Structural characterization and oligomerization of PB1-F2, a proapoptotic influenza A virus protein

Recently, a novel 87-amino acid influenza A virus protein with proapoptotic properties, PB1-F2, has been reported that originates from an alternative reading frame in the PB1 polymerase gene and is encoded in most known human influenza A virus isolates. Here we characterize the molecular structure of a biologically active synthetic version of the protein (sPB1-F2). Western blot analysis, chemical cross-linking, and NMR spectroscopy afforded direct evidence of the inherent tendency of sPB1-F2 to undergo oligomerization mediated by two distinct domains located in the N and C termini, respectively. CD and (1)H NMR spectroscopic analyses indicate that the stability of structured regions in the molecule clearly depends upon the hydrophobicity of the solvent. In aqueous solutions, the behavior of sPB1-F2 is typical of a largely random coil peptide that, however, adopts alpha-helical structure upon the addition of membrane mimetics. (1)H NMR analysis of three overlapping peptides afforded, for the first time, direct experimental evidence of the presence of a C-terminal region with strong alpha-helical propensity comprising amino acid residues Ile(55)-Lys(85) connected via an essentially random coil structure to a much weaker helix-like region, located in the N terminus between residues Trp(9) and Lys(20). The C-terminal helix is not a true amphipathic helix and is more compact than previously predicted. It corresponds to a positively charged region previously shown to include the mitochondrial targeting sequence of PB1-F2. The consequences of the strong oligomerization and helical propensities of the molecule are discussed and used to formulate a hypothetical model of its interaction with the mitochondrial membrane.     

Increase in expression levels and resistance to sulfhydryl oxidation of peroxiredoxin isoforms in amyloid beta-resistant nerve cells

Peroxiredoxins (Prxs) are a ubiquitously expressed family of thiol peroxidases that reduce hydrogen peroxide, peroxynitrite, and hydroperoxides using a highly conserved cysteine. There is substantial evidence that oxidative stress elicited by amyloid beta (Abeta) accumulation is a causative factor in the pathogenesis of Alzheimer disease (AD). Here we show that Abeta-resistant PC12 cell lines exhibit increased expression of multiple Prx isoforms with reduced cysteine oxidation. Abeta-resistant PC12 cells also display higher levels of thioredoxin and thioredoxin reductase, two enzymes critical for maintaining Prx activity. PC12 cells and rat primary hippocampal neurons transfected with wild type Prx1 exhibit increased Abeta resistance, whereas mutant Prx1, lacking a catalytic cysteine, confers no protection. Using an antibody that specifically recognizes sulfinylated and sulfonylated Prxs, it is demonstrated that primary rat cortical nerve cells exposed to Abeta display a time-dependent increase in cysteine oxidation of the catalytic site of Prxs that can be blocked by the addition of the thiol-antioxidant N-acetylcysteine. In support of previous findings, expression of Prx1 is higher in post-mortem human AD cortex tissues than in age-matched controls. In addition, two-dimensional gel electrophoresis and mass spectrometry analysis revealed that Prx2 exists in a more oxidized state in AD brains than in control brains. These findings suggest that increased Prx expression and resistance to sulfhydryl oxidation in Abeta-resistant nerve cells is a compensatory response to the oxidative stress initiated by chronic pro-oxidant Abeta exposure.     

The Light Quanta Modulated Physiological Response of Brassica Juncea Seedlings Subjected to Ni(II) Stress

This work is a study of the inter‐relationship between parameters that principally affect the metal up‐take in the plant. The relationships between the concentration of metal in the growth medium, C_s, the concentration of metal absorbed by the plant, C_p, and the total biomass achieved, M, all of which are factors relevant to the efficiency of phytoremediation of the plant, have been investigated via the macro‐physiological response of Brassica juncea seedlings to Ni(II) stress. The factorial growth experiments treated the Ni(II) concentration in the agar gel and the diurnal light quanta (DLQ) as independently variable parameters. Observations included the evidence of light enhancement of Ni toxicity at the root as well as at the whole plant level, the shoot mass index as a possible indicator of shoot metal sequestration in B. juncea, the logarithmic variation of C_p with C_s and the power‐law dependence of M on C_p. The sum total of these observations indicates that for the metal accumulator B. juncea with regard to its capacity to accumulate Ni, the overall metabolic nature of the plant is important – neither rapid biomass increase nor a high metal concentration capability favor the removal of high metal mass from the medium, but rather the plant with the moderate photosynthetically driven biomass growth and moderate metal concentrations demonstrated the ability to remove the maximum mass of metal from the medium. The implications of these observations in the context of the perceived need in phytoremediation engineering to maximize C_p and M simultaneously in the same plant, are discussed.     

Sulfated hyaluronic acid and dexamethasone possess a synergistic potential in the differentiation of osteoblasts from human bone marrow stromal cells

The development of novel bioactive biomaterials is urgently needed to meet the needs of an aging population. Both sulfated hyaluronic acid and dexamethasone are candidates for the functionalization of bone grafts, as they have been shown to enhance the differentiation of osteoblasts from bone marrow stromal cells in vitro and in vivo. However, the underlying mechanisms are not fully understood. Furthermore, studies combining different approaches to assess synergistic potentials are rare. In this study, we aim to gain insights into the mode of action of both sulfated hyaluronic acid and dexamethasone by a comprehensive analysis of the cellular fraction, released matrix vesicles, and the extracellular matrix, combining classical biochemical assays with mass spectrometry–based proteomics, supported by novel bioinformatical computations. We found elevated differentiation levels for both treatments, which were further enhanced by a combination of sulfated hyaluronic acid and dexamethasone. Single treatments revealed specific effects on osteogenic differentiation. Dexamethasone activates signalling pathways involved in the differentiation of osteoblasts, for example, CXC-motif chemokine receptor type 4 and mitogen-activated protein kinases. The effects of sulfated hyaluronic acid were predominantly linked to an alteration in the composition of the extracellular matrix, affecting the synthesis, secretion, and/or activity of fibrillary (fibronectin and thrombospondin-2) and nonfibrillary (transglutaminase-2, periostin, and lysyloxidase) extracellular matrix components, including proteases and their inhibitors (matrix metalloproteinase-2, tissue inhibitor of metalloproteinase-3). The effects were treatment specific, and less additive or contrary effects were found. Thus, we anticipate that the synergistic action of the treatment-specific effects is the key driver in elevated osteogenesis.     

Crystal structures and enzymatic properties of three formyltransferases from archaea: environmental adaptation and evolutionary relationship

Formyltransferase catalyzes the reversible formation of formylmethanofuran from N(5)-formyltetrahydromethanopterin and methanofuran, a reaction involved in the C1 metabolism of methanogenic and sulfate-reducing archaea. The crystal structure of the homotetrameric enzyme from Methanopyrus kandleri (growth temperature optimum 98 degrees C) has recently been solved at 1.65 A resolution. We report here the crystal structures of the formyltransferase from Methanosarcina barkeri (growth temperature optimum 37 degrees C) and from Archaeoglobus fulgidus (growth temperature optimum 83 degrees C) at 1.9 A and 2.0 A resolution, respectively. Comparison of the structures of the three enzymes revealed very similar folds. The most striking difference found was the negative surface charge, which was -32 for the M. kandleri enzyme, only -8 for the M. barkeri enzyme, and -11 for the A. fulgidus enzyme. The hydrophobic surface fraction was 50% for the M. kandleri enzyme, 56% for the M. barkeri enzyme, and 57% for the A. fulgidus enzyme. These differences most likely reflect the adaptation of the enzyme to different cytoplasmic concentrations of potassium cyclic 2,3-diphosphoglycerate, which are very high in M. kandleri (>1 M) and relatively low in M. barkeri and A. fulgidus. Formyltransferase is in a monomer/dimer/tetramer equilibrium that is dependent on the salt concentration. Only the dimers and tetramers are active, and only the tetramers are thermostable. The enzyme from M. kandleri is a tetramer, which is active and thermostable only at high concentrations of potassium phosphate (>1 M) or potassium cyclic 2,3-diphosphoglycerate. Conversely, the enzyme from M. barkeri and A. fulgidus already showed these properties, activity and stability, at much lower concentrations of these strong salting-out salts.     

Fast high-throughput screening of temoporfin-loaded liposomal formulations prepared by ethanol injection method

A new strategy for fast, convenient high-throughput screening of liposomal formulations was developed, utilizing the automation of the so-called ethanol-injection method. This strategy was illustrated by the preparation and screening of the liposomal formulation library of a potent second-generation photosensitizer, temoporfin. Numerous liposomal formulations were efficiently prepared using a pipetting robot, followed by automated size characterization, using a dynamic light scattering plate reader. Incorporation efficiency of temoporfin and zeta potential were also detected in selected cases. To optimize the formulation, different parameters were investigated, including lipid types, lipid concentration in injected ethanol, ratio of ethanol to aqueous solution, ratio of drug to lipid, and the addition of functional phospholipid. Step-by-step small liposomes were prepared with high incorporation efficiency. At last, an optimized formulation was obtained for each lipid in the following condition: 36.4 mg·mL(-1) lipid, 13.1 mg·mL(-1) mPEG(2000)-DSPE, and 1:4 ethanol:buffer ratio. These liposomes were unilamellar spheres, with a diameter of approximately 50?nm, and were very stable for over 20 weeks. The results illustrate this approach to be promising for fast high-throughput screening of liposomal formulations.     

Loading of Arabidopsis centromeric histone CENH3 occurs mainly during G2 and requires the presence of the histone fold domain

The centromeric histone H3 (CENH3) substitutes histone H3 within the nucleosomes of active centromeres in all eukaryotes. CENH3 deposition at centromeres is needed to assemble the kinetochore, a complex of conserved proteins responsible for correct chromosome segregation during nuclear division. Histones of regular nucleosomes are loaded during replication in S phase, while CENH3 deposition deviates from this pattern in yeast, human, and Drosophila melanogaster cells. Little is known about when and how CENH3 targets centromeric loci. Therefore, we determined the location and quantity of recombinant enhanced yellow fluorescent protein (EYFP)-CENH3 in mitotic root and endopolyploid leaf nuclei of transgenic Arabidopsis thaliana cells. Our data indicate significant loading of A. thaliana CENH3 during G2 (before splitting into sister kinetochores) rather than during the S or M phase of the cell cycle. The histone fold domain of the C-terminal part of CENH3 is sufficient to target A. thaliana centromeres. A. thaliana EYFP-CENH3 can recognize and target three different centromeric repeats of Arabidopsis lyrata but not field bean (Vicia faba) centromeres.     

Protein kinase A-mediated phosphorylation of connexin36 in mouse retina results in decreased gap junctional communication between AII amacrine cells

Gap junctions in AII amacrine cells of mammalian retina participate in the coordination of the rod and cone signaling pathway involved in visual adaptation. Upon stimulation by light, released dopamine binds to D(1) receptors on AII amacrine cells leading to increased intracellular cAMP (cyclic adenosine monophosphate) levels. AII amacrine cells express the gap junctional protein connexin36 (Cx36). Phosphorylation of Cx36 has been hypothesized to regulate gap junctional activity of AII amacrine cells. However, until now in vivo phosphorylation of Cx36 has not been reported. Indeed, it had been concluded that Cx36 in bovine retina is not phosphorylated, but in vitro phosphorylation for Cx35, the bass ortholog of Cx36, had been shown. To clarify this experimental discrepancy, we examined protein kinase A (PKA)-induced phosphorylation of Cx36 in mouse retina as a possible mechanism to modulate the extent of gap junctional coupling. The cytoplasmic domains of Cx36 and the total Cx36 protein were phosphorylated in vitro by PKA. Mass spectroscopy revealed that all four possible PKA consensus motifs were phosphorylated; however, domains point mutated at the sites in question showed a prevalent usage of Ser-110 and Ser-293. Additionally, we demonstrated that Cx36 was phosphorylated in cultured mouse retina. Furthermore, activation of PKA increased the level of phosphorylation of Cx36. cAMP-stimulated, PKA-mediated phosphorylation of Cx36 protein was accompanied by a decrease of tracer coupling between AII amacrine cells. Our results link increased phosphorylation of Cx36 to down-regulation of permeability through gap junction channels mediating light adaptation in the retina.     

Use of gentian violet to differentiate in vitro and ex vitro- formed roots during acclimatization of grapevine

Corrigendum

Use of gentian violet to differentiate in vitro and ex vitro- formed roots during acclimatization of grapevine