Ångstrom-Depth Resolution with Chemical Specificity at the Liquid-Vapor Interface.

The determination of depth profiles across interfaces is of primary importance in many scientific and technological areas. Photoemission spectroscopy is in principle well suited for this purpose, yet a quantitative implementation for investigations of liquid-vapor interfaces is hindered by the lack of understanding of electron-scattering processes in liquids. Previous studies have shown, however, that core-level photoelectron angular distributions (PADs) are altered by depth-dependent elastic electron scattering and can, thus, reveal information on the depth distribution of species across the interface. Here, we explore this concept further and show that the experimental anisotropy parameter characterizing the PAD scales linearly with the average distance of atoms along the surface normal obtained by molecular dynamics simulations. This behavior can be accounted for in the low-collision-number regime. We also show that results for different atomic species can be compared on the same length scale. We demonstrate that atoms separated by about 1 Å along the surface normal can be clearly distinguished with this method, achieving excellent depth resolution.

Adaptive hybrid optimal quantum control for imprecisely characterized systems.

Optimal quantum control theory carries a huge promise for quantum technology. Its experimental application, however, is often hindered by imprecise knowledge of the input variables, the quantum system's parameters. We show how to overcome this by adaptive hybrid optimal control, using a protocol named Ad-HOC. This protocol combines open- and closed-loop optimal control by first performing a gradient search towards a near-optimal control pulse and then an experimental fidelity estimation with a gradient-free method. For typical settings in solid-state quantum information processing, adaptive hybrid optimal control enhances gate fidelities by an order of magnitude, making optimal control theory applicable and useful.

Multimode circuit quantum electrodynamics with hybrid metamaterial transmission lines.

Quantum transmission lines are central to superconducting and hybrid quantum computing. In this work we show how coupling them to a left-handed transmission line allows circuit QED to reach a new regime: multimode ultrastrong coupling. Out of the many potential applications of this novel device, we discuss the preparation of multipartite entangled states and the simulation of the spin-boson model where a quantum phase transition is reached up to finite size effects.

Characterization and optimization of phospholipase A2 catalyzed synthesis of phosphatidylcholine.

The phospholipase A2 (PLA2) catalyzed synthesis and hydrolysis of phosphatidylcholine (PC) was studied in a water activity controlled organic medium. The aim of the study was to find the conditions most favorable for the synthetic reaction. To do this, the impact of various parameters such as water activity, substrate concentration and temperature on enzyme activity and equilibrium yield was determined. The PC to lysophosphatidylcholine (LPC) ratio at equilibrium increases with decreasing water activity and increasing fatty acid concentration, as can be expected from the law of mass action of an esterification reaction. The enzyme activity on the other hand decreases under conditions that favor the esterification. The best yield in the synthetic reaction is 60% at a water activity of 0.11 and an oleic acid concentration of 1.8 M. That is to our knowledge the highest yield ever reported in this reaction. Both the hydrolysis and synthesis reaction follow Michaelis-Menten kinetics, the apparent Km values are the same for PC and LPC, namely 4.9 mM. Vmax is 82.5 and 10.4 nmol h(-1) mg(-1) for the hydrolysis and synthesis reaction, respectively. Studies on PLA2 at water activity controlled conditions resulted in a more complete understanding of the enzymatic reaction and allowed to find the conditions most favorable for the synthetic reaction.

Characterisation and mitochondrial localisation of AUH, an AU-specific RNA-binding enoyl-CoA hydratase.

AU-rich elements function as instability elements which direct rapid mRNA degradation. AUH protein exhibits an AU-specific RNA-binding property and an intrinsic enoyl-CoA hydratase activity and may therefore function to link mRNA decay to metabolic processes (. Proc. Natl. Acad. Sci. USA 92, 2051-2055). The sequence encoding the murine protein, muAUH, was established by cloning, and the corresponding polypeptide predicted to have a molecular mass of 37kDa. As shown for the human protein, muAUH is expressed in a 32kDa form and there is 94% homology between the two species. Recombinant muAUH was shown to be an RNA-binding enoyl-CoA hydratase. All murine cells studied contained a single AUH transcript of approx. 1.7kb and an investigation of tissue-specific expression revealed highest levels in kidney, skeletal muscle, heart, liver and spleen. It was further determined, using immunoelectron microscopy, that AUH is located in the mitochondria of mouse cells.

Electron microscopic immunocytochemistry. Silver enhancement of colloidal gold marker allows double labeling with the same primary antibody.

Electron microscopic sections, immunocytochemically labeled with colloidal gold, can be prepared for double labeling by applying the "EM-silver enhancement" procedure. This method, a photographic, so-called physical, development, increases the size of the gold marker to a predeterminable value and thereby inactivates the anti-species antibody present on the gold grain, thus allowing the labeling of a second antigen with antibody raised in the same species.

Light and electron microscopic in situ hybridization: non-radioactive labeling and detection, double hybridization, and combined hybridization-immunocytochemistry.

We performed light and electron microscopic in situ hybridization, according to the same protocol and without pretreatment of sections, on Lowicryl- and LR Gold-embedded cells. Digoxigenin (DIG)- or biotin-labeled riboprobes were visualized by direct or indirect immunodetection using commercially available gold-antibody conjugates with 0.8-10-nm gold grains. At the ultrastructural level, the main findings were that DIG-labeled probes gave a slightly higher labeling intensity (grains per signal) than biotin. The direct detection method produced a more compact signal, which led to better resolution at medium and high magnifications. Labeling intensities of all gold grain sizes were essentially equal. Grain sizes of 5 nm and larger were highly preferable because available enhancement methods are unsatisfactory for ultrasmall grains. The optimized immunodetection protocols are suitable for double hybridization with two different probes and for combined hybridization and immunocytochemistry.

Induction of leukotriene production by bleomycin and asparaginase in mast cells in vitro and in patients in vivo.

Bleomycin and asparaginase are widely used antineoplastic agents which may induce allergic or inflammatory side-effects. Mast cells are implicated as effector cells in allergic and inflammatory responses. The aim of this study was to establish whether bleomycin or asparaginase modulate leukotriene production in vitro and in vivo. Leukotriene C4 (LTC4) production by murine bone marrow-derived mast cells (BMMC) was determined by radioimmunoassay (RIA). Leukotriene production in patients was assessed by determining leukotriene E4 and N-acetyl-leukotriene E4 in urine by means of combined HPLC and RIA. Bleomycin induced an up to 2.1-fold increase in LTC4 production both in unstimulated and in calcium ionophore-stimulated mast cells. In 3 of 7 patients treated with bleomycin, a greater than 2-fold increase in endogenous leukotriene production was observed. This effect was associated with febrile responses and was most pronounced in a patient who developed an Adult Respiratory Distress Syndrome (ARDS). Asparaginase increased leukotriene production up to 10-fold in stimulated but not in unstimulated BMMC. In a patient who developed an anaphylactic reaction after treatment with asparaginase, a pronounced increase in urinary leukotriene concentration was observed. In contrast to bleomycin or asparaginase, a number of other cytostatic agents did not significantly change leukotriene production by BMMC. Our data indicate that some of the inflammatory and allergic side-effects of bleomycin and asparaginase could be mediated by leukotrienes, a possible source of which may be mast cells.

Characteristics of the poliovirus replication complex.

In the infected cell, the poliovirus replication complex (RC) is found in the center of a rosette formed by many virus-induced vesicles. The RC is attached to the vesicular membranes and contains a compact central part which encloses the replication forks of the replicative intermediate and all proteins necessary for strand elongation. The growing plus strands of the replicative intermediate protrude from the central part of the RC, but are still enclosed by membraneous structures of the rosette. After completion, progeny 36S RNA is set free at the surface of the rosette. In an in vitro transcription system, isolated replication complex-containing rosettes are active in initiation, elongation and maturation (release) of plus strand progeny RNA. Full functionality of the RC depends on an intact structural framework of all membraneous components of the rosette.

In situ hybridization for light and electron microscopy: a comparison of methods for the localization of viral RNA using biotinylated DNA and RNA probes.

We report procedures for in situ hybridization at the light and electron microscopic level for localization of viral RNA in poliovirus-infected, Lowicryl-embedded cells. We compare specificity and signal intensity of biotinylated, double-stranded DNA and single-stranded, strand-specific RNA probes, both corresponding to the same region of the poliovirus genome. The hybrids were detected with antibiotin antibodies or streptavidin with colloidal gold as a marker. Hybridization with the RNA probe was more sensitive and gave lower background than with DNA. Detection with immunogold proved to be by far more sensitive than with streptavidin. The hybridization and detection protocols for the DNA and the RNA probes could be applied without modification to light microscopic semi-thick sections as well as to electron microscopic ultrathin sections.

Protein (western) blotting.

The different steps involved in protein (Western) blotting and subsequent analysis of the proteins are reviewed. Electrophoretic separation of proteins, procedures of transfer to membranes, immunological and nonimmunological protein detection systems, and characterization of protein-nucleic acid and protein-protein interactions are described. Emphasis is on the sensitivity of the methods described and on possible variations that allow the individual steps of Western blotting to be adapted to specific questions.

Electron microscopic silver enhancement for double labeling with antibodies raised in the same species.

In immunoelectron microscopy (IEM), simultaneous labeling of two or more antigens on the same section is desirable for many applications. If the antibodies (Ab) to be used are raised in the same species, as is usually the case with monoclonal antibodies (MAb), the difficulty arises that the labeled secondary, antispecies Ab used in the first labeling step traps the primary Ab directed against the second antigen, thus leading to a nonspecific signal for the second antigen.

Colloidal gold staining and immunoprobing on the same Western blot.

Proteins, blotted from polyacrylamide gels onto nitrocellulose sheets (Western Blots) can be stained nonspecifically with a variety of dyes, or they can be identified individually by probing with appropriate antibodies. These procedures may be performed on duplicate blots, staining the total protein pattern on one blot and using the second blot for the immune reaction (1,2). This chapter describes how to combine both methods on one blot, i.e., staining the blot first for total protein, followed by an indirect immune reaction (3).

Electron microscopic silver enhancement for double labeling with antibodies raised in the same species.

In immunoelectron microscopy (IEM), simultaneous labeling of two or more antigens on the same section is desirable for many applications. If the antibodies (Ab) to be used are raised in the same species, as is usually the case with monoclonal antibodies (MAb), the difficulty arises that the labeled secondary, antispecies Ab used in the first labeling step traps the primary Ab directed against the second antigen, thus leading to a nonspecific signal for the second antigen.

Colloidal gold staining and immunoprobing on the same Western blot.

Proteins, blotted from polyacrylamide gels onto nitrocellulose sheets (Western Blots) can be stained nonspecifically with a variety of dyes, or they can be identified individually by probing with appropriate antibodies. These procedures may be performed on duplicate blots, staining the total protein pattern on one blot and using the second blot for the immune reaction (1,2). This chapter describes how to combine both methods on one blot, i.e., staining the blot first for total protein, followed by an indirect immune reaction (3).