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To handle friction forces occurring in industrial linear motors, this work pursues compensation by using a disturbance observer (DOB). First, the classical DOB is examined for stability and its behavior in combination with a PID controller in open and closed loops. It could be shown that the disturbance rejection can be optimized independent of the setpoint tracking transfer function due to the DOB. Furthermore, a switching mode DOB (SMDOB) is introduced, which adaptively adjusts the convergence time for observing the disturbance force to the slider trajectory. The positive effect of the SMDOB on the reduction of the control error is demonstrated by simulations as well as by experiments on a test bench. The evaluation of the control error of a sinusoidal trajectory shows that adding the SMDOB to the PID controller reduces the mean control error by a factor of 2.5.

Coatings on wooden substrates for furniture, flooring and interior are a main application for radiation curing lacquer; Increased adhesion is an important target for development of UV coatings on wood. [...]

Cellulose–water interactions are crucial to understand biological processes as well as to develop tailor made cellulose-based products. However, the main challenge to study these interactions is the diversity of natural cellulose fibers and alterations in their supramolecular structure. Here, we study the humidity response of different, well-defined, ultrathin cellulose films as a function of industrially relevant treatments using different techniques. As treatments, drying at elevated temperature, swelling, and swelling followed by drying at elevated temperatures were chosen. The cellulose films were prepared by spin coating a soluble cellulose derivative, trimethylsilyl cellulose, onto solid substrates followed by conversion to cellulose by HCl vapor. For the highest investigated humidity levels (97%), the layer thickness increased by ca. 40% corresponding to the incorporation of 3.6 molecules of water per anhydroglucose unit (AGU), independent of the cellulose source used. The aforementioned treatments affected this ratio significantly with drying being the most notable procedure (2.0 and 2.6 molecules per AGU). The alterations were investigated in real time with X-ray reflectivity and quartz crystal microbalance with dissipation, equipped with a humidity module to obtain information about changes in the thickness, roughness, and electron density of the films and qualitatively confirmed using grazing incidence small angle X-ray scattering measurements using synchrotron irradiation.

The use of information via communication within the control design is an important aspect for realizing dependable and efficient autonomous driving functions. In this contribution, we focus on the assisted start-up of a vehicle convoy at an intersection. It is illustrated how vehicle-to-vehicle (V2V) communication can be used in the control design in order to satisfy both demands – an increase of the intersection's throughput and a guaranteed collision avoidance. The key idea is to first design an overshoot-free closed loop with a general speed-dependent target distance. Subsequently, this function can be extended to meet safety requirements in the presence of delays and constraints. Input and speed constraints are included in the control design to address the passenger comfort and the compliance with road traffic regulations. The benefit of the networked controller is demonstrated by simulations.

Metropolitan area network (MAN) serves as a transitory technology between the current local area network (LAN) systems and the broadband integrated services digital network (B-ISDN). It uses fiber optics to link corporate locations, schools or government agencies, as compared with a LAN, which interconnects terminals within the same building or adjacent buildings. MAN is compatible with B-ISDN because of its linkage with asynchronous transfer mode networks. MAN's network architecture is of a dual-fiber communication bus that provides redundancy and self-healing capabilities.

Background: To clarify the optimum mesh-tack ratio MTR (mesh area in cm2divided by the number of fixation tacks) in laparoscopic ventral and incisional hernia repair, we compared IPOM Plus procedures with more intensive mesh fixation to those with standard mesh fixation. Methods: In a retrospective cohort study, 84 patients (mean hernia width 6.6?±?4.4 cm) intraoperatively received an intensive mesh fixation I-IPOM Plus with MTR?=?4:1 (e.g. ,150 cm2mesh fixed by 50 tacks) and 74 patients (mean hernia width 6.7?±?3.4 cm) received a standard mesh fixation S-IPOM Plus with MTR?>?4:1 (e.g., 150 cm2mesh fixed by 30 tacks) at a community hospital between 2014 and 2017. Outcomes in recurrence rates, immediate and chronic postoperative pain, as well as long-term functionality of the abdominal wall were then evaluated. Results: After a mean follow-up time of 34 months, a 2.3% recurrence rate in I-IPOM Plus patients and a 13.5% recurrence rate in S-IPOM Plus patients were recorded (p?=?0.018). The recurrence was associated with large hernia?>?10 cm (OR 3.7, 95% CI 1.3–5.4) and MTR?>?5 (OR 2.4, 95% CI 1.1–3.8) in the multivariate analysis. There was a positive correlation between immediate postoperative pain intensity measured on day 7 and number of fixation tacks placed (I-IPOM Plus: mean 4.5?±?2.5 VAS versus S-IPOM Plus: mean 2.7?±?2.0 VAS, p?=?0.001). However, there were no outcome differences in terms of length of immediate postoperative pain experience, sick leave duration, chronic pain rate and long-term abdominal wall functionality between these two groups. Conclusion: For ventral and incisional hernia patients with multiple recurrence risk factors, a mesh-tack ratio MTR?=?4:1 should be applied in laparoscopic IPOM Plus procedures.

Issues concerning descriptions of stockyards used in the cement industry for storing materials with abrasive properties are discussed. Particular attention is given to the storing of petcoke, of synthetic gypsum and to types of storage room designs.

The undrilled, offshore Bamaga Basin (pre-Jurassic) is overlain by two previously explored sedimentary basins: Carpentaria (Jurassic–Cretaceous) and Karumba (Tertiary). In 1984, Duyken-1, the only well in the Gulf of Carpentaria, 120km southwest of the Bamaga Basin, tested the Carpentaria and Karumba successions but not the deeper Bamaga Basin, which at the time was unrecognised. 2D regional seismic data acquired in 2012, and a second infill seismic survey in 2014, confirmed the presence of a sedimentary succession in the Bamaga, probably of early–middle/late Paleozoic age, and identified an intriguing, complex structural history and large potential petroleum traps. Basin modelling in the centre of the Bamaga Basin where the sedimentary section is deepest shows that the sediments are mature for petroleum generation with the hydrocarbon phase being either oil or gas, although gas is more likely, especially below 2000m. Analysis of the available data indicates that in the oil generation zone there are six targets with the potential to hold prospective resources (3U) of more than 250 million stock barrels (MMstb) of oil each, the largest of which could hold as much as 663 MMstb of oil. In the gas generation zone, there are nine targets with the potential to hold prospective resources (3U) of 1 trillion cubic feet (Tcf) or more gas each, the largest of which could hold as much as 2.5 Tcf gas. The water depth (60m) and closeness to shore (150km) make it operationally and commercially attractive.

AbstractCoarse-grained molecular dynamics (CGMD) simulation technique (MARTINI force field) is applied to monitor the aggregation of helical peptides representing the transmembrane sequence and its extension of bone marrow stromal cell antigen 2 (BST-2). One of the peptides is coupled with a protein transducing domain (PTD) of nine arginine residues (R9) at its N-terminal side as well as a peptide, pep11**, which has been shown to bind to human papilloma virus 16 (HPV16) E6 oncoprotein. A short hydrophobic stretch of the transmembrane domain (TMD) of BST-2 aggregates the fastest and inserts into a lipid membrane. An aggregate of R9-pep11** attaches to the membrane viasimultaneous contact of many arginine residues. Monomers from the aggregates of the shortest of the hydrophobic TMDs dissolve into the opposing leaflet when the aggregate spans the bilayer. A ‘flipping’ of the individual monomeric peptides is not observed.Communicated by Ramaswamy H. Sarma

Long-range, terrestrial quantum networks require high-brightness single-photon sources emitting in the telecom C-band for maximum transmission rates. For solid-state quantum emitters, the underlying pumping process, i.e., coherent or incoherent excitation schemes, impacts several photon properties such as photon indistinguishability, single-photon purity, and photon number coherence. These properties play a major role in quantum communication applications, the latter in particular for quantum cryptography. Here, we present a versatile telecom C-band single-photon source that is operated coherently and incoherently using two complementary pumping schemes. The source is based on a quantum dot coupled to a circular Bragg grating cavity, whereas coherent (incoherent) operation is performed via the novel SUPER scheme (phonon-assisted excitation). In this way, high end-to-end-efficiencies (ηend) of 5.36% (6.09%) are achieved simultaneously with a small multiphoton contribution g(2)(0) of 0.076 ± 0.001 [g(2)(0) of 0.069 ± 0.001] for coherent (incoherent) operation.

In Germany, the public is exposed to pro and counter arguments regarding different electricity generation technologies. To assess the attitudinal consequences of these arguments, we presented a balanced set of seven pro and seven counter arguments concerning one of six electricity-generating technologies (i.e., coal power stations, gas power stations, onshore wind power stations, offshore wind power stations, open space photovoltaics, or biomass power plants) to respondents with heterogeneous socio-demographic characteristics. We asked them to rate the strength of each argument and report their perceived familiarity with each argument. Based on the respondents’ answers, we examined the tendencies that underlie the process of evaluating arguments using different theoretical approaches. We found that persuasiveness ratings are driven by arguments’ compatibility with respondents’ initial attitudes, arguments’ quality (i.e., strong, moderate, or weak), and respondents’ perceived familiarity with the arguments. Furthermore, we determined the extent to which respondents’ initial attitudes toward an electricity-generating technology, measured immediately before evaluation of 14 conflicting arguments, changed after exposure to the arguments. Unlike former studies on attitude polarization, we examined conditional probabilities instead of the absolute level of global attitude change or the marginal probabilities of attitude change and persistence. This allowed for more nuanced (re)examination of the issue and showed, among other results, that attitude polarization is the exception rather than the rule.

Interaction of E5 of papillomavirus-16 based on its three transmembrane domains (TMDs) with a peptide mimicking the fourth TMD (TMD-A) of the 16 kDa c subunit of the human vacuolar H+-ATPase, ATP6V0C, and one of its mutant is investigated. Docking reveals binding of the peptide between the second and third TMD of E5. A series of hydrophobic residues are responsible for the contact. Estimated weak binding energies based on potential of mean force calculations reveal marginal differences of the estimated binding energies between wild type (WT) and mutant peptide. Also differences in estimated binding energies of dimers of the individual TMDs of E5 with the WT peptide are marginal. Correlation of rotational data derived from coarse-grained molecular dynamics simulations of the peptides and the protein as well as from the principal component analysis reveal that the binding of TMD-A with TMD3 is enthalpy driven and binding with TMD2 is guided by entropic conditions.

Epidermal growth factor (EGF) and homodimeric vascular endothelial growth factor (VEGF) bind to cell surface receptors. They are responsible for cell growth and angiogenesis, respectively. Docking of the individual proteins as monomeric units using ZDOCK 2.3.2 reveals a partial blocking of the receptor binding site of VEGF by EGF. The receptor binding site of EGF is not affected by VEGF. The calculated binding energy is found to be intermediate between the binding energies calculated for Alzheimer’s Aß42 and the barnase/barstar complex.

Nonstructural protein 4A (NS4A) of Dengue virus (DENV) is a membrane protein involved in rearrangements of the endoplasmic reticulum membrane that are required for formation of replication vesicles. NS4A is composed most likely of three membrane domains. The N- and C-terminal domains are supposed to traverse the lipid membrane whereas the central one is thought to reside on the membrane surface, thus forming a u-shaped protein. All three membrane domains are proposed to be helical by secondary structure prediction programs. After performing multi nanosecond molecular dynamics (MD) simulations at various temperatures (300, 310, and 315.15 K) with each of the individual domains, they are used in a docking approach to define putative association motifs of the transmembrane domains (TMDs). Two structures of the u-shaped protein are generated by separating two assembled TMDs linking them with the membrane-attached domain. Lipid undulation is monitored with the structures embedded in a fully hydrated lipid bilayer applying multiple 200 ns MD simulations at 310 K. An intact structure of the protein supports membrane undulation. The strong unwinding of the helices in the domain-linking section of one of the structures lowers its capability to induce membrane curvature. Unwinding of the link region is due to interactions of two tryptophan residues, Trp-96 and 104. These results provide first insights into the membrane-altering properties of DENV NS4A.

Abstract

A new multicolor fluorescence in situ hybridization (mFISH) probe set is presented, and its possible applications are highlighted in 25 clinical cases. The so-called heterochromatin-M-FISH (HCM-FISH) probe set enables a one-step characterization of the large heterochromatic regions within the human genome. HCM-FISH closes a gap in the now available mFISH probe sets, as those do not normally cover the acrocentric short arms; the large pericentric regions of chromosomes 1, 9, and 16; as well as the band Yq12. Still, these regions can be involved in different kinds of chromosomal rearrangements such as translocations, insertions, inversions, amplifications, and marker chromosome formations. Here, examples are given for all these kinds of chromosomal aberrations, detected as constitutional rearrangements in clinical cases. Application perspectives of the probe set in tumors as well as in evolutionary cytogenetic studies are given.

In this paper a novel direct method for mechanical testing of single fibers and fiber joints is presented. Additional to the measurement of joint strength it is also possible to investigate loading geometry, deformation during testing, bending stiffness of fibers as well as bonding energy. Furthermore biaxial loading of fiber-fiber joints is possible. Initial experiments have shown that mode II and III shear strength of fiber-fiber joints can be measured with this setup. Initial results for fibers and fiber joints of unbleached softwood kraft pulp are presented. The resulting values are 1.1724·10-9to 3.8798·10-11Nm² for bending stiffness, 3.9180·10-10to 2.8793·10-11kJ/bond for bonding energy, 6.54 mN for mode II and 1.057 mN for mode III mean shear strength. Mode II mean shear strength under biaxial load with a preload of 20 mN perpendicular to the loading direction of the fiber-fiber joint was found to be 2.52 mN. These values are compared to results of other measurement techniques and discussed.

This paper describes the origin of the idea, term and concept of sustainable development. Driving forces were fundamental crises, which were essentially crises of the energy system: the idea originated during the crisis of the medieval agrarian solar system; the term emerged when the agrarian energy system could not satisfy the growing energy demand of the emerging industrial age at the end of the 18th century; the concept originated at the end of the 20th century, when the fossil fuel energy system came close to its ecological limits and society was in search of a concept reconciling ecological, economic and social goals for present and future generations. Currently, we are witnessing a slow transition to a new, probably a post-fossil energy system. Therefore, managing the present and future in a sustainable way is a task that will accompany humanity on its way into the future.

The secretory protein chromogranin A (CHGA) plays a necessary role in formation of catecholamine storage vesicles and gives rise to a catecholamine release–inhibitory fragment. Because genetic variation in the proximal human CHGApromoter predicts autonomic function and blood pressure, we explored how a common genetic variant alters transcription of the gene.

Efficacy and safety of an intensified dosing (ID) regimen of enteric-coated mycophenolate sodium (EC-MPS), which achieves higher mycophenolic acid exposure early posttransplantation, were evaluated in comparison with a standard dosing (SD) regimen.

Non-nephrotoxic immunosuppressive strategies that allow reduction of calcineurin-inhibitor exposure without compromising safety or efficacy remain a goal in kidney transplantation. Immunosuppression based on the mammalian-target-of-rapamycin inhibitor everolimus was assessed as a strategy for elimination of calcineurin-inhibitor exposure and optimisation of renal-graft function while maintaining efficacy.

ABSTRACTBacterial type IV secretion systems are macromolecule transporters with essential functions for horizontal gene transfer and for symbiotic and pathogenic interactions with eukaryotic host cells. Helicobacter pylori, the causative agent of type B gastritis, peptic ulcers, gastric adenocarcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma, uses the Cag type IV secretion system to inject its effector protein CagA into gastric cells. This protein translocation results in altered host cell gene expression profiles and cytoskeletal rearrangements, and it has been linked to cancer development. Interactions of CagA with host cell proteins have been studied in great detail, but little is known about the molecular details of CagA recognition as a type IV secretion substrate or of the translocation process. Apart from components of the secretion apparatus, we previously identified several CagA translocation factors that are either required for or support CagA translocation. To identify protein-protein interactions between these translocation factors, we used a yeast two-hybrid approach comprising all cagpathogenicity island genes. Among several other interactions involving translocation factors, we found a strong interaction between the coupling protein homologue Cagβ (HP0524) and the Cag-specific translocation factor CagZ (HP0526). We show that CagZ has a stabilizing effect on Cagβ, and we demonstrate protein-protein interactions between the cytoplasmic part of Cagβ and CagA and between CagZ and Cagβ, using immunoprecipitation and pull-down assays. Together, our data suggest that these interactions represent a substrate-translocation factor complex at the bacterial cytoplasmic membrane.

Bacterial type IV secretion systems are macromolecule transporters with essential functions for horizontal gene transfer and for symbiotic and pathogenic interactions with eukaryotic host cells. Helicobacter pylori, the causative agent of type B gastritis, peptic ulcers, gastric adenocarcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma, uses the Cag type IV secretion system to inject its effector protein CagA into gastric cells. This protein translocation results in altered host cell gene expression profiles and cytoskeletal rearrangements, and it has been linked to cancer development. Interactions of CagA with host cell proteins have been studied in great detail, but little is known about the molecular details of CagA recognition as a type IV secretion substrate or of the translocation process. Apart from components of the secretion apparatus, we previously identified several CagA translocation factors that are either required for or support CagA translocation. To identify protein-protein interactions between these translocation factors, we used a yeast two-hybrid approach comprising all cag pathogenicity island genes. Among several other interactions involving translocation factors, we found a strong interaction between the coupling protein homologue Cagβ (HP0524) and the Cag-specific translocation factor CagZ (HP0526). We show that CagZ has a stabilizing effect on Cagβ, and we demonstrate protein-protein interactions between the cytoplasmic part of Cagβ and CagA and between CagZ and Cagβ, using immunoprecipitation and pull-down assays. Together, our data suggest that these interactions represent a substrate-translocation factor complex at the bacterial cytoplasmic membrane.

Copyright of Fuss & Sprunggelenk is the property of Elsevier GmbH, Urban & Fischer Verlag and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Fuss & Sprunggelenk is the property of Elsevier GmbH, Urban & Fischer Verlag and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

An HPTLC method has been established for analysis of α-solanine and α-chaconine in potatoes during industrial processing. Chromatography is performed on silica gel plates in a horizontal developing chamber. Post-chromatographic derivatization by dipping the plates into antimony(III) chloride solution and subsequent heating results in red chromatographic bands. The reaction products are detected and quantified densitometrically by reflectance scanning at 560 nm. The limit of quantification is approximately 30 ng per zone. Depending on the matrix of the sample, the detection limit for both compounds is between 5 and 20 ng per zone. The calibration plots for α-solanine and α-chaconine are linear in the range 30 to 700 ng. Polynomial regression with low relative standard deviation is possible from 30 to 1500 ng. Easy sample preparation, ruggedness, and high selectivity are outstanding advantages of this method. The method is applicable to samples at all stages of processing - raw, peeled, blanched, cooked, and dried potatoes, and byproducts such as peel, waste mash, and processing waters. After steam-peeling, the total glycoalkaloid (TGA) concentration in the analyzed cultivar Karlenawas reduced to approximately 25% of its original content in the whole potato, which can increase to more than 1000 mg TGA kg-1fresh weight. Because of further leaching during processing, the commercial potato flakes tested contained less than 6 mg TGA kg-1reconstituted mashed potatoes. Food with TGA concentrations of up to 100 mg kg-1is regarded as completely safe. The ratio of α-chaconine to α-solanine was between 4:1 to 2:1 for all the samples analyzed.

ZusammenfassungEs werden Konzepte zur Regelung eines HCCI-Brennverfahrens (Homogeneous Charge Compression Ignition) mit interner Abgasrückführung (Trapping) bei Otto-Motoren vorgestellt. Die Konzepte basieren auf Verbrennungsmerkmalen, die aus dem Brennraumdrucksignal gewonnen werden. Zur Beeinflussung der Verbrennung stehen die Stellgrößen des Luft- und Kraftstoffsystems zur Verfügung. Die Arbeiten erfolgen an Motoren mit variablem Ventiltrieb mit unterschiedlicher Ausprägung der Variabilität und einem System zur Kraftstoffdirekteinspritzung. Im ersten Schritt wird von einer vollvariablen Ventilsteuerung ausgegangen, die eine zylinderindividuelle Verstellung der Gaswechselventile ermöglicht. Getrieben vom Einsatz kostenoptimaler Ventilsysteme wird in einem zweiten Schritt eine Regelungsstrategie vorgestellt, die diese Vorrausetzung der Zylinderindividualität fallen lässt und auf eine Ventilstrategie setzt, die mit konventionellen Phasenstellern realisierbar ist.

Basiliximab, a monoclonal CD25 antibody has proofed effective in reducing acute rejection episodes in adults in various immunosuppressive regimens. The effect of basiliximab in the pediatric population is controversial.

For around 20 years, the sustainability concept and its implementation have been discussed by society at large (Kobiowu et al., 2005; Goldblatt, 2005; Marechal et al., 2005; Backstrand, 2006; Spangenberg, 2005) and, among others, by the green economics community (Lunn, 2006; Kennet and Heinemann, 2006b; Dobson and Bell, 2005a; Barry, 2005). Sustainability is regarded as a solution to society's present and future problems. Energy already plays an important role for sustainable development in the Brundtland Commission's report. We developed a new Indicator for Sustainable Development (ISD) in order to analyse the German energy system and a quantitative sustainable development pathway on the basis of scientific work by the German Federal Government. Our analysis shows that developments in the energy sector can be described with the help of measurable sustainability indicators and that the indicators identify the need for political action in the energy sector and thus provide a starting point for political measures.

Mycophenolic acid (MPA) pharmacokinetics using the mycophenolate mofetil (MMF) formulation are known to differ between patients receiving tacrolimus or cyclosporine, but only limited data exist concerning concomitant use of tacrolimus and enteric-coated mycophenolate sodium (EC-MPS).

Numerous bacterial pathogens use type III secretion systems (T3SSs) or T4SSs to inject or translocate virulence proteins into eukaryotic cells. Several different reporter systems have been developed to measure the translocation of these proteins. In this study, a peptide tag-based reporter system was developed and used to monitor the injection of T3S and T4S substrates. The glycogen synthase kinase (GSK) tag is a 13-residue phosphorylatable peptide tag derived from the human GSK-3{szligbeta} kinase. Translocation of a GSK-tagged protein into a eukaryotic cell results in host cell protein kinase-dependent phosphorylation of the tag, which can be detected with phosphospecific GSK-3{szligbeta} antibodies. A series of expression plasmids encoding Yop-GSK fusion proteins were constructed to evaluate the ability of the GSK tag to measure the injection of Yops by the Yersinia pestis T3SS. GSK-tagged YopE, YopH, LcrQ, YopK, YopN, and YopJ were efficiently phosphorylated when translocated into HeLa cells. Similarly, the injection of GSK-CagA by the Helicobacter pylori T4SS into different cell types was measured via phosphorylation of the GSK tag. The GSK tag provides a simple method to monitor the translocation of T3S and T4S substrates.

The p7 protein is a small ion-channel-forming membrane polypeptide encoded by the hepatitis C virus which consists of two transmembrane α-helices, TM1 and TM2, and can be blocked by long-alkyl-chain iminosugar derivatives. The length of TM1 and TM2 was estimated by employing different secondary structure prediction algorithms and is proposed to span from Ala-10 to Leu-32 for TM1 and from Trp-36 to Pro-58 for TM2. A configurational search protocol based on simulated annealing combined with short restrained molecular dynamics simulations is used in addition to protein−protein docking to investigate the packing of TM1/TM2. Full p7 oligomeric bundles were generated, and in the most plausible models serines and threonines are facing the hydrophilic pore. In these models, His-17 would be a pore-facing residue, suggesting that p7 may be sensitive to pH in respect to its function.

Viren manipulieren den Stoffwechsel einer infizierten Zelle zu ihren Gunsten. Dafür nutzen sie kleine Proteine, die Ionenkanäle bilden. Doch wie lagern sich diese Proteine zu funktionellen, membrandurchspannenden Einheiten zusammen? Leitfähigkeitsmessungen und Computersimulation sind zwei der vielen Methoden, die dazu beitragen, die Vorgänge zu verstehen und letztlich neue antivirale Wirkstoffe zu entwerfen. Viren manipulieren den Stoffwechsel einer infizierten Zelle zu ihren Gunsten. Dafür nutzen sie kleine Proteine, die Ionenkanäle bilden. Doch wie lagern sich diese Proteine zu funktionellen, membrandurchspannenden Einheiten zusammen? Leitfähigkeitsmessungen und Computersimulation sind zwei der vielen Methoden, die dazu beitragen, die Vorgänge zu verstehen und letztlich neue antivirale Wirkstoffe zu entwerfen.

DNA rearrangement permits bacteria to regulate gene content and expression. In Helicobacter pylori, cagY, which contains an extraordinary number of direct DNA repeats, encodes a surface-exposed subunit of a (type IV) bacterial secretory system. Examining potential DNA rearrangements involving the cagY repeats indicated that recombination events invariably yield in-frame open reading frames, producing alternatively expressed genes. In individual hosts, H. pylori cell populations include strains that produce CagY proteins that differ in size, due to the predicted in-frame deletions or duplications, and elicit minimal or no host antibody recognition. Using repetitive DNA, H. pylori rearrangements in a host-exposed subunit of a conserved bacterial secretion system may permit a novel form of antigenic evasion.

Helicobacter pylori causes one of the most common, chronic bacterial infections and is a primary cause of severe gastric disorders. To unravel the bacterial factors necessary for the process of gastric colonization and pathogenesis, signature tagged mutagenesis (STM) was adapted to H. pylori. The Mongolian gerbil (Meriones unguiculatus) was used as model system to screen a set of 960 STM mutants. This resulted in 47 H. pylori genes, assigned to 9 different functional categories, representing a set of biological functions absolutely essential for gastric colonization, as verified and quantified for many mutants by competition experiments. Identification of previously known colonization factors, such as the urease and motility functions validated this method, but also novel and several hypothetical genes were found. Interestingly, a secreted collagenase, encoded by hp0169, could be identified and functionally verified as a new essential virulence factor for H. pylori stomach colonization. Furthermore, comB4, encoding a putative ATPase being part of a DNA transformation-associated type IV transport system of H. pylori was found to be absolutely essential for colonization, but natural transformation competence was apparently not the essential function. Thus, this first systematic STM application identified a set of previously unknown H. pylori colonization factors and may help to potentiate the development of novel therapies against gastric Helicobacter infections.

Alois Alzheimer evaluated five cases of Alzheimer's disease in the early 20th century. We focused on the family of “Johann F.,” Alzheimer's second patient, who died in October 1910 at age 57 years, and whose brain pathology is typical of a subgroup of Alzheimer's disease, the so‐called “plaque‐only type.” It was perhaps Emil Kraepelin's personal knowledge of this patient and the histological data of the other four cases that influenced Kraepelin to coin the term Alzheimer's disease. The church archive in Passau has a genealogical database drawn from sacramental registers dating from approximately 1580 to 1900. The genealogical data of the “Johann F.” family, which comes from villages in Lower Bavaria, extends as far back as 1670. We found documentation starting around 1830 about cause of death in the church records, which shows a familial predisposition to dementia. Affected family members include the mother, maternal grandfather, maternal great‐aunt, maternal great‐grandfather as well as three of Johann F.'s eight siblings. The offspring (children and grandchildren) of these affected siblings also were affected by mental illness. We conclude that “Johann F.” represents the index case of a family with a predisposition to presenile dementia with variable age of onset (30s to 60s).

Multiple forms of GnRH within individual brains may have different functions. However, some vertebrates such as salmonids continue to reproduce even though they have lost or do not express 1 of the 3 forms of GnRH found in most other teleosts. We examined a basal salmonid, lake whitefish, to determine the mechanism by which a reduction in the number of GnRH forms occurs. We identified for the first time 3 distinct GnRHs in a salmonid. One form is novel and is designated whitefish GnRH. The primary structure is pGlu-His-Trp-Ser-Tyr-Gly-Met-Asn-Pro-Gly-NH2. HPLC and RIA were used for purification followed by Edman degradation for sequence determination. Mass spectroscopy was used to confirm the sequence and amidation of the peptide. The other 2 forms, salmon GnRH and chicken GnRH-II, are identical to the 2 forms found in salmon, which evolved later than whitefish. Synthetic whitefish GnRH is biologically active, as it increased mRNA expression of growth hormone and the α-subunit for LH and thyroid-stimulating hormone in dispersed fish pituitary cells. Our data support the hypothesis that the ancestral salmonid had a third GnRH form when the genome doubled (tetraploidization), but the third form was lost later in some salmonids due to chromosomal rearrangements. We suggest that the salmon GnRH form compensated for the loss of the third form.

Viral ion channels are short auxiliary membrane proteins with a length of ca. 100 amino acids. They are found in enveloped viruses from influenza A, influenza B and influenza C (Orthomyxoviridae), and the human immunodeficiency virus type 1 (HIV-1, Retroviridae). The channels are called M2 (influenza A), NB (influenza B), CM2 (influenza C) and Vpu (HIV-1). Recently, in Paramecium bursaria chlorella virus (PBCV-1, Phycodnaviridae), a K selective ion channel has been discovered. The viral channels form homo oligomers to allow an ion flux and represent miniaturised systems. Proton conductivity of M2 is established; NB, Vpu and the potassium channel from PBC-1 conduct ions; for CM2 ion conductivity is still under proof. This review summarises the current knowledge of these short viral membrane proteins. Their discovery is outlined and experimental evidence for their structure and function is discussed. Studies using computational methods are presented as well as investigations of drug-protein interactions.

ABSTRACTHelicobacter pyloriproduces a number of proteins associated with the outer membrane, including adhesins and the vacuolating cytotoxin. These proteins are supposed to integrate into the outer membrane by β-barrel structures, characteristic of the family of autotransporter proteins. By using the SOMPES (shuttle vector-based outer membrane protein expression) system for outer membrane protein production, we were able to functionally express inH. pylorithe cholera toxin B subunit genetically fused to the C-terminal VacA domain. We demonstrate that the fusion protein is translocated to the H. pyloriouter membrane and that the CtxB domain is exposed on the H. pylorisurface. Thus, we provide the first experimental evidence that the C-terminal β-domain of VacA can transport a foreign passenger protein to theH. pylorisurface and hence acts as a functional autotransporter.

A series of novel resorcin[4]arenes with extended π systems have been synthesised and developed as potassium-selective transporters. Resorcin[4]arenes that feature crown ether moieties function as efficient carriers of K⁺ across bulk liquid membranes showing enhanced selectivity over the other alkali metal ions relative to a model system (benzo[15]crown-5). Incorporation of functionalities suitable for pore formation, in addition to an extra annulus of aromatic residues, gives molecules which have remarkable ion-channel-mimicking behaviour in a biological lipid bilayer with outstanding K⁺/Na⁺ selectivity.

Nanosecond molecular dynamics simulations in a fully solvated phospholipid bilayer have been performed on single transmembrane α-helices from three putative ion channel proteins encoded by viruses: NB (from influenza B), CM2 (from influenza C), and Vpu (from HIV-1). α-Helix stability is maintained within a core region of ca. 28 residues for each protein. Helix perturbations are due either to unfavorable interactions of hydrophobic residues with the lipid headgroups or to the need of the termini of short helices to extend into the surrounding interfacial environment in order to form H-bonds. The requirement of both ends of a helix to form favorable interactions with lipid headgroups and/or water may also lead to tilting and/or kinking of a transmembrane α-helix. Residues that are generally viewed as poor helix formers in aqueous solution (e.g., Gly, Ile, Val) do not destabilize helices, if located within a helix that spans a lipid bilayer. However, helix/bilayer mismatch such that a helix ends abruptly within the bilayer core destabilizes the end of the helix, especially in the presence of Gly and Ala residues. Hydrogen bonding of polar side-chains with the peptide backbone and with one another occurs when such residues are present within the bilayer core, thus minimizing the energetic cost of burying such side-chains. © 2000 John Wiley & Sons, Inc. Biopoly 53: 529–538, 2000

We have purified and characterized a factor, from the conditioned medium of neural stem cell cultures, which is required for fibroblast growth factor 2's (FGF-2) mitogenic activity on neural stem cells. This autocrine/paracrine cofactor is a glycosylated form of cystatin C (CCg), whose N-glycosylation is required for its activity. We further demonstrated that, both in vitro and in vivo, neural stem cells undergoing cell division are immunopositive for cystatin C. Finally, we showed in vivo functional activity of CCg by demonstrating that the combined delivery of FGF-2 and CCg to the adult dentate gyrus stimulated neurogenesis. We propose that the process of neurogenesis is controlled by the cooperation between trophic factors and autocrine/paracrine cofactors, of which CCg is a prototype.

The study of the nuclear pore in vertebrates would benefit from a strategy to directly identify new nucleoporins and interactions between those nucleoporins. We have developed a novel two-step “organelle trap” assay involving affinity selection and in vitro pore assembly. In the first step, soluble proteins derived from Xenopusegg extracts are applied to a column containing a ligand of interest. The bound proteins are then tagged by biotinylation and eluted. In the second step, potential nucleoporins are selected for by virtue of their ability to assemble into annulate lamellae, a cytoplasmic mimic of nuclear pores. The incorporated proteins are then recognized by their biotin tag. Here we use the lectin wheat germ agglutinin (WGA) as ligand; WGA inhibits nuclear transport and has been shown to directly bind three known nucleoporins from Xenopusextract, Nup62, Nup98, and Nup214, all of which containN-acetylglucosamine residues. Under reduced-stringency conditions, three additional proteins bind to WGA–Sepharose and are revealed by the organelle trap assay. We identified all three as partner nucleoporins. Two were discovered to be XenopusNup93 and Nup205. The third is a novel vertebrate nucleoporin, Nup188. This new vertebrate protein, XenopusNup188, exists in a complex with xNup93 and xNup205. The Nup93-Nup188-Nup205 complex does not bind directly to WGA but binds indirectly via theN-acetylglucosamine–modified nucleoporins. A gene encoding human Nup188 was also identified. The discovery of vertebrate Nup188, related to a yeast nucleoporin, and its novel protein–protein interactions illustrates the power of the two-step organelle trap assay and identifies new building blocks for constructing the nuclear pore.

The N-linked glycan present on corticotropin releasing factor binding protein (CRFBP) recombinantly expressed in CHO cells has been characterized using a variety of techniques including sequential hydrolysis with specific exoglycosidases monitored with reversed-phase high performance liquid chromatography (RP-HPLC), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization mass spectrometry (ES-MS). Determining the intact glycan mass and comparing possible glycan compositions with entries in the Complex Carbohydrate Database facilitated the choice of enzymes. Tandem mass spectrometry (MS/MS) was used to confirm the structure of the glycan. Using this approach, complementary information could be obtained through specific glycosidase reactions monitored with MS and MSnfragmentation of the intact or enzymatically-modified glycopeptides. No evidence was found for the presence of sites of O-linked glycosylation. In addition, to understand better the role of the glycan on CRFBP, we characterized the in vitrobinding affinity of a mutant non-glycosylated [Gln180] CRFBP and an enzymatically deglycosylated form of CRFBP to human corticotropin releasing factor (hCRF).

Helicobacter pyloriinfection of the gastric mucosa is accompanied by an activated histamine metabolism. Histamine plays a central role in the regulation of gastric acid secretion and is involved in the pathogenesis of gastroduodenal ulcerations. Histidine decarboxylase (HDC) is the rate-limiting enzyme for histamine production, and its activity is regulated through transcriptional mechanisms. The present study investigated the effect of H. pyloriinfection on the transcriptional activity of the human HDC (hHDC) promoter in a gastric epithelial cell line (AGS) and analyzed the underlying molecular mechanisms. Our studies demonstrate that H. pyloriinfection potently transactivated the hHDC promoter. The H. pylori-responsive element of the hHDC gene was mapped to the sequence +1 to +27 base pairs, which shows no homology to known cis-acting elements and also functions as a gastrin-responsive element. H. pyloriregulates the activity of this element via a Raf-1/MEK/ERK pathway, which was activated in a Ras-independent manner. Furthermore, we found that H. pylori-induced transactivation of the hHDC promoter was independent of the cagpathogenicity island and the vacuolating cytotoxin A gene and therefore may be exerted through (a) new virulence factor(s). A better understanding of H. pylori-directed hHDC transcription can provide novel insights into the molecular mechanisms of H. pylori-dependent gene regulation in gastric epithelial cells and may lead to new therapeutic approaches.

All poxviruses studied encode a type 1B topoisomerase that introduces transient nicks into DNA and thereby relaxes DNA supercoils. Here we present a study of the protein domains of the topoisomerase of the poxvirus molluscum contagiosum (MCV), which allows us to specify DNA contacts made by different domains. Partial proteolysis of the enzyme revealed two stable domains separated by a protease-sensitive linker. A fragment encoding the linker and carboxyl-terminal domain (residues 82-323) was overexpressed in Escherichia coliand purified. MCV topoisomerase (MCV-TOP)(82-323) could relax supercoiled plasmids in vitro,albeit with a slower rate than the wild-type enzyme. MCV-TOP(82-323) was sensitive to sequences in the favored 5′-(T/C)CCTT-3′ recognition site and also flanking DNA, indicating that some of the sequence-specific contacts are made by residues 82-323. Assays of initial binding and covalent catalysis by MCV-TOP(82-323) identified the contacts flanking the 5′-CCCTT-3′ sequence at +10, +9, −2, and −3 to be important. Tests with substrates containing a 5-bridging phosphorothiolate that trap the cleaved complex revealed that correct contacts to the flanking sequences were important in the initial cleavage step. MCV-TOP(82-323) differed from the full-length protein in showing reduced sensitivity to mutations at a position within the 5′-(T/C)CCTT-3′ recognition site, consistent with a model in which the amino-terminal domain contacts this region. These findings provide insight into the division of labor within the MCV-TOP enzyme.

The transforming growth factor β (TGFβ) superfamily of cytokines elicit diverse biological responses by interacting with two distinct, but structurally related transmembrane receptor serine kinases (type I and type II). The binding of these dimeric ligands to the type II receptor is the first event in transmembrane signaling for this family. Here we report the 1.5 Å resolution crystal structure of the extracellular ligand-binding domain of the type II activin receptor (ActRII-ECD), which reveals a fold similar to that of a class of toxins known as three-finger toxins. This fold is primarily dictated by disulfide bonds formed by eight conserved cysteines, with a characteristic spacing, and thus is likely to be shared by most of the type I and II receptors for the TGFβ family. Sequence comparison with an evolutionarily distant activin binding-protein identifies several conserved residues, including two hydrophobic clusters that may form binding surfaces for activin and the type I receptor.