Membrane-associated phosphoinositides-specific phospholipase C forms from <Emphasis Type="Italic">Catharanthus roseus</Emphasis> transformed roots

We have previously reported that Catharanthus roseus transformed roots contain at least two phosphatidylinositol 4,5-bisphosphate-phospholipase C (PLC) activities, one soluble and the other membrane associated. Detergent, divalent cations, and neomycin differentially regulate these activities and pure protein is required for a greater understanding of the function and regulation of this enzyme. In this article we report a partia purification of membrane-associated PLC. We found that there are at least two forms of membrane-associated PLC in transformed roots of C. roseus. These forms were separated on the basis of their affinity for heparin. One form shows an affinity for heparin and elutes at approx 600 mM KCl. This form has a molecular mass of 67 kDa by size exclusion chromatography and Western blot analysis, whereas the other form does not bind to heparin and has a molecular mass of 57 kDa. Possible differential regulation of these forms during transformed root growth is discussed.     

A solution approach for deriving alternative fuel station infrastructure requirements

When an alternative fuel is introduced, the infrastructure through which that fuel is made available to the market is often underdeveloped. Transportation service providers relying on such infrastructures are unlikely to adopt alternative fuel vehicles as it may impose long detours for refueling. In this paper, we design and apply a new solution approach to derive minimum infrastructure requirements, in terms of the number of alternative fuel stations. The effectiveness of our approach is demonstrated by applying it to the case of introducing liquefied natural gas (LNG) as a transportation fuel in The Netherlands. From this case, we learn that, depending on the driving range of the LNG trucks and the size of area on which those trucks operate, a minimum of 5–12 LNG fuel stations is necessary to render LNG trucks economically and environmentally beneficial.     

A Polyketide Synthase Gene Required for Biosynthesis of the Aflatoxin-like Toxin, Dothistromin

Dothistromin is a polyketide toxin, produced by a fungal forest pathogen, with structural similarity to the aflatoxin precursor versicolorin B. Biochemical and genetic studies suggested that there are common steps in the biosynthetic pathways for these metabolites and showed similarities between some of the genes. A polyketide synthase gene (pksA) was isolated from dothistromin-producing Dothistroma septosporum by hybridization with an aflatoxin ortholog from Aspergillus parasiticus. Inactivation of this gene in D. septosporum resulted in mutants that could not produce dothistromin but that could convert exogenous aflatoxin precursors, including norsolorinic acid, into dothistromin. The mutants also had reduced asexual sporulation compared to the wild type. So far four other genes are known to be clustered immediately alongside pksA. Three of these (cypA, moxA, avfA) are predicted to be orthologs of aflatoxin biosynthetic genes. The other gene (epoA), located between avfA and moxA, is predicted to encode an epoxide hydrolase, for which there is no homolog in either the aflatoxin or sterigmatocystin gene clusters. The pksA gene is located on a small chromosome of ~1.3 Mb in size, along with the dothistromin ketoreductase (dotA) gene.     

Binding cavities and druggability of intrinsically disordered proteins

To assess the potential of intrinsically disordered proteins (IDPs) as drug design targets, we have analyzed the ligand-binding cavities of two datasets of IDPs (containing 37 and 16 entries, respectively) and compared their properties with those of conventional ordered (folded) proteins. IDPs were predicted to possess more binding cavity than ordered proteins at similar length, supporting the proposed advantage of IDPs economizing genome and protein resources. The cavity number has a wide distribution within each conformation ensemble for IDPs. The geometries of the cavities of IDPs differ from the cavities of ordered proteins, for example, the cavities of IDPs have larger surface areas and volumes, and are more likely to be composed of a single segment. The druggability of the cavities was examined, and the average druggable probability is estimated to be 9% for IDPs, which is almost twice that for ordered proteins (5%). Some IDPs with druggable cavities that are associated with diseases are listed. The optimism versus obstacles for drug design for IDPs is also briefly discussed.     

Interplay between glutathione,Atx1 and copper. 1. Copper(I) glutathionate induced dimerization of Atx1

Copper is both an essential element as a catalytic cofactor and a toxic element because of its redox properties. Once in the cell, Cu(I) binds to glutathione (GSH) and various thiol-rich proteins that sequester and/or exchange copper with other intracellular components. Among them, the Cu(I) chaperone Atx1 is known to deliver Cu(I) to Ccc2, the Golgi Cu–ATPase, in yeast. However, the mechanism for Cu(I) incorporation into Atx1 has not yet been unraveled. We investigated here a possible role of GSH in Cu(I) binding to Atx1. Yeast Atx1 was expressed in Escherichia coli and purified to study its ability to bind Cu(I). We found that with an excess of GSH [at least two GSH/Cu(I)], Atx1 formed a Cu(I)-bridged dimer of high affinity for Cu(I), containing two Cu(I) and two GSH, whereas no dimer was observed in the absence of GSH. The stability constants (log β) of the Cu(I) complexes measured at pH 6 were 15–16 and 49–50 for CuAtx1 and Cu₂^I(GS⁻)₂(Atx1)₂, respectively. Hence, these results suggest that in vivo the high GSH concentration favors Atx1 dimerization and that Cu₂^I(GS⁻)₂(Atx1)₂ is the major conformation of Atx1 in the cytosol.