Characterization of functionally independent domains in the human ubiquitin conjugating enzyme UbcH2

AbstractUbcH2 encodes a human ubiquitin conjugating enzyme (E2) able to conjugate ubiquitin to histone H2A in an E3 independent manner in vitro, which indicates that UbcH2 directly interacts with its substrates. To identify parts of the enzyme that are capable of binding H2A, we expressed several deletion mutants of UbcH2 in E. coli and tested the ability of the affinity purified mutant proteins to ubiquitinate H2A in the presence of bacterial expressed E1 and ubiquitin. With this in vitro assay we identified a C-terminal part of UbcH2 to be important for the interaction with H2A. Transfer of this C-terminal domain to another human E2, which is unable to catalyze ubiquitination of histones, leads to a fully active hybrid human ubiquitin conjugating enzyme capable of H2A ubiquitination. These results demonstrate that UbcH2 consists of two functionally independent domains. A N-terminal core domain with ubiquitin conjugating activity, and a C-terminal domain which interacts with substrate proteins

An End-to-End Performance Comparison of Seven Permissioned Blockchain Systems

The emergence of more and more blockchain solutions with innovative approaches to optimising performance, scalability, privacy and governance complicates performance analysis. Reasons for the difficulty of benchmarking blockchains include, for example, the high number of system parameters to configure and the effort to deploy a blockchain network. In addition, performance data, which mostly comes from system vendors, is often intransparent. We investigate and evaluate the performance of seven permissioned blockchain systems using different parameter settings in a reproducible manner. We employ an end-to-end approach, where the clients sending the transactions are fully involved in the data collection approach. Our results highlight the peculiarities and limitations of the systems under investigation. Due to the insights given, our work forms the basis for continued research to optimise the performance of blockchain systems.Comment: 14 pages, 5 figures, 20 tables, Middleware Conferenc

Recent approaches in the development and enhancement of self-regulated learning

Bedingungen heutiger Schule und heutigen Studiums bringen eine erhebliche Zunahme des außerschulischen Lernens außerhalb von Präsenzphasen mit sich, was eine erhebliche Lernbelastung für Schüler und Studenten bedeutet. Für die Unterstützung und Förderung der Kompetenz zu selbstgesteuertem Lernen wurden in neuerer Zeit einige Programme entwickelt, die hier in ihren wesentlichsten Prinzipien knapp dargestellt und kritisch danach bewertet werden sollen, ob sie tatsächlich eine eigenständige Lernweggestaltung und Lernüberprüfung ermöglichen können. (DIPF/Orig.)Conditions and context of contemporary schooling and studying lead to a considerable increase in intensity and amount of learning beyond school or university. College and university students thus are confronted with operations beyond capacity. Some recent work has focussed on the development of remedial and supportive learning and schooling programs to foster and assist self-guided study. Some of these programs are sketched in their main principles. The programs are further evaluated for their factual assistance and impact on the planning, checking and control of learning by means of independent learner-guided self-regulation

Processing Gradual Information with Fuzzy Arden Syntax

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Treatment of Semantic Heterogeneity in Information Retrieval

"Nowadays, users of information services are faced with highly decentralised, heterogeneous document sources with different content analysis. Semantic heterogeneity occurs e.g. when resources using different systems for content description are searched using a single query system. This report describes several approaches of handling semantic heterogeneity used in projects of the German Social Science Information Centre." (author's abstract

Protein-disulfide isomerase- and protein thiol-dependent dehydroascorbate reduction and ascorbate accumulation in the lumen of the endoplasmic reticulum.

The transport and intraluminal reduction of dehydroascorbate was investigated in microsomal vesicles from various tissues. The highest rates of transport and intraluminal isotope accumulation (using radiolabeled compound and a rapid filtration technique) were found in hepatic microsomes. These microsomes contain the highest amount of protein-disulfide isomerase, which is known to have a dehydroascorbate reductase activity. The steady-state level of intraluminal isotope accumulation was more than 2-fold higher in hepatic microsomes prepared from spontaneously diabetic BioBreeding/Worcester rats and was very low in fetal hepatic microsomes although the initial rate of transport was not changed. In these microsomes, the amount of protein-disulfide isomerase was similar, but the availability of protein thiols was different and correlated with dehydroascorbate uptake. The increased isotope accumulation was accompanied by a higher rate of dehydroascorbate reduction and increased protein thiol oxidation in microsomes from diabetic animals. The results suggest that both the activity of protein-disulfide isomerase and the availability of protein thiols as reducing equivalents can play a crucial role in the accumulation of ascorbate in the lumen of the endoplasmic reticulum. These findings also support the fact that dehydroascorbate can act as an oxidant in the protein-disulfide isomerase-catalyzed protein disulfide formation

Uncoupled redox systems in the lumen of the endoplasmic reticulum. Pyridine nucleotides stay reduced in an oxidative environment.

The redox state of the intraluminal pyridine nucleotide pool was investigated in rat liver microsomal vesicles. The vesicles showed cortisone reductase activity in the absence of added reductants, which was dependent on the integrity of the membrane. The intraluminal pyridine nucleotide pool could be oxidized by the addition of cortisone or metyrapone but not of glutathione. On the other hand, intraluminal pyridine nucleotides were slightly reduced by cortisol or glucose 6-phosphate, although glutathione was completely ineffective. Redox state of microsomal protein thiols/disulfides was not altered either by manipulations affecting the redox state of pyridine nucleotides or by the addition of NAD(P)+ or NAD(P)H. The uncoupling of the thiol/disulfide and NAD(P)+/NAD(P)H redox couples was not because of their subcompartmentation, because enzymes responsible for the intraluminal oxidoreduction of pyridine nucleotides were distributed equally in smooth and rough microsomal subfractions. Instead, the phenomenon can be explained by the negligible representation of glutathione reductase in the endoplasmic reticulum lumen. The results demonstrated the separate existence of two redox systems in the endoplasmic reticulum lumen, which explains the contemporary functioning of oxidative folding and of powerful reductive reactions