Anterograde Signalling by Nitric Oxide: Characterisation and In Vitro Reconstitution of an Identified Nitrergic Synapse

Nitric oxide (NO) is recognized as a signaling molecule in the CNS where it is a candidate retrograde neurotransmitter. Here we provide direct evidence that NO mediates slow excitatory anterograde transmission between the NO synthase (NOS)-expressing B2 neuron and an NO-responsive follower neuron named B7nor. Both are motoneurons located in the buccal ganglia of the snail Lymnaea stagnalis where they participate in feeding behavior. Transmission between B2 and B7nor is blocked by inhibiting NOS and is suppressed by extracellular scavenging of NO. Furthermore, focal application of NO to the cell body of the B7nor neuron causes a depolarization that mimics the effect of B2 activity. The slow interaction between the B2 and B7nor neurons can be re-established when the two neurons are cocultured, and it shows the same susceptibility to NOS inhibition and NO scavenging. In cell culture we have also examined spatial aspects of NO signaling. We show that before the formation of an anatomical connection, the presynaptic neuron can cause depolarizing potentials in the follower neuron at distances up to 50 micro(m). The strength of the interaction increases when the distance between the cells is reduced. Our results suggest that NO can function as both a synaptic and a nonsynaptic signaling molecul

Associative memory stored by functional novel pathway rather than modifications of preexisting neuronal pathways

Associative conditioning involves changes in the processing pathways activated by sensory information to link the conditioned stimulus (CS) to the conditioned behavior. Thus, conditioning can recruit neuronal elements to form new pathways for the processing of the CS and/or can change the strength of existing pathways. Using a behavioral and systems level electrophysiological approach on a tractable invertebrate circuit generating feeding in the mollusk Lymnaea stagnalis, we identified three independent pathways for the processing of the CS amyl acetate used in appetitive conditioning. Two of these pathways, one suppressing and the other stimulating feeding, mediate responses to the CS in naive animals. The effects ofthese two pathways on feeding behavior are unaltered by conditioning. In contrast, the CS response ofa third stimulatory pathway is significantly enhanced after conditioning, becoming an importantcontributor to the overall CS response. This is unusual because, in most of the previous examples in which naive animals already respond to the CS, memory formation results from changes in the strength of pathways that mediate the existing response. Here, we show that, in the molluscan feeding system, both modified and unmodified pathways are activated in parallel by the CS after conditioning, and it is their integration that results in the conditioned respons

Development and Introduction of an Information System to Support Business Processes

Diplomová práca sa zameriava na vývoj informačného systému určeného pre správu vnútropodnikových procesov spoločnosti IJM Group, s.r.o. Zobrazuje teoretické východiská, na ktoré nadväzuje ťažisková časť diplomovej práce rozoberajúca návrh, vývoj a implementáciu samotného systému.Master’s thesis is focusing on the development of informatic system designed for the intra-enterprise processes of IJM Group s. r. o. company. The main part of the thesis dealing with the development and implementation of the system is linked to theoretical options.

Web Pages Design

Vytvorenie aplikácie, ktorá umožňuje užívateľovi intuitívnu tvorbu webovej prezentácie bez akejkoľvek predchádzajúcej znalosti programovacích jazykov. Vytvorená webová prezentácia je použitá ako vzdelávací systém pre určitú skupinu užívateľov v oblasti multi level marketingu.To create an application that allows users intuitive formation of web presentations without any prior knowledge of programming languages. The created web presentation is used as an educational system for a group of users in multi-level marketing.

Timed and targeted differential regulation of nitric oxide synthase (NOS) and anti-NOS genes by reward conditioning leading to long-term memory formation

In a number of neuronal models of learning, signaling by the neurotransmitter nitric oxide (NO), synthesized by the enzyme neuronal NO synthase (nNOS), is essential for the formation of long-term memory (LTM). Using the molluscan model system Lymnaea, we investigate here whether LTM formation is associated with specific changes in the activity of members of the NOS gene family: Lym-nNOS1, Lym-nNOS2, and the antisense RNA-producing pseudogene (anti-NOS). We show that expression of the Lym-nNOS1 gene is transiently upregulated in cerebral ganglia after conditioning. The activation of the gene is precisely timed and occurs at the end of a critical period during which NO is required for memory consolidation. Moreover, we demonstrate that this induction of the Lym-nNOS1 gene is targeted to an identified modulatory neuron called the cerebral giant cell (CGC). This neuron gates the conditioned feeding response and is an essential part of the neural network involved in LTM formation. We also show that the expression of the anti-NOS gene, which functions as a negative regulator of nNOS expression, is downregulated in the CGC by training at 4 h after conditioning, during the critical period of NO requirement. This appears to be the first report of the timed and targeted differential regulation of the activity of a group of related genes involved in the production of a neurotransmitter that is necessary for learning, measured in an identified neuron of known function. We also provide the first example of the behavioral regulation of a pseudogene

Suppression of nitric oxide (NO)-dependent behavior by double-stranded RNA-mediated silencing of a neuronal NO synthase gene

We have used double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) to disrupt neuronal nitric oxide (NO) synthase (nNOS) gene function in the snail Lymnaea stagnalis and have detected a specific behavioral phenotype. The injection of whole animals with synthetic dsRNA molecules targeted to the nNOS-encoding mRNA reduces feeding behavior in vivo and fictive feeding in vitro and interferes with NO synthesis by the CNS. By showing that synthetic dsRNA targeted to the nNOS mRNA causes a significant and long-lasting reduction in the levels of Lym-nNOS mRNA, we verify that specific RNAi has occurred. Importantly, our results establish that the expression of nNOS gene is essential for normal feeding behavior. They also show that dsRNA can be used in the investigation of functional gene expression in the context of whole animal behavior, regardless of the availability of targeted mutation technologies

A P2X receptor from the tardigrade species Hypsibius dujardini with fast kinetics and sensitivity to zinc and copper

<p>Abstract</p> <p>Background</p> <p>Orthologs of the vertebrate ATP gated P2X channels have been identified in <it>Dictyostelium </it>and green algae, demonstrating that the emergence of ionotropic purinergic signalling was an early event in eukaryotic evolution. However, the genomes of a number of animals including <it>Drosophila melanogaster </it>and <it>Caenorhabditis elegans</it>, both members of the Ecdysozoa superphylum, lack P2X-like proteins, whilst other species such as the flatworm <it>Schistosoma mansoni </it>have P2X proteins making it unclear as to what stages in evolution P2X receptors were lost. Here we describe the functional characterisation of a P2X receptor (<it>Hd</it>P2X) from the tardigrade <it>Hypsibius dujardini </it>demonstrating that purinergic signalling is preserved in some ecdysozoa.</p> <p>Results</p> <p>ATP (EC₅₀~44.5 μM) evoked transient inward currents in <it>Hd</it>P2X with millisecond rates of activation and desensitisation. <it>Hd</it>P2X is antagonised by pyridoxal-phosphate-6-azophenyl-2',4' disulfonic acid (IC₅₀15.0 μM) and suramin (IC₅₀22.6 μM) and zinc and copper inhibit ATP-evoked currents with IC₅₀values of 62.8 μM and 19.9 μM respectively. Site-directed mutagenesis showed that unlike vertebrate P2X receptors, extracellular histidines do not play a major role in coordinating metal binding in <it>Hd</it>P2X. However, H306 was identified as playing a minor role in the actions of copper but not zinc. Ivermectin potentiated responses to ATP with no effect on the rates of current activation or decay.</p> <p>Conclusion</p> <p>The presence of a P2X receptor in a tardigrade species suggests that both nematodes and arthropods lost their P2X genes independently, as both traditional and molecular phylogenies place the divergence between Nematoda and Arthropoda before their divergence from Tardigrada. The phylogenetic analysis performed in our study also clearly demonstrates that the emergence of the family of seven P2X channels in human and other mammalian species was a relatively recent evolutionary event that occurred subsequent to the split between vertebrates and invertebrates. Furthermore, several characteristics of <it>Hd</it>P2X including fast kinetics with low ATP sensitivity, potentiation by ivermectin in a channel with fast kinetics and distinct copper and zinc binding sites not dependent on histidines make <it>Hd</it>P2X a useful model for comparative structure-function studies allowing a better understanding of P2X receptors in higher organisms.</p

THE EXTERNAL STATE DEBT OF UKRAINE: FORMATION, MANAGEMENT AND SERVICING

Introduction. State debt is a substantial instrument of the macroeconomic and the fiscal policy of the state and it affects a social and an economic condition in Ukraine. At the same time, the rapid growth in volumes of a gross external debt in absolute value and in ratio to GDP increases the currency risks of the country. Significant debt to the international organizations and the foreign governments threatens the country’s economic security. Undeveloped market of government securities complicates the borrowing of loans and lack of the comprehensive legal support and a perfect institutional mechanism hamper a process of the effective management of the state debt. Purpose. The purpose of the article is to research a practice of forming and servicing of the external state debt borrows, to identify the priority directions of improving the management of the external state debt. Results. It was substantiated that in Ukraine the growth of the state debt and expends of its servicing are gaining more menacing traits. It was determined that the gross external debt includes the state external debt of the government and the National Bank, as well as private banks, national and joint-stock companies, etc. Ukraine’s gross external debt as of the end of 2017 amounted to 116,578 million USD and it is current decreasing of the total volume of 3,060 million USD compared to the beginning of the year. It was investigated that according to the results of 2010-2017, the aggregate volume of the external state debt increased by 40% or 14,229 million USD to a level of 48,989 million USD. According to indicators of growth rates of the external state debt of 2010-2017, the trend is characterized by slowing in the growth of the debt since 2011-2013, rapid increase in 2015 due to economic and political aggravations in Ukraine and decreasing in growth rate in 2016- 2017, which is a result of restructuring of the country’s debt to foreign private creditors. It was emphasized that rapid reduce of the volumes of gold and foreign exchange reserves of Ukraine during the period of the new economic crisis of 2014 and a gradual steady increasing in volumes in subsequent years are both logical. The years of the most intense debt growth with the simultaneous gradual increase in the volumes of gold and foreign exchange reserves can be admitted 2014-2017, which are characterized by aggravation of the economic instability of the country due to high currency borrowing risks and the significant debt burden on the budget. We determined that an indicator of Ukraine’s external state debt per 1 person in 2017 amounted to 2,744 USD, exceeding the maximum permissible limit more than 10 times. We noted that one of the actual threats to the national security of Ukraine is the ineffective management of the state debt. Conclusions. External loans in the short term increase the financial potential of a borrowing country however, in the long term they contribute to reduction of the national income and deepen financial dependence on other countries. System of the management of the external state debt remains ineffective and leads to shifting of the debt burden for servicing and repayment of the state debt upon taxpayers and subsequent generations. The policy of such borrowings is a significant threat to the fiscal security, financial stability and further socio-economic development of the state. The above mentioned demonstrate objective need for further scientific research in order to improve the system of the management and servicing of the external state debt

Mutation of the Glucosinolate Biosynthesis Enzyme Cytochrome P450 83A1 Monooxygenase Increases Camalexin Accumulation and Powdery Mildew Resistance

Small secondary metabolites, including glucosinolates and the major phytoalexin camalexin, play important roles in immunity in Arabidopsis thaliana. We isolated an Arabidopsis mutant with increased resistance to the powdery mildew fungus Golovinomyces cichoracearum and identified a mutation in the gene encoding cytochrome P450 83A1 monooxygenase (CYP83A1), which functions in glucosinolate biosynthesis. The cyp83a1-3 mutant exhibited enhanced defense responses to G. cichoracearum and double mutant analysis showed that this enhanced resistance requires NPR1, EDS1, and PAD4, but not SID2 or EDS5. In cyp83a1-3 mutants, the expression of genes related to camalexin synthesis increased upon G. cichoracearum infection. Significantly, the cyp83a1-3 mutant also accumulated higher levels of camalexin. Decreasing camalexin levels by mutation of the camalexin synthetase gene PAD3 or the camalexin synthesis regulator AtWRKY33 compromised the powdery mildew resistance in these mutants. Consistent with these observations, overexpression of PAD3 increased camalexin levels and enhanced resistance to G. cichoracearum. Taken together, our data indicate that accumulation of higher levels of camalexin contributes to increased resistance to powdery mildew