Dynamical diffraction effects on the geometric phase of inhomogeneous strain fields.

In specimens with an inhomogeneous displacement field in electron beam direction dynamical diffraction effects lead to complex non-linear properties of the diffracted electron wave. Consequently, the diffracted beam's phase contains information about the inhomogeneous displacement field. These phases are experimentally and theoretically investigated under different excitation errors and specimen thicknesses as well as for different depths of the displacement field. An inclined InGaAs layer with a larger lattice constant than the surrounding GaAs matrix serves as controlled displacement field, which is inhomogeneous in electron beam direction with a continuously changing depth. The phase and amplitude of the diffracted beam are measured by dark-field electron holography. The measurements agree with calculations performed by numerical propagation of the electron wave using the Darwin-Howie-Whelan equations. A strong dependency on the excitation conditions is found showing that the interplay between dynamical effects and the strain field must be considered in the interpretation of the geometric phase.

Gated interference for time-resolved electron holography.

Pump-probe measurements of periodic processes require a temporal gating for the time-dependent signal. For this purpose we propose to take advantage of the sensitivity of interferometric techniques like electron holography to instabilities. We have realized such a gating by synchronized disturbances of the measurement setup and successfully conducted measurements of electric fields with microsecond time resolution.

Electron holography on HfO2/HfO2-x bilayer structures with multilevel resistive switching properties.

Unveiling the physical nature of the oxygen-deficient conductive filaments (CFs) that are responsible for the resistive switching of the HfO2-based resistive random access memory (RRAM) devices represents a challenging task due to the oxygen vacancy related defect nature and nanometer size of the CFs. As a first important step to this goal, we demonstrate in this work direct visualization and a study of physico-chemical properties of oxygen-deficient amorphous HfO2-x by carrying out transmission electron microscopy electron holography as well as energy dispersive x-ray spectroscopy on HfO2/HfO2-x bilayer heterostructures, which are realized by reactive molecular beam epitaxy. Furthermore, compared to single layer devices, Pt/HfO2/HfO2-x /TiN bilayer devices show enhanced resistive switching characteristics with multilevel behavior, indicating their potential as electronic synapses in future neuromorphic computing applications.

Averaging scheme for atomic resolution off-axis electron holograms.

All micrographs are limited by shot-noise, which is intrinsic to the detection process of electrons. For beam insensitive specimen this limitation can in principle easily be circumvented by prolonged exposure times. However, in the high-resolution regime several instrumental instabilities limit the applicable exposure time. Particularly in the case of off-axis holography the holograms are highly sensitive to the position and voltage of the electron-optical biprism. We present a novel reconstruction algorithm to average series of off-axis holograms while compensating for specimen drift, biprism drift, drift of biprism voltage, and drift of defocus, which all might cause problematic changes from exposure to exposure. We show an application of the algorithm utilizing also the possibilities of double biprism holography, which results in a high quality exit-wave reconstruction with 75 pm resolution at a very high signal-to-noise ratio.

Creating arrays of electron vortices.

We demonstrate the production of an ordered array of electron vortices making use of an electron optical setup consisting of two electrostatic biprisms. The biprism filaments are oriented nearly orthogonal with respect to each other in a transmission electron microscope. Matching the position of the filaments, we can choose to form different topological features in the electron wave. We outline the working principle of the setup and demonstrate first experimental results. This setup partially bridges the gap between angular momentum carried by electron spin, which is intrinsic and therefore present in any position of the wave, and angular momentum carried by the vortex character of the wave, which can be extrinsic depending on the axis around which it is measured.

Local estimation of lattice constants in HRTEM images.

A model-based approach to estimate lattice constants from an atomically resolved HRTEM image is presented. The approach only utilizes the inherent periodicity of these images and does not require a centrosymmetric structure of the specimen. This allows the evaluation of, for instance, wurtzite-based materials like InGaN/GaN heterostructures. The lattice constants are determined within precisions below 3 pm from areas only a few unit cells large. This makes this method suitable for further strain/compositional analysis. Furthermore, the impact of the approximations of the true detector's covariance matrices on the assessment of the model-based approach is investigated, and insights into the quality of these noise models of the detector are gained.

The glycosyl phosphatidylinositol anchor of human T-cadherin binds lipoproteins.

T-cadherin (T-cad) is a Ca(2+)-dependent cell adhesion glycoprotein bound to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. T-cad expressed on vascular smooth muscle cells (SMC) binds lipoproteins on blot. To analyze the molecular basis for the interaction of T-cad with lipoproteins we expressed recombinant human T-cad in HEK293 cells. Whereas membrane-bound T-cad from SMC and T-cad transfected HEK293 cells bind lipoproteins, T-cadherin proteins cleaved from the cell surface by phosphatidylinositol-specific phospholipase C (PI-PLC) do not. The lipoprotein-binding function is also lacking both for a recombinant human T-cad expressed in HEK293 cells without the GPI signal sequence, and for a human T-cad form expressed in Escherichia coli that contains the signal sequence for GPI attachment but is not modified with a GPI. We conclude that the GPI moiety of T-cadherin is necessary and sufficient to mediate lipoprotein binding.

LDL binds to surface-expressed human T-cadherin in transfected HEK293 cells and influences homophilic adhesive interactions.

T-cadherin (T-cad) is an unusual glycosylphosphatidylinositol-anchored member of the cadherin family of cell adhesion molecules. Binding of low density lipoproteins (LDLs) to T-cad can be demonstrated on Western blots of smooth muscle cell lysates, membranes and purified proteins. Using HEK293 cells transfected with human T-cad cDNA (T-cad+), we have investigated the adhesion properties of expressed mature and precursor proteins and examined the postulate that LDL represents a physiologically relevant ligand for T-cad. T-cad+ exhibits an increased Ca(2+)-dependent aggregation (vs. control) that was reduced by selective proteolytic cleavage of precursor T-cad and abolished after either proteolytic or phosphatidylinositol-specific phospholipase C (PI-PLC) cleavage of both mature and precursor proteins, indicating that both proteins function in intercellular adhesion. T-cad+ exhibited a significantly increased specific cell surface-binding of [(125)I]-LDL that was sensitive to PI-PLC pre-treatment of cells. Ca(2+)-dependent intercellular adhesion of T-cad+ was significantly inhibited by LDL. Our results support the suggestion that LDL is a physiologically relevant ligand for T-cad.

[The public health office and its partners: perspectives on cooperation between public health offices and self-help organizations]. / Das Gesundheitsamt und seine Partner: Perspektiven der Zusammenarbeit zwischen Gesundheitsämtern und Selbsthilfeorganisationen.

Besides the three existing health sector entities in Germany: hospitals, established doctors and public health departments, a fourth area has developed, consisting of self-help organisations with their self-help groups. Today self-help organisations have a very extensive knowledge about special diseases and handicaps. Combined with the psychological and social assistance which they afford, their knowledge helps the affected persons considerably. Nevertheless, this practical experience cannot replace the professionals' know-how. Within the local "Gemeinschaftsaufgabe Gesundheitsförderung" (common task of health promotion) the co-operation between laymen and professionals may be particularly supported by the Public Health Offices.

The predicted secondary structure of the G-type glutamine amidotransferase is compatible with TIM-barrel topology.

Glutamine amidotransferase (GAT) subunits or domains catalyze an important partial reaction in many complex biosynthetic reactions. The structure of one member of the F-type GATs is known, but the structure of the unrelated G-type is still unknown. Because many protein sequences are available for anthranilate synthase component II (product of the trpG gene), we have predicted its average secondary structure by a joint prediction method [Niermann and Kirschner (1991a) Protein Engng, 4, 359-370]. The predicted eight beta-strands and seven alpha-helices follow an 8-fold cyclic repetition of a beta-strand-loop-alpha-helix-loop module with helix alpha 7 missing. This pattern of secondary structure suggests that the G-type GAT domain has an 8-fold beta alpha-barrel topology, as found first in triose phosphate isomerase (TIM-barrel). This model is supported by the location of known catalytically essential residues in loops between beta-strands and alpha-helices. Evidence from published sequencing and mutational studies on selected members of the GAT superfamily (carbamoyl phosphate, imidazoleglycerol phosphate, GMP and CTP synthases) support both the secondary structure prediction and the TIM-barrel topology.

Three-dimensional structure of the bifunctional enzyme phosphoribosylanthranilate isomerase: indoleglycerolphosphate synthase from Escherichia coli refined at 2.0 A resolution.

The three-dimensional structure of the monomeric bifunctional enzyme N-(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from Escherichia coli has been refined at 2.0 A resolution, using oscillation film data obtained from synchrotron radiation. The model includes the complete protein (452 residues), two phosphate ions and 628 water molecules. The final R-factor is 17.3% for all observed data between 15 and 2 A resolution. The root-mean-square deviations from ideal bond lengths and bond angles are 0.010 A and 3.2 degrees, respectively. The structure of N-(5'-phosphoribosyl)anthranilate isomerase: indole-3-glycerol-phosphate synthase from E. coli comprises two beta/alpha-barrel domains that superimpose with a root-mean-square deviation of 2.03 A for 138 C alpha-pairs. The C-terminal domain (residues 256 to 452) catalyses the PRAI reaction and the N-terminal domain (residues 1 to 255) catalyses the IGPS reaction, two sequential steps in tryptophan biosynthesis. The enzyme has the overall shape of a dumb-bell, resulting in a surface area that is considerably larger than normally observed for monomeric proteins of this size. The active sites of the PRAI and the IGPS domains, both located at the C-terminal side of the central beta-barrel, contain equivalent binding sites for the phosphate moieties of the substrates N-(5'-phosphoribosyl) anthranilate and 1-(o-carboxyphenylamino)-1-deoxyribulose-5-phosphate. These two phosphate binding sites are identical with respect to their positions within the tertiary structure of the beta/alpha-barrel, the conformation of the residues involved in phosphate binding and the hydrogen-bonding network between the phosphate ions and the protein. The active site cavities of both domains contain similar hydrophobic pockets that presumably bind the anthranilic acid moieties of the substrates. These similarities of the tertiary structures and the active sites of the two domains provide evidence that N-(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from E. coli results from a gene duplication event of a monomeric beta/alpha-barrel ancestor.

Striking structural and functional similarities suggest that intestinal sucrase-isomaltase, human lysosomal alpha-glucosidase and Schwanniomyces occidentalis glucoamylase are derived from a common ancestral gene.

Sequence comparison of the primary structure of the yeast Schwanniomyces occidentalis glucoamylase (GAM) with GAMs in different microorganisms did not reveal significant similarities. By contrast, striking similarities were, surprisingly, found with 3 mammalian secretory and integral membrane proteins: the 2 subunits of intestinal brush border sucrase-isomaltase and human lysosomal alpha-glucosidase. The similarities among these proteins are found as clusters of up to 8 amino acids and distributed all over the protein sequences. The major sequence differences are found in the N-terminal regions accounting, probably, for the different cellular locations of these proteins. The high level of similarities between sucrase, isomaltase, Sch. occidentalis GAM and human lysosomal alpha-glucosidase suggest that these proteins are derived from the same ancestral gene. To our knowledge, this is the first report that describes similarities between a yeast secretory protein and mammalian secretory and integral membrane proteins.

Improving the prediction of secondary structure of 'TIM-barrel' enzymes.

The information contained in aligned sets of homologous protein sequences should improve the score of secondary structure prediction. Seven different enzymes having the (beta/alpha)8 or TIM-barrel fold were used to optimize the prediction with regard to this class of enzymes. The alpha-helix, beta-strand and loop propensities of the Garnier-Osguthorpe-Robson method were averaged at aligned residue positions, leading to a significant improvement over the average score obtained from single sequences. The increased accuracy correlates with the average sequence variability of the aligned set. Further improvements were obtained by using the following averaged properties as weights for the averaged state propensities: amphipathic moment and alpha-helix; hydropathy and beta-strand; chain flexibility and loop. The clustering of conserved residues at the C-terminal ends of the beta-strands was used as an additional positive weight for beta-strand propensity and increased the prediction of otherwise unpredicted beta-strands decisively. The automatic weighted prediction method identifies greater than 95% of the secondary structure elements of the set of seven TIM-barrel enzymes.

Properties and primary structure of the L-malate dehydrogenase from the extremely thermophilic archaebacterium Methanothermus fervidus.

L-Malate dehydrogenase from the extremely thermophilic mathanogen Methanothermus fervidus was isolated and its phenotypic properties were characterized. The primary structure of the protein was deducted from the coding gene. The enzyme is a homomeric dimer with a molecular mass of 70 kDa, possesses low specificity for NAD+ or NADP+ and catalyzes preferentially the reduction of oxalacetate. The temperature dependence of the activity as depicted in the Arrhenius and van't Hoff plots shows discontinuities near 52 degrees C, as was found for glyceraldehyde-3-phosphate dehydrogenase from the same organism. With respect to the primary structure, the archaebacterial L-malate dehydrogenase deviates strikingly from the eubacterial and eukaryotic enzymes. The sequence similarity is even lower than that between the L-malate dehydrogenases and L-lactate dehydrogenases of eubacteria and eukaryotes. The phylogenetic meaning of this relationship is discussed.

Analysis of the alpha-amylase gene of Schwanniomyces occidentalis and the secretion of its gene product in transformants of different yeast genera.

We have cloned and characterized the alpha-amylase gene (AMY1) of the yeast Schwanniomyces occidentalis. A cosmid gene library of S. occidentalis DNA was screened in Saccharomyces cerevisiae for alpha-amylase secretion. The positive clone contained a DNA fragment harbouring an open reading frame of 1536 nucleotides coding for a 512-amino-acid polypeptide with a calculated Mr of 56,500. The deduced amino acid sequence reveals significant similarity to the sequence of the Saccharomycopsis fibuligera and Aspergillus oryzae alpha-amylases. The AMY l gene was found to be expressed from its original promoter in S. cerevisiae, Kluyveromyces lactis and Schizo-saccharomyces pombe leading to an active secreted gene product and thus enabling the different yeast transformants to grow on starch as a sole carbon source.

Nucleotide sequence of the glyceraldehyde-3-phosphate dehydrogenase gene from the mesophilic methanogenic archaebacteria Methanobacterium bryantii and Methanobacterium formicicum. Comparison with the respective gene structure of the closely related extreme thermophile Methanothermus fervidus.

The genes for glyceraldehyde-3-phosphate dehydrogenase (gap genes) from the mesophilic methanogenic archaebacteria Methanobacterium formicicum and Methanobacterium bryantii were cloned and sequenced. The deduced amino acid sequences show 95% identity to each other and about 70% identity to the glyceraldehyde-3-phosphate dehydrogenase from the thermophilic methanogenic archaebacterium Methanothermus fervidus. Although the sequence similarity between the archaebacterial glyceraldehyde-3-phosphate dehydrogenase and the homologous enzyme of eubacteria and eukaryotes is low, an equivalent secondary-structural arrangement can be deduced from the profiles of the physical parameters hydropathy, chain flexibility and amphipathy. In order to find possible thermophile-specific structural features of the enzyme from M. fervidus, a comparative primary-sequence analysis was performed. Amino acid exchanges leading, to a stabilization of the main-chain conformation, could be found throughout the sequence of the thermophile enzyme. Striking features of the thermophile sequence are the preference for isoleucine, especially in beta-sheets, and a low arginine/lysine ratio of 0.54.

Prediction of secondary structure by evolutionary comparison: application to the alpha subunit of tryptophan synthase.

The amino acid sequences of the a subunits of tryptophan synthase from ten different microorganisms were aligned by standard procedures. The alpha helices, beta strands and turns of each sequence were predicted separately by two standard prediction algorithms and averaged at homologous sequence positions. Additional evidence for conserved secondary structure was derived from profiles of average hydropathy and chain flexibility values, leading to a joint prediction. There is good agreement between (1) predicted beta strands, maximal hydropathy and minimal flexibility, and (2) predicted loops, great chain flexibility, and protein segments that accept insertions of various lengths in individual sequences. The a subunit is predicted to have eight repeated beta-loop-alpha-loop motifs with an extra N-terminal alpha helix and an intercalated segment of highly conserved residues. This pattern suggests that the territory structure of the a subunit is an eightfold alpha/beta barrel. The distribution of conserved amino acid residues and published data on limited proteolysis, chemical modification, and mutagenesis are consistent with the alpha/beta barrel structure. Both the active site of the a subunit and the combining site for the beta 2 subunit are at the end of the barrel formed by the carboxyl-termini of the beta strands.