Insights to the Structural Basis for the Stereospecificity of the Escherichia coli Phytase,AppA

AppA, the Escherichia coli periplasmic phytase of clade 2 of the histidine phosphatase (HP2) family, has been well-characterized and successfully engineered for use as an animal feed supplement. AppA is a 1D-6-phytase and highly stereospecific but transiently accumulates 1D-myo-Ins(2,3,4,5)P₄ and other lower phosphorylated intermediates. If this bottleneck in liberation of orthophosphate is to be obviated through protein engineering, an explanation of its rather rigid preference for the initial site and subsequent cleavage of phytic acid is required. To help explain this behaviour, the role of the catalytic proton donor residue in determining AppA stereospecificity was investigated. Four variants were generated by site-directed mutagenesis of the active site HDT amino acid sequence motif containing the catalytic proton donor, D304. The identity and position of the prospective proton donor residue was found to strongly influence stereospecificity. While the wild-type enzyme has a strong preference for 1D-6-phytase activity, a marked reduction in stereospecificity was observed for a D304E variant, while a proton donor-less mutant (D304A) displayed exclusive 1D-1/3-phytase activity. High-resolution X-ray crystal structures of complexes of the mutants with a non-hydrolysable substrate analogue inhibitor point to a crucial role played by D304 in stereospecificity by influencing the size and polarity of specificity pockets A and B. Taken together, these results provide the first evidence for the involvement of the proton donor residue in determining the stereospecificity of HP2 phytases and prepares the ground for structure-informed engineering studies targeting the production of animal feed enzymes capable of the efficient and complete dephosphorylation of dietary phytic acid.     

Correlation between arterial blood volume obtained by arterial spin labelling and cerebral blood volume in intracranial tumours

Objective  

To compare measurements of the arterial blood volume (aBV), a perfusion parameter calculated from arterial spin labelling (ASL), and cerebral blood volume (CBV), calculated from dynamic susceptibility contrast (DSC) MRI. In the clinic, CBV is used for grading of intracranial tumours.     

Identification of 1,3-diiminoisoindoline carbohydrazides as potential antimalarial candidates

A series of inhibitors of plant enzymes of the non-mevalonate pathway from herbicide research efforts at BASF were screened for antimalarial activity in a cell-based assay. A 1,3-diiminoisoindoline carbohydrazide was found to inhibit the growth of Plasmodium falciparum with an IC(50) value <100 nM. Synthesis of a variety of derivatives allowed an improvement of the initial antimalarial activity down to IC(50) =18 nM for the most potent compound, the establishment of a structure-activity relationship, and the evaluation of the cytotoxic profile of the diiminoisoindolines. Furthermore, interesting configurational and conformational aspects for this class of compounds were studied by computational and X-ray crystal structure analysis. Some of the compounds can act as tridentate ligands, forming 2:1 ligand-iron(III) complexes, which also display antimalarial activity in the nanomolar IC(50) range, paired with low cytotoxicity.     

Proline Fingerprint in Intrinsically Disordered Proteins

NMR spectroscopy is one of the main techniques used for high‐resolution studies of intrinsically disordered proteins (IDPs), permitting mapping of the structural and dynamic features of all the amino acids constituting the polypeptide at atomic resolution. Only proline residues are less straightforward to characterize because they lack any amide proton, thus rendering them not directly visible in the commonly used 2D ¹H,¹⁵N correlation experiments. However, proline residues are highly abundant in IDPs and can mediate important functions. In this work we present an easy and effective way to obtain fingerprints of proline residues in IDPs at high resolution.     

Co-gasification of coal and wood in a dual fluidized bed gasifier

In the last decade the reduction of CO₂ emissions from fossil fuels became a worldwide topic. Co-gasification of coal and wood provides an opportunity to combine the advantages of the well-researched usage of fossil fuels such as coal with CO₂-neutral biomass. Gasification itself is a technology with many advantages. The producer gas can be used in many ways; for electric power generation in a gas engine or gas turbine, for Fischer-Tropsch synthesis of liquid fuels and also for production of gaseous products such as synthetic natural gas (bio SNG). Moreover, the use of the producer gas in fuel cells is under investigation. The mixture of coal and wood leads to the opportunity to choose the gas composition as best befits the desired process. Within this study the focus of investigation was of gasification of coal and wood in various ratios and the resulting changes in producer gas composition. Co-gasification of coal and wood leads to linear producer gas composition changes with linear changing load ratios (coal/wood). Hydrogen concentrations rise with increasing coal ratio, while CO concentrations decrease. Due to the lower sulfur and nitrogen content of wood, levels of the impurities NH₃ and H₂S in the producer gas fall with decreasing coal ratio. It is also shown that the majority of sulfur is released in the gasification zone and, therefore, no further cleaning of the flue gas is necessary. All mixture ratios, from 100 energy% to 0 energy% coal, performed well in the 100 kW dual fluidized bed gasifier. Although the gasifier was originally designed for wood, an addition of coal as fuel in industrial sized plants based on the same technology should pose no problems.     

Linking Ageing to Development Agendas in Sub-Saharan Africa: Challenges and Approaches

International calls and frameworks for policies on ageing in sub-Saharan African countries, encapsulated in the UN Madrid Plan of Action on Ageing (2002) and the African Union Policy Framework and Plan of Action on Ageing (2003), have resulted in little concrete policy action. The lack of progress calls for critical reflection on the status of policy debates and arguments on ageing in the sub-region. In a context of acute development challenges and resource constraints, the paper links the impasse in policy action to a fundamental lack of clarity about how rationales and approaches for policy on ageing relate to core national development agendas. It then explicates four steps required to elucidate these connections, namely: (a) A full appreciation of key aspects of mainstream development agendas; (b) identification of ambiguities in calls for policy on ageing; (c) pinpointing of key perspectives, arguments and queries for redressing the ambiguities; and (d) addressing ensuing information needs. We argue that advocacy and research on ageing in sub-Saharan Africa need to consider the framework proposed in the paper urgently, in order to advance policy and debate on ageing in the region.     

Plasma Lipid Profiling Contributes to Untangle the Complexity of Moyamoya Arteriopathy

Moyamoya arteriopathy (MA) is a rare cerebrovascular disorder characterized by ischemic/hemorrhagic strokes. The pathophysiology is unknown. A deregulation of vasculogenic/angiogenic/inflammatory pathways has been hypothesized as a possible pathophysiological mechanism. Since lipids are implicated in modulating neo-vascularization/angiogenesis and inflammation, their deregulation is potentially involved in MA. Our aim is to evaluate angiogenic/vasculogenic/inflammatory proteins and lipid profile in plasma of MA patients and control subjects (healthy donors HD or subjects with atherosclerotic cerebrovascular disease ACVD). Angiogenic and inflammatory protein levels were measured by ELISA and a complete lipidomic analysis was performed on plasma by mass spectrometry. ELISA showed a significant decrease for MMP-9 released in plasma of MA. The untargeted lipidomic analysis showed a cumulative depletion of lipid asset in plasma of MA as compared to HD. Specifically, a decrease in membrane complex glycosphingolipids peripherally circulating in MA plasma with respect to HD was observed, likely suggestive of cerebral cellular recruitment. The quantitative targeted approach demonstrated an increase in free sphingoid bases, likely associated with a deregulated angiogenesis. Our findings indicate that lipid signature could play a central role in MA and that a detailed biomarker profile may contribute to untangle the complex, and still obscure, pathogenesis of MA.     

Active beating modes of two clamped filaments driven by molecular motors

Biological cilia pump the surrounding fluid by asymmetric beating that is driven by dynein motors between sliding microtubule doublets. The complexity of biological cilia raises the question about minimal systems that can re-create similar patterns of motion. One such system consists of a pair of microtubules that are clamped at the proximal end. They interact through dynein motors that cover one of the filaments and pull against the other one. Here, we study theoretically the static shapes and the active dynamics of such a system. Using the theory of elastica, we analyse the shapes of two filaments of different lengths with clamped ends. Starting from equal lengths, we observe a transition similar to Euler buckling leading to a planar shape. When further increasing the length ratio, the system assumes a non-planar shape with spontaneously broken chiral symmetry after a secondary bifurcation and then transitions to planar again. The predicted curves agree with experimentally observed shapes of microtubule pairs. The dynamical system can have a stable fixed point, with either bent or straight filaments, or limit cycle oscillations. The latter match many properties of ciliary motility, demonstrating that a two-filament system can serve as a minimal actively beating model.     

Influence of regioirregular structural units on the crystallization of isotactic polypropylene

Seven samples of isotactic polypropylene were examined to study the influence on the formation of the γ crystalline phase of possible regiodefects along the chain. Wide‐angle X‐ray diffraction allowed the determination of the percentage of the γ phase in the samples and ¹³C‐NMR spectroscopy was used to correlate the development of the γ phase with the existence of regioirregular structural units along the chain. Furthermore, it was possible to appraise the contributions given by the different families of lamellae to the small‐angle X‐ray diffraction patterns. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 79: 375–384, 2001