Point mutations in the stem region and the fourth AAA domain of cytoplasmic dynein heavy chain partially suppress the phenotype of NUDF/LIS1 loss in Aspergillus nidulans

Cytoplasmic dynein performs multiple cellular tasks but its regulation remains unclear. The dynein heavy chain has a N-terminal stem that binds to other subunits and a C-terminal motor unit that contains six AAA (ATPase associated with cellular activities) domains and a microtubule-binding site located between AAA4 and AAA5. In Aspergillus nidulans, NUDF (a LIS1 homolog) functions in the dynein pathway, and two nudF6 partial suppressors were mapped to the nudA dynein heavy chain locus. Here we identified these two mutations. The nudAL1098F mutation resides in the stem region, and nudAR3086C is in the end of AAA4. These mutations partially suppress the phenotype of nudF deletion but do not suppress the phenotype exhibited by mutants of dynein intermediate chain and Arp1. Surprisingly, the stronger DeltanudF suppressor, nudAR3086C, causes an obvious decrease in the basal level of dynein's ATPase activity and an increase in dynein's distribution along microtubules. Thus, suppression of the DeltanudF phenotype may result from mechanisms other than simply the enhancement of dynein's ATPase activity. The fact that a mutation in the end of AAA4 negatively regulates dynein's ATPase activity but partially compensates for NUDF loss indicates the importance of the AAA4 domain in dynein regulation in vivo.

Effect of constant and variable domain glycosylation on pharmacokinetics of therapeutic antibodies in mice

Previous studies on the effect of glycosylation on the elimination rate of antibodies have produced conflicting results. Here, we performed pharmacokinetic studies in mice with two preparations of a monoclonal IgG1 antibody enriched for complex type or high mannose type oligosaccharides at the Fc glycosylation site. No significant difference in the serum half-life was found between the two antibody glycoforms, nor was any difference observed in the serum half-lives of different complex type glycoforms. To evaluate the influence of glycosylation within the variable domain, a second monoclonal antibody, glycosylated in both the Fc and Fv domains, was separated into fractions containing different amounts of Fv-associated sialic acid and administered to mice. Again, no significant difference was found in the clearance rates of variants carrying different amounts of Fv-associated sialic acid or lacking Fv-glycosylation. These results suggest that glycosylation has little or no impact on the pharmacokinetic behavior of these two monoclonal antibodies in mice.

Time-domain models for power system stability and unbalance

It is an important and difficult challenge to protect modern interconnected power system from blackouts. Applying advanced power system protection techniques and increasing power system stability are ways to improve the reliability and security of power systems. Phasor-domain software packages such as Power System Simulator for Engineers (PSS/E) can be used to study large power systems but cannot be used for transient analysis. In order to observe both power system stability and transient behavior of the system during disturbances, modeling has to be done in the time-domain. This work focuses on modeling of power systems and various control systems in the Alternative Transients Program (ATP). ATP is a time-domain power system modeling software in which all the power system components can be modeled in detail. Models are implemented with attention to component representation and parameters. The synchronous machine model includes the saturation characteristics and control interface. Transient Analysis Control System is used to model the excitation control system, power system stabilizer and the turbine governor system of the synchronous machine. Several base cases of a single machine system are modeled and benchmarked against PSS/E. A two area system is modeled and inter-area and intra-area oscillations are observed. The two area system is reduced to a two machine system using reduced dynamic equivalencing. The original and the reduced systems are benchmarked against PSS/E. This work also includes the simulation of single-pole tripping using one of the base case models. Advantages of single-pole tripping and comparison of system behavior against three-pole tripping are studied. Results indicate that the built-in control system models in PSS/E can be effectively reproduced in ATP. The benchmarked models correctly simulate the power system dynamics. The successful implementation of a dynamically reduced system in ATP shows promise for studying a small sub-system of a large system without losing the dynamic behaviors. Other aspects such as relaying can be investigated using the benchmarked models. It is expected that this work will provide guidance in modeling different control systems for the synchronous machine and in representing dynamic equivalents of large power systems.

Two dimensional Lattice Boltzmann simulation of fluid flow through an idealized micro-structure of disordered media

This technical report discusses the application of Lattice Boltzmann Method (LBM) in the fluid flow simulation through porous filter-wall of disordered media. The diesel particulate filter (DPF) is an example of disordered media. DPF is developed as a cutting edge technology to reduce harmful particulate matter in the engine exhaust. Porous filter-wall of DPF traps these soot particles in the after-treatment of the exhaust gas. To examine the phenomena inside the DPF, researchers are looking forward to use the Lattice Boltzmann Method as a promising alternative simulation tool. The lattice Boltzmann method is comparatively a newer numerical scheme and can be used to simulate fluid flow for single-component single-phase, single-component multi-phase. It is also an excellent method for modelling flow through disordered media. The current work focuses on a single-phase fluid flow simulation inside the porous micro-structure using LBM. Firstly, the theory concerning the development of LBM is discussed. LBM evolution is always related to Lattice gas Cellular Automata (LGCA), but it is also shown that this method is a special discretized form of the continuous Boltzmann equation. Since all the simulations are conducted in two-dimensions, the equations developed are in reference with D2Q9 (two-dimensional 9-velocity) model. The artificially created porous micro-structure is used in this study. The flow simulations are conducted by considering air and CO2 gas as fluids. The numerical model used in this study is explained with a flowchart and the coding steps. The numerical code is constructed in MATLAB. Different types of boundary conditions and their importance is discussed separately. Also the equations specific to boundary conditions are derived. The pressure and velocity contours over the porous domain are studied and recorded. The results are compared with the published work. The permeability values obtained in this study can be fitted to the relation proposed by Nabovati [8], and the results are in excellent agreement within porosity range of 0.4 to 0.8.

Monitoring protein-protein interactions between the mammalian integral membrane transporters and PDZ-interacting partners using a modified split-ubiquitin membrane yeast two-hybrid system

PDZ-binding motifs are found in the C-terminal tails of numerous integral membrane proteins where they mediate specific protein-protein interactions by binding to PDZ-containing proteins. Conventional yeast two-hybrid screens have been used to probe protein-protein interactions of these soluble C termini. However, to date no in vivo technology has been available to study interactions between the full-length integral membrane proteins and their cognate PDZ-interacting partners. We previously developed a split-ubiquitin membrane yeast two-hybrid (MYTH) system to test interactions between such integral membrane proteins by using a transcriptional output based on cleavage of a transcription factor from the C terminus of membrane-inserted baits. Here we modified MYTH to permit detection of C-terminal PDZ domain interactions by redirecting the transcription factor moiety from the C to the N terminus of a given integral membrane protein thus liberating their native C termini. We successfully applied this "MYTH 2.0" system to five different mammalian full-length renal transporters and identified novel PDZ domain-containing partners of the phosphate (NaPi-IIa) and sulfate (NaS1) transporters that would have otherwise not been detectable. Furthermore this assay was applied to locate the PDZ-binding domain on the NaS1 protein. We showed that the PDZ-binding domain for PDZK1 on NaS1 is upstream of its C terminus, whereas the two interacting proteins, NHERF-1 and NHERF-2, bind at a location closer to the N terminus of NaS1. Moreover NHERF-1 and NHERF-2 increased functional sulfate uptake in Xenopus oocytes when co-expressed with NaS1. Finally we used MYTH 2.0 to demonstrate that the NaPi-IIa transporter homodimerizes via protein-protein interactions within the lipid bilayer. In summary, our study establishes the MYTH 2.0 system as a novel tool for interactive proteomics studies of membrane protein complexes.

PHYLOGENETIC ANALYSES, GENE DOSAGE EFFECT AND SELECTION IN MEMBERS OF THE LATERAL BOUNDARY DOMAIN (LBD) GENES TRANSCRIPTION FACTOR FAMILY

Wood formation is an economically and environmentally important process and has played a significant role in the evolution of terrestrial plants. Despite its significance, the molecular underpinnings of the process are still poorly understood. We have previously shown that four Lateral Boundary Domain (LBD) transcription factors have important roles in the regulation of wood formation with two (LBD1 and LBD4) involved in secondary phloem and ray cell development and two (LBD15 and LBD18) in secondary xylem formation. Here, we used comparative phylogenetic analyses to test potential roles of the four LBD genes in the evolution of woodiness. We studied the copy number and variation in DNA and amino acid sequences of the four LBDs in a wide range of woody and herbaceous plant taxa with fully sequenced and annotated genomes. LBD1 showed the highest gene copy number across the studied species, and LBD1 gene copy number was strongly and significantly correlated with the level of ray seriation. The lianas, cucumber and grape, with multiseriate ray cells showed the highest gene copy number (12 and 11, respectively). Because lianas’ growth habit requires significant twisting and bending, the less lignified ray parenchyma cells likely facilitate stem flexibility and maintenance of xylem conductivity. We further demonstrate conservation of amino acids in the LBD18 protein sequences that are specific to woody taxa. Neutrality tests showed evidence for strong purifying selection on these gene regions across various orders, indicating adaptive convergent evolution of LBD18. Structural modeling demonstrates that the conserved amino acids have a significant impact on the tertiary protein structure and thus are likely of significant functional importance.

Use of concatamers to study GABAA receptor architecture and function: application to delta-subunit-containing receptors and possible pitfalls

Many membrane proteins, including the GABA(A) [GABA (gamma-aminobutyric acid) type A] receptors, are oligomers often built from different subunits. As an example, the major adult isoform of the GABA(A) receptor is a pentamer built from three different subunits. Theoretically, co-expression of three subunits may result in many different receptor pentamers. Subunit concatenation allows us to pre-define the relative arrangement of the subunits. This method may thus be used to study receptor architecture, but also the nature of binding sites. Indeed, it made possible the discovery of a novel benzodiazepine site. We use here subunit concatenation to study delta-subunit-containing GABA(A) receptors. We provide evidence for the formation of different functional subunit arrangements in recombinant alpha(1)beta(3)delta and alpha(6)beta(3)delta receptors. As with all valuable techniques, subunit concatenation has also some pitfalls. Most of these can be avoided by carefully titrating and minimizing the length of the linker sequences joining the two linked subunits and avoiding inclusion of the signal sequence of all but the N-terminal subunit of a multi-subunit construct. Maybe the most common error found in the literature is that low expression can be overcome by simply overloading the expression system with genetic information. As some concatenated constructs result by themselves in a low level of expression, this erroneous assembly leading to receptor function may be promoted by overloading the expression system and leads to wrong conclusions.

Domain Name Transfer before Slovak and Czech Courts

In Europe, a disagreement persists in the courts about the possibility of plaintiffs to request a domain name transfer in domain name disputes. In the last ten years, Slovak and Czech courts also produced some jurisprudence on this issue. Interestingly, the BGH’s influential opinion in the shell.de decision, which denied domain name transfer as an available remedy under German law back in 2002, wasn’t initially followed. To the contrary, several Slovak and Czech decisions of lower courts allowed a domain name transfer using two different legal bases. This seemingly settled case law was rejected a few months ago by the globtour.cz decision of the Czech Supreme Court, which refused domain name transfers for the time being

Aspekte der strukturellen Organisation der Basalmembran und der Mauscornea

Thin and ultrathin cryosections of mouse cornea were labeled with affinity-purified antibodies directed against either laminin, its central segments (domain 1), the end of its long arm (domain 3), the end of one of its short arms (domain 4), nidogen, or low density heparan sulfate proteoglycan. All basement membrane proteins are detected by indirect immunofluorescence exclusively in the epithelial basement membrane, in Descemet's membrane, and in small amorphous plaques located in the stroma. Immunoelectron microscopy using the protein A-gold technique demonstrated laminin domain 1 and nidogen in a narrow segment of the lamina densa at the junction to the lamina lucida within the epithelial basement membrane. Domain 3 shows three preferred locations at both the cellular and stromal boundaries of the epithelial basement membrane and in its center. Domain 4 is located predominantly in the lamina lucida and the adjacent half of the lamina densa. The low density heparan sulfate proteoglycan is found all across the basement membrane showing a similar uniform distribution as with antibodies against the whole laminin molecule. In Descemet's membrane an even distribution was found with all these antibodies. It is concluded that within the epithelial basement membrane the center of the laminin molecule is located near the lamina densa/lamina lucida junction and that its long arm favors three major orientations. One is close to the cell surface indicating binding to a cell receptor, while the other two are directed to internal matrix structures. The apparent codistribution of laminin domain 1 and nidogen agrees with biochemical evidence that nidogen binds to this domain.

Cloning and characterization of two bistructural S-layer-RTX proteins from Campylobacter rectus

Campylobacter rectus is an important periodontal pathogen in humans. A surface-layer (S-layer) protein and a cytotoxic activity have been characterized and are thought to be its major virulence factors. The cytotoxic activity was suggested to be due to a pore-forming protein toxin belonging to the RTX (repeats in the structural toxins) family. In the present work, two closely related genes, csxA and csxB (for C. rectus S-layer and RTX protein) were cloned from C. rectus and characterized. The Csx proteins appear to be bifunctional and possess two structurally different domains. The N-terminal part shows similarity with S-layer protein, especially SapA and SapB of C. fetus and Crs of C. rectus. The C-terminal part comprising most of CsxA and CsxB is a domain with 48 and 59 glycine-rich canonical nonapeptide repeats, respectively, arranged in three blocks. Purified recombinant Csx peptides bind Ca2+. These are characteristic traits of RTX toxin proteins. The S-layer and RTX domains of Csx are separated by a proline-rich stretch of 48 amino acids. All C. rectus isolates studied contained copies of either the csxA or csxB gene or both; csx genes were absent from all other Campylobacter and Helicobacter species examined. Serum of a patient with acute gingivitis showed a strong reaction to recombinant Csx protein on immunoblots.

Smell and taste of chewing gum affect frequency domain EEG source localizations

We investigated brain electric field signatures of subjective feelings after chewing regular gum or gum base without flavor. 19-channel eyes-closed EEG from 20 healthy males before and after 5 minutes of chewing the two gum types in random sequence was source modeled in the frequency domain using the FFT-Dipole-Approximation. 3-dimensional brain locations and strengths (Global Field Power, GFP) of the equivalent sources of five frequency bands were computed as changes from pre-chewing baseline. Gum types differed (ANOVA) in pre-post changes of source locations for the alpha-2 band (to anterior and right after regular gum, opposite after gum base) and beta-2 band (to anterior and inferior after regular gum, opposite after gum base), and of GFP for delta-theta, alpha-2 and beta-1 (regular gum: increase, gum base: decrease). Subjective feeling changed to more positive values after regular gum than gum base (ANOVA).—Thus, chewing gum with and without taste-smell activates different brain neuronal populations.

Structural rearrangements of the central region of the morbillivirus attachment protein stalk domain trigger F protein refolding for membrane fusion

It is unknown how receptor binding by the paramyxovirus attachment proteins (HN, H, or G) triggers the fusion (F) protein to fuse with the plasma membrane for cell entry. H-proteins of the morbillivirus genus consist of a stalk ectodomain supporting a cuboidal head; physiological oligomers consist of non-covalent dimer-of-dimers. We report here the successful engineering of intermolecular disulfide bonds within the central region (residues 91-115) of the morbillivirus H-stalk; a sub-domain that also encompasses the putative F-contacting section (residues 111-118). Remarkably, several intersubunit crosslinks abrogated membrane fusion, but bioactivity was restored under reducing conditions. This phenotype extended equally to H proteins derived from virulent and attenuated morbillivirus strains and was independent of the nature of the contacted receptor. Our data reveal that the morbillivirus H-stalk domain is composed of four tightly-packed subunits. Upon receptor binding, these subunits structurally rearrange, possibly inducing conformational changes within the central region of the stalk, which, in turn, promote fusion. Given that the fundamental architecture appears conserved among paramyxovirus attachment protein stalk domains, we predict that these motions may act as a universal paramyxovirus F-triggering mechanism.

Agency and ownership are independent components of 'sensing the self' in the auditory-verbal domain

'Sensing the self' relies on the ability to distinguish self-generated from external stimuli. It requires functioning mechanisms to establish feelings of agency and ownership. Agency is defined causally, where the subjects action is followed by an effect. Ownership is defined by the features of the effect, independent from the action. In our study, we manipulated these qualities separately. 13 right-handed healthy individuals performed the experiment while 76-channel EEG was recorded. Stimuli consisted of visually presented words, read aloud by the subject. The experiment consisted of six conditions: (a) subjects saw a word, read it aloud, heard it in their own voice; (b) like a, but the word was heard in an unfamiliar voice; (c) subject heard a word in his/her own voice without speaking; (d) like c, but the word was heard in an unfamiliar voice; (e) like a, but subjects heard the word with a delay; (f) subjects read without hearing. ERPs and difference maps were computed for all conditions. Effects were analysed topographically. The N100 (86-172 ms) displayed significant main effects of agency and ownership. The topographies of the two effects shared little common variance, suggesting independent effects. Later effects (174-400 ms) of agency and ownership were topographically similar, suggesting common mechanisms. Replicating earlier studies, significant N100 suppression was observed, with a topography resembling the agency effect. 'Sensing the self' appears to recruit from at least two very distinct processes: an agency assessment that represents causality and an ownership assessment that compares stimulus features with memory content.

Lessons from the Multilateral Trading System for Reforming the Architecture of the International Environmental Regime

Recent studies on environmental regimes suggest that important lessons and policy recommendations may be drawn from the functioning of the multilateral trading regime. This brief compares the needs and goals of the trade and environment regimes, and discusses how insights from over sixty years of experience of the multilateral trading system might provide ideas for redesigning the architecture of the international environmental regime. It further calls for a better dialogue and improved complementarities between the two fields in order to enhance coherence within international law.

Single-stranded DNA-binding proteins regulate the abundance of LIM domain and LIM domain-binding proteins.

The LIM domain-binding protein Ldb1 is an essential cofactor of LIM-homeodomain (LIM-HD) and LIM-only (LMO) proteins in development. The stoichiometry of Ldb1, LIM-HD, and LMO proteins is tightly controlled in the cell and is likely a critical determinant of their biological actions. Single-stranded DNA-binding proteins (SSBPs) were recently shown to interact with Ldb1 and are also important in developmental programs. We establish here that two mammalian SSBPs, SSBP2 and SSBP3, contribute to an erythroid DNA-binding complex that contains the transcription factors Tal1 and GATA-1, the LIM domain protein Lmo2, and Ldb1 and binds a bipartite E-box-GATA DNA sequence motif. In addition, SSBP2 was found to augment transcription of the Protein 4.2 (P4.2) gene, a direct target of the E-box-GATA-binding complex, in an Ldb1-dependent manner and to increase endogenous Ldb1 and Lmo2 protein levels, E-box-GATA DNA-binding activity, and P4.2 and beta-globin expression in erythroid progenitors. Finally, SSBP2 was demonstrated to inhibit Ldb1 and Lmo2 interaction with the E3 ubiquitin ligase RLIM, prevent RLIM-mediated Ldb1 ubiquitination, and protect Ldb1 and Lmo2 from proteasomal degradation. These results define a novel biochemical function for SSBPs in regulating the abundance of LIM domain and LIM domain-binding proteins.