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

Processing Gradual Information with Fuzzy Arden Syntax

Abstract Th

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

Characterization of digital annular pulleys and their entheses: an ultrasonographic study with anatomical and histological correlations

Objectives: Digital annular pulleys (DAP) are important anatomical structures for finger function. The anatomy, histology, and imaging assessment of DAP, particularly at the level of their entheses is still not clearly defined. The advent of high-frequency ultrasound (US) transducers opened new perspectives in evaluating sub-millimeter scale structures, such as pulleys, paving the way for their global assessment. The study aimed at characterizing DAP from an anatomical, histological, and US perspective, focusing on the detection and complete description of pulley entheses. Methods: US assessment and gross anatomy dissection were conducted on 20 cadaveric hands to study DAP thickness and structure including enthesis identification. The results of the US and anatomical measurements were correlated. DAP entheses identified by US were characterized via histological analysis. DAP in 20 healthy controls (HC) were detected and measured by US. The A1, A2, and A4 DAP entheses were assessed using a new dynamic maneuver to better evaluate those structures. Results: 1200 DAP (400 cadaveric, 800 HC) were analyzed. The cadaveric study demonstrated strong correlation between anatomical and US measurement of DAP (r = 0.96). At histological level, DAP entheses at the volar plate, sesamoid bones, or phalangeal ridges contained fibrous and fibrocartilaginous tissue. The US assessment of A1, A2, and A4 DAP in HC allowed the identification of 718/720 (99.73%) entheses. Conclusion: US is an effective tool to detect and study DAP. DAP entheses reveal both fibrous and fibrocartilaginous characteristics. A newly described maneuver to optimize DAP enthesis visualization enhances their detection by US

Learning Lightprobes for Mixed Reality Illumination

This paper presents the first photometric registration pipeline for Mixed Reality based on high quality illumination estimation by convolutional neural network (CNN) methods. For easy adaptation and deployment of the system, we train the CNN using purely synthetic images and apply them to real image data. To keep the pipeline accurate and efficient, we propose to fuse the light estimation results from multiple CNN instances, and we show an approach for caching estimates over time. For optimal performance, we furthermore explore multiple strategies for the CNN training. Experimental results show that the proposed method yields highly accurate estimates for photo-realistic augmentations