin remodeling. Our results describe a novel interaction between the PKA pathway and MAPK signaling cascades and suggest that MK5, but not MK2, is implicated in PKA-induced microfilament rearrangement.     

Modifications of EEG Related to Repeated Realizations of the Emotional Stroop Task

Multichannel recording of EEG in 11 subjects, who were three times subjected to the emotional Stroop task (in the presence of words with negative emotional loading, neutral words, and inhibitory verbal stimuli), demonstrated that the spectral power of the high-frequency subcomponent of the alpha-rhythm in the left hemisphere increased sequentially under these conditions. Modifications of EEG, in general, were indicative of noticeable transformation of informational flows related to the learning effect and decrease of the cognitive “pressure” due to habituation to the effects of emotionally neutral and inhibitory verbal stimuli. Data related to the EEG pattern agree with the results of measurements of a behavioral index (decrease in the time of sensorimotor reaction).     

Specificity determinants of a novel Nck interaction with the juxtamembrane domain of the epidermal growth factor receptor

Nck is a ubiquitously expressed adaptor protein containing Src homology 2 (SH2) and Src homology 3 (SH3) domains. It integrates downstream effector proteins with cell membrane receptors, such as the epidermal growth factor receptor (EGFR). EGFR plays a critical role in cellular proliferation and differentiation. The 45-residue juxtamembrane domain of EGFR (JM), located between the transmembrane and kinase domains, regulates receptor activation and trafficking to the basolateral membrane of polarized epithelia through a proline-rich motif that resembles a consensus SH3 domain binding site. We demonstrate here that the JM region can bind to Nck, showing a notable binding preference for the second SH3 domain. To elucidate the structural determinants for this interaction, we have determined the NMR solution structures of both the first and second Nck SH3 domains (Nck1-1 and Nck1-2). These domains adopt a canonical SH3 beta-barrel-like fold, containing five antiparallel strands separated by three loop regions and one 3 10-helical turn. Chemical shift perturbation studies have identified the residues that form the binding cleft of Nck1-2, which are primarily located in the RT and n-Src loops. JM binds to Nck1-2 with an affinity of approximately 80 microM through a positively charged sequence near the N-terminus, as opposed to the polyproline sequence. The two Nck SH3 domains exhibit both steric and electrostatic differences in their RT-Src and n-Src loops, and a model of the Nck1-2 domain complexed with the JM highlights the factors that define the putative binding mode for this ligand.     

Relations between the mitogen-activated protein kinase and the cAMP-dependent protein kinase pathways: comradeship and hostility

Inter- and intracellular communications and responses to environmental changes are pivotal for the orchestrated and harmonious operation of multi-cellular organisms. These well-tuned functions in living organisms are mediated by the action of signal transduction pathways, which are responsible for receiving a signal, transmitting and amplifying it, and eliciting the appropriate cellular responses. Mammalian cells posses numerous signal transduction pathways that, rather than acting in solitude, interconnect with each other, a phenomenon referred to as cross-talk. This allows cells to regulate the distribution, duration, intensity and specificity of the response. The cAMP/cAMP-dependent protein kinase (PKA) pathway and the mitogen-activated protein kinase (MAPK) cascades modulate common processes in the cell and multiple levels of cross-talk between these signalling pathways have been described. The first- and best-characterized interconnections are the PKA-dependent inhibition of the MAPKs ERK1/2 mediated by RAF-1, and PKA-induced activation of ERK1/2 interceded through B-RAF. Recently, novel interactions between components of these pathways and new mechanisms for cross-talk have been elucidated. This review discusses both known and novel interactions between compounds of the cAMP/PKA and MAPKs signalling pathways in mammalian cells.     

Identification of a poly-cyclopropylglycine–containing peptide via bioinformatic mapping of radical S-adenosylmethionine enzymes

Peptide-derived natural products are a large class of bioactive molecules that often contain chemically challenging modifications. In the biosynthesis of ribosomally synthesized and posttranslationally modified peptides (RiPPs), radical-SAM (rSAM) enzymes have been shown to catalyze the formation of ether, thioether, and carbon-carbon bonds on the precursor peptide. The installation of these bonds typically establishes the skeleton of the mature RiPP. To facilitate the search for unexplored rSAM-dependent RiPPs for the community, we employed a bioinformatic strategy to screen a subfamily of peptide-modifying rSAM enzymes which are known to bind up to three [4Fe-4S] clusters. A sequence similarity network was used to partition related families of rSAM enzymes into >250 clusters. Using representative sequences, genome neighborhood diagrams were generated using the Genome Neighborhood Tool. Manual inspection of bacterial genomes yielded numerous putative rSAM-dependent RiPP pathways with unique features. From this analysis, we identified and experimentally characterized the rSAM enzyme, TvgB, from the tvg gene cluster from Halomonas anticariensis. In the tvg gene cluster, the precursor peptide, TvgA, is comprised of a repeating TVGG motif. Structural characterization of the TvgB product revealed the repeated formation of cyclopropylglycine, where a new bond is formed between the γ-carbons on the precursor valine. This novel RiPP modification broadens the functional potential of rSAM enzymes and validates the proposed bioinformatic approach as a practical broad search tool for the discovery of new RiPP topologies.