M. Sulc

Scanning force microscopy of domain structures in Pb(Zn1/3Nb2/3)O3-8% PbTiO3 and Pb(Mg1/3Nb2/3)O3-29% PbTiO3

Domain structures of unpoled as well as poled (along [001]- and [110]-direction) Pb(Zn1/3Nb2/3)O3 (PZN)-8% PbTiO3 (PT) and Pb(Mg1/3Nb2/3)O3 (PMN)-29% PT single crystals have been investigated by scanning force microscopy (SFM) in the piezoresponse mode, at room temperature. Antiparallel domain structures have been detected mostly in unpoled crystals of both materials, with a fingerprint pattern in (001)-oriented PZN-8% PT crystal. The ferroelastic domain wall has been identified in poled (110)-oriented PZN-8% PT crystal. “Writing” of ferroelectric domains has been performed by applying a dc voltage to the SFM tip. Local re-poling has been observed for all unpoled as well as for poled (001)-oriented crystals at the voltage ±60 V. Local electrical switching was successful in poled (110)-oriented PMN-29% PT at higher voltage (±120 V) but was not successful in poled (110)-oriented PZN-8% PT crystal. Domain-engineered crystals poled in [110]-direction seem to exhibit more stable (in the sense of local re-polin...

Results from the OSQAR photon-regeneration experiment : No light shining through a wall

A new method to amplify the photon-axion conversions in a magnetic field is proposed using a buffer gas at a specific pressure in a photon-regeneration experiment. As a first result, new bounds for mass and coupling constant for laboratory experiments aiming to detect any hypothetical scalars and pseudoscalars, which can couple to photons were obtained, excluding with 95% confidence level, the recently withdrawn PVLAS result.

Laser Doppler vibrometry for evaluating the piezoelectric coefficient d33 on thin film

The problem of electromechanical film characterization, and, in particular, the determination of the piezoelectric activities of thin films deposited on substrates, is of fundamental importance in the development of structures for microelectromechanical system (MEMS) applications. The design and the architecture of the piezoelectric MEMS are directly related to the mechanical and the piezoelectric performances of the material. In this article, we present and compare some results obtained on different experimental setup for the determination of the d33 coefficient. We have optimized the experimental conditions using a laser Doppler vibrometer. The main problem is the contribution of the bending effect of the substrates on the d33 coefficient, which is an intrinsic property of the film. We show that the d33 values are directly related to parameters such as the top electrode diameter and the substrate holder. The results are in agreement with those obtained with the conventional double beam interferometer us...

Both the N-terminal Loop and Wing W2 of the Forkhead Domain of Transcription Factor Foxo4 Are Important for DNA Binding

FoxO4 belongs to the “O” subset of forkhead transcription factors, which participate in various cellular processes. The forkhead DNA binding domain (DBD) consists of three-helix bundle resting on a small antiparallel β-sheet from which two extended loops protrude and create two wing-like structures. The wing W2 of FoxO factors contains a 14-3-3 protein-binding motif that is phosphorylated by protein kinase B in response to insulin or growth factors. In this report, we investigated the role of the N-terminal loop (portion located upstream of first helix H1) and the C-terminal region (loop known as wing W2) of the forkhead domain of transcription factor FoxO4 in DNA binding. Although the deletion of either portion partly reduces the FoxO4-DBD binding to the DNA, the simultaneous deletion of both regions inhibits DNA binding significantly. Förster resonance energy transfer measurements and molecular dynamics simulations suggest that both studied N- and C-terminal regions of FoxO4-DBD directly interact with DNA. In the presence of the N-terminal loop the protein kinase B-induced phosphorylation of wing W2 by itself has negligible effect on DNA binding. On the other hand, in the absence of this loop the phosphorylation of wing W2 significantly inhibits the FoxO4-DBD binding to the DNA. The binding of the 14-3-3 protein efficiently reduces DNA-binding potential of phosphorylated FoxO4-DBD regardless of the presence of the N-terminal loop. Our results show that both N- and C-terminal regions of forkhead domain are important for stability of the FoxO4-DBD·DNA complex.

Development of THGEM-based photon detectors for Cherenkov Imaging Counters

The development of a large size gaseous detector of single photons, able to stably operate at high gain and high rate, and to provide good time resolution and insensitivity to magnetic field would be beneficial to future Cherenkov Imaging Counters. The detector could be based on the use of a multilayer architecture of THGEM electron multipliers coupled to a solid state CsI photocathode. A systematic study of the response of THGEM-based counters versus the geometrical parameters has been performed and the main results will be presented. Small photon detector prototypes have been built and preliminary data obtained detecting single photoelectrons are presented as well. The key aspect of photoelectron extraction from the photocathode is illustrated presenting both simulation and dedicated measurement results.

The 14-3-3 Protein Affects the Conformation of the Regulatory Domain of Human Tyrosine Hydroxylase†

Tyrosine hydroxylase (TH) catalyzes the first step in the biosynthesis of catecholamines. Regulation of TH enzyme activity is controlled through the posttranslational modification of its regulatory domain. The regulatory domain of TH can be phosphorylated at four serines (8, 19, 31, and 40) by a variety of protein kinases. Phosphorylation of Ser19 does not by itself increase TH activity but induces its binding to the 14-3-3 protein. That leads to the enhancement of TH activity with a still not fully understood mechanism. The main goal of this work was to investigate whether the 14-3-3 protein binding affects the conformation of the regulatory domain of human TH isoform 1 (TH1R). Site-directed mutagenesis was used to generate five single-tryptophan mutants of TH1R with the Trp residue located at five different positions within the domain (positions 14, 34, 73, 103, and 131). Time-resolved tryptophan fluorescence measurements revealed that phosphorylation of Ser19 and Ser40 does not itself induce any significant structural changes in regions surrounding inserted tryptophans. On the other hand, the interaction between the 14-3-3 protein and phosphorylated TH1R decreases the solvent exposure of tryptophan residues at positions 14 and 34 and induces distinct structural change in the vicinity of Trp73. The 14-3-3 protein binding also reduces the sensitivity of phosphorylated TH1R to proteolysis by protecting its N-terminal part (first 33 residues). Circular dichroism measurements showed that TH1R is an unstructured protein with a low content of secondary structure and that neither phosphorylation nor the 14-3-3 protein binding changes its secondary structure.

14-3-3 Protein Masks the DNA Binding Interface of Forkhead Transcription Factor FOXO4

The role of 14-3-3 proteins in the regulation of FOXO forkhead transcription factors is at least 2-fold. First, the 14-3-3 binding inhibits the interaction between the FOXO and the target DNA. Second, the 14-3-3 proteins prevent nuclear reimport of FOXO factors by masking their nuclear localization signal. The exact mechanisms of these processes are still unclear, mainly due to the lack of structural data. In this work, we used fluorescence spectroscopy to investigate the mechanism of the 14-3-3 protein-dependent inhibition of FOXO4 DNA-binding properties. Time-resolved fluorescence measurements revealed that the 14-3-3 binding affects fluorescence properties of 5-(((acetylamino)ethyl)amino) naphthalene-1-sulfonic acid moiety attached at four sites within the forkhead domain of FOXO4 that represent important parts of the DNA binding interface. Observed changes in 5-(((acetylamino)ethyl)amino) naphthalene-1-sulfonic acid fluorescence strongly suggest physical contacts between the 14-3-3 protein and labeled parts of the FOXO4 DNA binding interface. The 14-3-3 protein binding, however, does not cause any dramatic conformational change of FOXO4 as documented by the results of tryptophan fluorescence experiments. To build a realistic model of the FOXO4·14-3-3 complex, we measured six distances between 14-3-3 and FOXO4 using Förster resonance energy transfer time-resolved fluorescence experiments. The model of the complex suggests that the forkhead domain of FOXO4 is docked within the central channel of the 14-3-3 protein dimer, consistent with our hypothesis that 14-3-3 masks the DNA binding interface of FOXO4.

Alkaloid cluster gene ccsA of the ergot fungus Claviceps purpurea encodes chanoclavine I synthase, a flavin adenine dinucleotide-containing oxidoreductase mediating the transformation of N-methyl-dimethylallyltryptophan to chanoclavine I.

ABSTRACT Ergot alkaloids are indole-derived secondary metabolites synthesized by the phytopathogenic ascomycete Claviceps purpurea. In wild-type strains, they are exclusively produced in the sclerotium, a hibernation structure; for biotechnological applications, submerse production strains have been generated by mutagenesis. It was shown previously that the enzymes specific for alkaloid biosynthesis are encoded by a gene cluster of 68.5 kb. This ergot alkaloid cluster consists of 14 genes coregulated and expressed under alkaloid-producing conditions. Although the role of some of the cluster genes in alkaloid biosynthesis could be confirmed by a targeted knockout approach, further functional analyses are needed, especially concerning the early pathway-specific steps up to the production of clavine alkaloids. Therefore, the gene ccsA, originally named easE and preliminarily annotated as coding for a flavin adenine dinucleotide-containing oxidoreductase, was deleted in the C. purpurea strain P1, which is able to synthesize ergot alkaloids in axenic culture. Five independent knockout mutants were analyzed with regard to alkaloid-producing capability. Thin-layer chromatography (TLC), ultrapressure liquid chromatography (UPLC), and mass spectrometry (MS) analyses revealed accumulation of N-methyl-dimethylallyltryptophan (Me-DMAT) and traces of dimethylallyltryptophan (DMAT), the first pathway-specific intermediate. Since other alkaloid intermediates could not be detected, we conclude that deletion of ccsA led to a block in alkaloid biosynthesis beyond Me-DMAT formation. Complementation with a ccsA/gfp fusion construct restored alkaloid biosynthesis. These data indicate that ccsA encodes the chanoclavine I synthase or a component thereof catalyzing the conversion of N-methyl-dimethylallyltryptophan to chanoclavine I.

Single-step affinity purification of recombinant proteins using a self-excising module from Neisseria meningitidis FrpC

Purification of recombinant proteins is often a challenging process involving several chromatographic steps that must be optimized for each target protein. Here, we developed a self‐excising module allowing single‐step affinity chromatography purification of untagged recombinant proteins. It consists of a 250‐residue‐long self‐processing module of the Neisseria meningitidis FrpC protein with a C‐terminal affinity tag. The N terminus of the module is fused to the C terminus of a target protein of interest. Upon binding of the fusion protein to an affinity matrix from cell lysate and washing out contaminating proteins, site‐specific cleavage of the Asp–Pro bond linking the target protein to the self‐excising module is induced by calcium ions. This results in the release of the target protein with only a single aspartic acid residue added at the C terminus, while the self‐excising affinity module remains trapped on the affinity matrix. The system was successfully tested with several target proteins, including glutathione‐S‐transferase, maltose‐binding protein, β‐galactosidase, chloramphenicol acetyltransferase, and adenylate cyclase, and two different affinity tags, chitin‐binding domain or poly‐His. Moreover, it was demonstrated that it can be applied as an alternative to two currently existing systems, based on the self‐splicing intein of Saccharomyces cerevisiae and sortase A of Staphylococcus aureus.

Calmodulin and S100A1 Protein Interact with N Terminus of TRPM3 Channel

Background: Calcium-binding proteins bind to intracellular termini of TRP channels. Results: Two Ca2+-dependent binding sites for CaM/S100A1 were revealed by biophysical methods on TRPM3 N terminus. Conclusion: CaM and S100A1 serve as ligands for TRPM3 channel. Significance: Two clusters of positively charged residues form mutual binding sites for CaM and S100A1 proteins on TRPM3 N terminus. Transient receptor potential melastatin 3 ion channel (TRPM3) belongs to the TRP family of cation-permeable ion channels involved in many important biological functions such as pain transduction, thermosensation, and mechanoregulation. The channel was reported to play an important role in Ca2+ homeostasis, but its gating mechanisms, functions, and regulation are still under research. Utilizing biophysical and biochemical methods, we characterized two independent domains, Ala-35–Lys-124 and His-291–Gly-382, on the TRPM3 N terminus, responsible for interactions with the Ca2+-binding proteins calmodulin (CaM) and S100A1. We identified several positively charged residues within these domains as having a crucial impact on CaM/S100A1 binding. The data also suggest that the interaction is calcium-dependent. We also performed competition assays, which suggested that CaM and S100A1 are able to compete for the same binding sites within the TRPM3 N terminus. This is the first time that such an interaction has been shown for TRP family members.