Ubiquitination is required for the retro-translocation of a short-lived luminal endoplasmic reticulum glycoprotein to the cytosol for degradation by the proteasome

In the endoplasmic reticulum (ER), an efficient "quality control system" operates to ensure that mutated and incorrectly folded proteins are selectively degraded. We are studying ER-associated degradation using a truncated variant of the rough ER-specific type I transmembrane glycoprotein, ribophorin I. The truncated polypeptide (RI332) consists of only the 332 amino-terminal amino acids of the protein corresponding to most of its luminal domain and, in contrast to the long-lived endogenous ribophorin I, is rapidly degraded. Here we show that the ubiquitin-proteasome pathway is involved in the destruction of the truncated ribophorin I. Thus, when RI332 that itself appears to be a substrate for ubiquitination was expressed in a mutant hamster cell line harboring a temperature-sensitive mutation in the ubiquitin-activating enzyme E1 affecting ubiquitin-dependent proteolysis, the protein is dramatically stabilized at the restrictive temperature. Moreover, inhibitors of proteasome function effectively block the degradation of RI332. Cell fractionation experiments indicate that RI332 accumulates in the cytosol when degradation is prevented by proteasome inhibitors but remains associated with the lumen of the ER under ubiquitination-deficient conditions, suggesting that the release of the protein into the cytosol is ubiquitination-dependent. Accordingly, when ubiquitination is impaired, a considerable amount of RI332 binds to the ER chaperone calnexin and to the Sec61 complex that could effect retro-translocation of the polypeptide to the cytosol. Before proteolysis of RI332, its N-linked oligosaccharide is cleaved in two distinct steps, the first of which might occur when the protein is still associated with the ER, as the trimmed glycoprotein intermediate efficiently interacts with calnexin and Sec61. From our data we conclude that the steps that lead a newly synthesized luminal ER glycoprotein to degradation by the proteasome are tightly coupled and that especially ubiquitination plays a crucial role in the retro-translocation of the substrate protein for proteolysis to the cytosol.     

Preliminary safety analysis of ex-vessel LOCA for the European HCPB TBM system

Ex-vessel loss of coolant accident caused by a double-ended pipe break of the helium coolant system inside port cell is considered as one of the most critical accident for the European Helium Cooled Pebble Beds Test Blanket Module (HCPB TBM) system. The resulting rapid helium blow-down causes an immediate block of the TBM cooling, which requires a prompt plasma shutdown. Even after the plasma shutdown the temperature can increase over the design limit and the accident sequence can lead up to a break of the TBM box protection after the failure of different protection systems. Thus air ingresses in the vacuum vessel from the damaged TBM system and steam from the surrounding ITER blanket and divertor structures. The evaluation of this sequence is very important for the definition of the correct protection strategy of the system. To consider all these different events a methodology has been developed in KIT combining different codes for a complete analysis of the accident. In particular, this paper shows an application of MELCOR code to model beryllium–steam reaction in a particular accidental sequence for the long term cooling.     

Subwavelength size determination by spatially modulated illumination virtual microscopy

A new approach for determining the sizes of individual, small fluorescent objects with diameters considerably below the optical resolution limit is described in which spatially modulated illumination (SMI) microscopy and 360-647-nm excitation wavelengths are used. The results of SMI virtual microscopy computer simulations indicate that, in this wavelength range, reliable measurements of sizes as small as approximately 20 nm are feasible if the low numbers of fluorescence photons that are usually detected from such small objects are taken into account. This method is based on the well-known fact that the modulation of the diffraction image in a SMI microscope is disturbed by the size of the object. Using appropriately calculated calibration functions, one can use this disturbance of the modulation to determine the size of the original object.     

Measurement of fetal cells in the maternal circulation

There is evidence that the quantitation of fetal-maternal hemorrhage may be useful in reducing some of the RhoGAM failures in preventing Rh sensitization. The Betke-Kleihauer technique is a well-established method that has been used to estimate the amount of transplacental hemorrhage. Recently, a kit (Fetaldex) has become available for use in the detection and quantitation of fetal-maternal hemorrhage. Using control blood smears, we compared the results of the Fetaldex kit with those results obtained by the Betke-Kleihauer technique. The data indicate that the Fetaldex method is as accurate as the Betke-Kleihauer technique in detecting and measuring fetal red cells in the maternal circulation.     

Cross-linked polystyrene-dithiocarbamate polymers: Use in heavy metal removal

Cross-linked polystyrene polymers were converted to dithiocarbamate polymers. These polymers were used to remove heavy metal cations from water. Dithiocarbamate polymers can be understood to be inherently more stable toward hydrolysis than xanthate polymers.     

5‐Hydroxymethyl‐2′‐Deoxyuridine Residues in the Thrombin Binding Aptamer: Investigating Anticoagulant Activity by Making a Tiny Chemical Modification

We report an investigation into analogues of the thrombin binding aptamer (TBA). Individual thymidines were replaced by the unusual residue 5‐hydroxymethyl‐2′‐deoxyuridine (hmU). This differs from the canonical thymidine by a hydroxyl group on the 5‐methyl group. NMR and CD data clearly indicate that all TBA derivatives retain the ability to fold into the “chair‐like” quadruplex structure. The presence of the hmU residue does not significantly affect the thermal stability of the modified aptamers compared to the parent, except for analogue H9 , which showed a marked increase in melting temperature. Although all TBA analogues showed decreased affinities to thrombin, H3 , H7 , and H9 proved to have improved anticoagulant activities. Our data open up the possibility to enhance TBA biological properties, simply by introducing small chemical modifications.     

Properties and Potential Antiproliferative Activity of Thrombin-Binding Aptamer (TBA) Derivatives with One or Two Additional G-Tetrads

In this paper, we study the biological properties of two TBA analogs containing one and two extra G-tetrads, namely TBAG3 and TBAG4, respectively, and two further derivatives in which one of the small loops at the bottom (TBAG41S) or the large loop at the top (TBAG4GS) of the TBAG4 structure has been completely modified by replacing all loop residues with abasic site mimics. The therapeutical development of the TBA was hindered by its low thermodynamic and nuclease stability, while its potential as an anticancer/antiproliferative molecule is also affected by the anticoagulant activity, being a side effect in this case. In order to obtain suitable TBA analogs and to explore the involvement of specific aptamer regions in biological activity, the antiproliferative capability against DU 145 and MDAMB 231 cancer cell lines (MTT), the anticoagulant properties (PT), the biological degradability (nuclease stability assay) and nucleolin (NCL) binding ability (SPR) of the above described TBA derivatives have been tested. Interestingly, none of the TBA analogs exhibits an anticoagulant activity, while all of them show antiproliferative properties to the same extent. Furthermore, TBAG4 displays extraordinary nuclease stability and promising antiproliferative properties against breast cancer cells binding NCL efficiently. These results expand the range of G4-structures targeting NCL and the possibility of developing novel anticancer and antiviral drugs.     

Chemical structure and compatibility of polyamide–chitin and chitosan blends

This work presents a comparative study of the compatibilization of four binary blends with slight differences in their chemical structures. The natural polymers chitin (QA) and chitosan (QN) are blended with polyamide 6 (PA6) and polyamide 66 (PA66). The results, obtained using differential scanning calorimetry, infrared spectroscopy, and light and scanning electron microscopy, gave the following compatibilization sequence: PA6/QN ≈ PA66/QN > PA6/QA > PA66/QA. This behavior could be related to the ability of QN to form hydrogen bonds and also to the capability of the packing of PA66. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 78: 850–857, 2000     

Leaders First,Countries After: Mediated Political Personalization in the International Arena

This study is the first comparative analysis of mediated political personalization in the international arena; its contribution to the research in the field is twofold: (a) through a longitudinal analysis, it shows that media coverage of foreign countries focuses increasingly on state leaders rather than on the countries per se; and (b) it accounts for variations in the level of mediated political personalization between pairs of countries: the greater the distance between a pair of countries, in terms of values, political interests, economic relations, and geographical distance, the more their news coverage of each other focuses on the foreign country's leader at the expense of other political aspects.