A crystallographic approach to structural transitions in icosahedral viruses

Viruses with icosahedral capsids, which form the largest class of all viruses and contain a number of important human pathogens, can be modelled via suitable icosahedrally invariant finite subsets of icosahedral 3D quasicrystals. We combine concepts from the theory of 3D quasicrystals, and from the theory of structural phase transformations in crystalline solids, to give a framework for the study of the structural transitions occurring in icosahedral viral capsids during maturation or infection. As 3D quasicrystals are in a one-to-one correspondence with suitable subsets of 6D icosahedral Bravais lattices, we study systematically the 6D-analogs of the classical Bain deformations in 3D, characterized by minimal symmetry loss at intermediate configurations, and use this information to infer putative viral-capsid transition paths in 3D via the cut-and-project method used for the construction of quasicrystals. We apply our approach to the Cowpea Chlorotic Mottle virus (CCMV) and show that the putative transition path between the experimentally observed initial and final CCMV structures is most likely to preserve one threefold axis. Our procedure suggests a general method for the investigation and prediction of symmetry constraints on the capsids of icosahedral viruses during structural transitions, and thus provides insights into the mechanisms underlying structural transitions of these pathogens.     

Acetobacter pasteurianus Strain AB0220: Cultivability and Phenotypic Stability Over 9?Years of Preservation

Acetobacter species are members of the α-subclass of Proteobacteria, which harbors a large number of bacteria recalcitrant to cultivation. Strain AB0220 was isolated from a superficial acetification system and preserved for 9 years by short and long time methods. Under short time preservation it was estimated that 540.54 number of generations occurred, whereas in long time preservation conditions the number of generations was 17.40. Ethanol oxidation to acetic acid was stable and confirmed, as well as acetate assimilation during long time preservation. Cultivability checks showed persistence of phenotypic traits (growth on ethanol and methanol, growth on different carbon sources and cellulose production) over the extended preservation time. 16S rRNA gene sequences analysis showed 100 % of similarity with A. pasteurianus (Accession number GQ240636). Stability of subcultures related to the culture age and subcultures frequency, tested by ERIC/PCR, confirmed the suitability of long term preservation at least over a period of 9 years.     

Neural Crest and Olfactory System: New Prospective

Sensory neurons in vertebrates are derived from two embryonic transient cell sources: neural crest (NC) and ectodermal placodes. The placodes are thickenings of ectodermal tissue that are responsible for the formation of cranial ganglia as well as complex sensory organs that include the lens, inner ear, and olfactory epithelium. The NC cells have been indicated to arise at the edges of the neural plate/dorsal neural tube, from both the neural plate and the epidermis in response to reciprocal interactions Moury and Jacobson (Dev Biol 141:243?C253, 1990). NC cells migrate throughout the organism and give rise to a multitude of cell types that include melanocytes, cartilage and connective tissue of the head, components of the cranial nerves, the dorsal root ganglia, and Schwann cells. The embryonic definition of these two transient populations and their relative contribution to the formation of sensory organs has been investigated and debated for several decades (Basch and Bronner-Fraser, Adv Exp Med Biol 589:24?C31, 2006; Basch et al., Nature 441:218?C222, 2006) review (Baker and Bronner-Fraser, Dev Biol 232:1?C61, 2001). Historically, all placodes have been described as exclusively derived from non-neural ectodermal progenitors. Recent genetic fate-mapping studies suggested a NC contribution to the olfactory placodes (OP) as well as the otic (auditory) placodes in rodents (Murdoch and Roskams, J Neurosci Off J Soc Neurosci 28:4271?C4282, 2008; Murdoch et al., J Neurosci 30:9523?C9532, 2010; Forni et al., J Neurosci Off J Soc Neurosci 31:6915?C6927, 2011b; Freyer et al., Development 138:5403?C5414, 2011; Katoh et al., Mol Brain 4:34, 2011). This review analyzes and discusses some recent developmental studies on the OP, placodal derivatives, and olfactory system.     

Nitric Oxide in Myogenesis and Therapeutic Muscle Repair

Nitric oxide is a short-lived intracellular and intercellular messenger. The first realisation that nitric oxide is important in physiology occurred in 1987 when its identity with the endothelium-derived relaxing factor was discovered. Subsequent studies have shown that nitric oxide possesses a number of physiological functions that are essential not only to vascular homeostasis but also to neurotransmission, such as in the processes of learning and memory and endocrine gland regulation, as well as inflammation and immune responses. The discovery in 1995 that a splice variant of the neuronal nitric oxide synthase is localised at the sarcolemma via the dystrophin?Cglycoprotein complex and of its displacement in Duchenne muscular dystrophy has stimulated a host of studies exploring the role of nitric oxide in skeletal muscle physiology. Recently, nitric oxide has emerged as a relevant messenger also of myogenesis that it regulates at several key steps, especially when the process is stimulated for muscle repair following acute and chronic muscle injuries. Here, we will review briefly the mechanisms and functions of nitric oxide in skeletal muscle and discuss its role in myogenesis, with specific attention to the promising nitric oxide-based approaches now being explored at the pre-clinical and clinical level for the therapy of muscular dystrophy.     

A novel role for phospholipase D as an endogenous negative regulator of platelet sensitivity

Platelet aggregation, secretion and thrombus formation play a critical role in primary hemostasis to prevent excessive blood loss. On the other hand, uncontrolled platelet activation leads to pathological thrombus formation resulting in myocardial infarction or stroke. Stimulation of heterotrimeric G-proteins by soluble agonists or immunoreceptor tyrosine based activation motif-coupled receptors that interact with immobilized ligands such as the collagen receptor glycoprotein (GP) VI lead to the activation of phospholipases that cleave membrane phospholipids to generate soluble second messengers. Platelets contain the phospholipases (PL) D1 and D2 which catalyze the hydrolysis of phosphatidylcholine to generate the second messenger phosphatidic acid (PA). The production of PA is abrogated by primary alcohols that have been widely used for the analysis of PLD-mediated processes. However, it is not clear if primary alcohols effectively reduce PA generation or if they induce PLD-independent cellular effects. In the present study we made use of the specific PLD inhibitor 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) and show for the first time, that FIPI enhances platelet dense granule secretion and aggregation of human platelets. Further, FIPI has no effect on cytosolic Ca(2+) activity but needs proper Rho kinase signaling to mediate FIPI-induced effects on platelet activation. Upon FIPI treatment the phosphorylation of the PKC substrate pleckstrin was prominently enhanced suggesting that FIPI affects PKC-mediated secretion and aggregation in platelets. Similar effects of FIPI were observed in platelets from mouse wild-type and Pld1(-/-) mice pointing to a new role for PLD2 as a negative regulator of platelet sensitivity.     

Discovery and Structure Activity Relationship of Small Molecule Inhibitors of Toxic β-Amyloid-42 Fibril Formation

Increasing evidence implicates Aβ peptides self-assembly and fibril formation as crucial events in the pathogenesis of Alzheimer disease. Thus, inhibiting Aβ aggregation, among others, has emerged as a potential therapeutic intervention for this disorder. Herein, we employed 3-aminopyrazole as a key fragment in our design of non-dye compounds capable of interacting with Aβ42 via a donor-acceptor-donor hydrogen bond pattern complementary to that of the β-sheet conformation of Aβ42. The initial design of the compounds was based on connecting two 3-aminopyrazole moieties via a linker to identify suitable scaffold molecules. Additional aryl substitutions on the two 3-aminopyrazole moieties were also explored to enhance π-π stacking/hydrophobic interactions with amino acids of Aβ42. The efficacy of these compounds on inhibiting Aβ fibril formation and toxicity in vitro was assessed using a combination of biophysical techniques and viability assays. Using structure activity relationship data from the in vitro assays, we identified compounds capable of preventing pathological self-assembly of Aβ42 leading to decreased cell toxicity.     

Cytosolic NADP-isocitrate dehydrogenase in Arabidopsis leaves and roots

NADP-dependent isocitrate dehydrogenase (NADP-ICDH) catalyses the production of NADPH, which is an essential component in the cellular homeostasis. In Arabidopsis, the kinetic parameters (K _m and V _max) of cytosolic NADP-ICDH were different in leaves and roots. In vitro applied H₂O₂ did not affect the NADP-ICDH activity in either organ, however, the reduced glutathione inhibited the activity in leaves but not in roots. On the other hand, S-nitrosoglutathione (a NO donor) and peroxynitrite depressed NADP-ICDH activity in leaves and roots.     

Heterologous production and characterisation of two distinct dihaem-containing membrane integral cytochrome b(561) enzymes from Arabidopsis thaliana in Pichia pastoris and Escherichia coli cells

Cytochrome (cyt) b(561) proteins are dihaem-containing membrane proteins, belonging to the CYBASC (cytochrome-b(561)-ascorbate-reducible) family, and are proposed to be involved in ascorbate recycling and/or the facilitation of iron absorption. Here, we present the heterologous production of two cyt b(561) paralogs from Arabidopsis thaliana (Acytb(561)-A, Acytb(561)-B) in Escherichia coli and Pichia pastoris, their purification, and initial characterisation. Spectra indicated that Acytb(561)-A resembles the best characterised member of the CYBASC family, the cytochrome b(561) from adrenomedullary chromaffin vesicles, and that Acytb(561)-B is atypical compared to other CYBASC proteins. Haem oxidation-reduction midpoint potential (E(M)) values were found to be fully consistent with ascorbate oxidation activities and Fe(3+)-chelates reductase activities. The ascorbate dependent reduction and protein stability of both paralogs were found to be sensitive to alkaline pH values as reported for the cytochrome b(561) from chromaffin vesicles. For both paralogs, ascorbate-dependent reduction was inhibited and the low-potential haem E(M) values were affected significantly by incubation with diethyl pyrocarbonate (DEPC) in the absence of ascorbate. Modification with DEPC in the presence of ascorbate left the haem E(M) values unaltered compared to the unmodified proteins. However, ascorbate reduction was inhibited. We concluded that the ascorbate-binding site is located near the low-potential haem with the Fe(3+)-chelates reduction-site close to the high-potential haem. Furthermore, inhibition of ascorbate oxidation by DEPC treatment occurs not only by lowering the haem E(M) values but also by an additional modification affecting ascorbate binding and/or electron transfer. Analytical gel filtration experiments suggest that both cyt b(561) paralogs exist as homodimers.     

A novel dendrimeric peptide with antimicrobial properties: structure-function analysis of SB056

The novel antimicrobial peptide with a dimeric dendrimer scaffold, SB056, was empirically optimized by high-throughput screening. This procedure produced an intriguing primary sequence whose structure-function analysis is described here. The alternating pattern of hydrophilic and hydrophobic amino acids suggests the possibility that SB056 is a membrane-active peptide that forms amphiphilic β-strands in a lipid environment. Circular dichroism confirmed that the cationic SB056 folds as β-sheets in the presence of anionic vesicles. Lipid monolayer surface pressure experiments revealed unusual kinetics of monolayer penetration, which suggest lipid-induced aggregation as a membranolytic mechanism. NMR analyses of the linear monomer and the dendrimeric SB056 in water and in 30% trifluoroethanol, on the other hand, yielded essentially unstructured conformations, supporting the excellent solubility and storage properties of this compound. However, simulated annealing showed that many residues lie in the β-region of the Ramachandran plot, and molecular-dynamics simulations confirmed the propensity of this peptide to fold as a β-type conformation. The excellent solubility in water and the lipid-induced oligomerization characteristics of this peptide thus shed light on its mechanism of antimicrobial action, which may also be relevant for systems that can form toxic β-amyloid fibrils when in contact with cellular membranes. Functionally, SB056 showed high activity against Gram-negative bacteria and some limited activity against Gram-positive bacteria. Its potency against Gram-negative strains was comparable (on a molar basis) to that of colistin and polymyxin B, with an even broader spectrum of activity than numerous other reference compounds.