Co-Crystal Structures of PKG Iβ (92–227) with cGMP and cAMP Reveal the Molecular Details of Cyclic-Nucleotide Binding

Cyclic GMP-dependent protein kinases (PKGs) are central mediators of the NO-cGMP signaling pathway and phosphorylate downstream substrates that are crucial for regulating smooth muscle tone, platelet activation, nociception and memory formation. As one of the main receptors for cGMP, PKGs mediate most of the effects of cGMP elevating drugs, such as nitric oxide-releasing agents and phosphodiesterase inhibitors which are used for the treatment of angina pectoris and erectile dysfunction, respectively. configuration, with a conserved threonine residue anchoring both cyclic phosphate and guanine moieties. The structure of CNBD-A in the absence of bound cyclic nucleotide was similar to that of the cyclic nucleotide bound structures. Surprisingly, isothermal titration calorimetry experiments demonstrated that CNBD-A binds both cGMP and cAMP with a relatively high affinity, showing an approximately two-fold preference for cGMP. conformation through its interaction with Thr193 and an unusual cis-peptide forming residues Leu172 and Cys173. Although these studies provide the first structural insights into cyclic nucleotide binding to PKG, our ITC results show only a two-fold preference for cGMP, indicating that other domains are required for the previously reported cyclic nucleotide selectivity

Cell-to-Cell Transformation in Escherichia coli: A Novel Type of Natural Transformation Involving Cell-Derived DNA and a Putative Promoting Pheromone

Escherichia coli is not assumed to be naturally transformable. However, several recent reports have shown that E. coli can express modest genetic competence in certain conditions that may arise in its environment. We have shown previously that spontaneous lateral transfer of non-conjugative plasmids occurs in a colony biofilm of mixed E. coli strains (a set of a donor strain harbouring a plasmid and a plasmid-free recipient strain). In this study, with high-frequency combinations of strains and a plasmid, we constructed the same lateral plasmid transfer system in liquid culture. Using this system, we demonstrated that this lateral plasmid transfer was DNase-sensitive, indicating that it is a kind of transformation in which DNase-accessible extracellular naked DNA is essential. However, this transformation did not occur with purified plasmid DNA and required a direct supply of plasmid from co-existing donor cells. Based on this feature, we have termed this transformation type as ‘cell-to-cell transformation’. Analyses using medium conditioned with the high-frequency strain revealed that this strain released a certain factor(s) that promoted cell-to-cell transformation and arrested growth of the other strains. This factor is heat-labile and protease-sensitive, and its roughly estimated molecular mass was between ∼9 kDa and ∼30 kDa, indicating that it is a polypeptide factor. Interestingly, this factor was effective even when the conditioned medium was diluted 10–5–10–6, suggesting that it acts like a pheromone with high bioactivity. Based on these results, we propose that cell-to-cell transformation is a novel natural transformation mechanism in E. coli that requires cell-derived DNA and is promoted by a peptide pheromone. This is the first evidence that suggests the existence of a peptide pheromone-regulated transformation mechanism in E. coli and in Gram-negative bacteria

Specifying safety-critical embedded systems with statecharts and Z: A case study

In this paper we introduce a formal approach for the specification of safety-critical embedded systems. The specification formalisms Z and statecharts are integrated under a suitable structural model. The combined approach uses the advantages of the formalisms while avoiding their disadvantages. The different formalisms yield different, compatible views on the system: the functional view describing data and data-transformation, the reactive view, describing the system's reaction upon external stimuli, and the structural view, describing the components of the system and their interaction. The combination is discussed presenting parts of a case study: a traffic light control system. The case study is oriented at original planning documents. Besides its safety- and real-time-aspects, the case study is particularly interesting because structuring and reuse is of considerable importance in this example

An Open Environment for the Integration of Heterogeneous Modelling Techniques and Tools

this paper

A Modular Framework for the Integration of Heterogeneous Notations and Tools

. We report on the notation and tool integration framework developed in the application-oriented research project Espress. The framework is based on a Z core and provides means of integrating formalisms on a syntactical, semantical as well as tool technical level. Currently, the framework integrates Z with the languages of Statemate [15], i.e. Statecharts to describe reactive behavior and activity charts to describe structural aspects. Our means of integration are, however, general enough to integrate other, esp. synchronous languages, as well, and reuse much of the existing tools in the process. 1 Introduction Every software-based system potentially benefits from the application of formal techniques. For the development of mission or even safety-critical embedded systems, their use is of particular advantage, because the potential damage operators and developers have to envisage in case of malfunction may be much worse than the additional costs of applying formal techniques...