Mouse stefins A1 and A2 (Stfa1 and Stfa2) differentiate between papain-like endo- and exopeptidases

AbstractStefin A (Stfa) acts as a competitive inhibitor of intracellular papain-like cysteine proteases which play important roles in normal cellular functions such as general protein turnover, antigen processing and ovarian follicular growth and maturation. In the mouse there are at least three different variants of Stfa (Stfa1, Stfa2 and Stfa3). Recent genetic studies identified structural polymorphisms in Stfa1 and Stfa2 as candidates for Aod1b, a locus controlling susceptibility to day three thymectomy (D3Tx)-induced autoimmune ovarian disease (AOD). To evaluate the functional significance of these polymorphisms, recombinant allelic proteins were expressed in Escherichia coli, purified and characterized. The polymorphisms do not markedly alter the folding characteristics of the two proteins. Stfa1 and Stfa2 both act as fast and tight binding inhibitors of endopeptidases papain and cathepsins L and S, however their interaction with exopeptidases cathepsins B, C and H was several orders of magnitude weaker compared to human, porcine and bovine Stfa. Notwithstanding, the Ki values for the interactions of Stfa1-b from AOD resistant C57BL/6J mice was 10-fold higher than that of the Stfa1-a allele from susceptible A/J mice for papain, cathepsins B, C and H but not L and S. In contrast, the inhibitory activities of Stfa2-a and Stfa2-b were found to be roughly equivalent for all targets peptidases

Ružička days : International conference 16th Ružička Days “Today Science – Tomorrow Industry” : Proceedings

Proceedings contains articles presented at Conference divided into sections: open lecture (1), chemical analysis and synthesis (3), chemical and biochemical engineering (8), food technology and biotechnology (8), medical chemistry and pharmacy (3), environmental protection (11) and meeting of young chemists (2)

A system for acquisition of AV material and synchronization with other content

Z novimi tehnologijami so se pojavili izobraževalni sistemi, ki nam omogočajo izobraževanje na daljavo, to pa je neodvisno od časa in prostora ter vedno bolj razširjeno. Imenujemo ga tudi e-izobraževanje. V njem se pojavljajo kratki filmi, predavanja, pisana beseda, predstavitve s prosojnicami in zvočni posnetki. Da bi bilo takšno izobraževanje usmerjeno k učencu oz. učenju in ne zgolj k uporabi tehnologije kot take, poskuša zajeti različne stile učenja oz. vse tri zaznavne modalitete: avditivno (slušno), vizualno (vidno) in kinestetično (gibalno). Vse učne tipe hkrati lahko odlično zajamemo z video predavanji v kombinaciji s powerpoint prosojnicami. Za pripravo takega materiala je običajno potrebna višjecenovna tehnologija in dodaten kader (snemalec). Ker pa na trgu vlada močna konkurenca, smo želeli predavatelju omogočiti zajem videa brez snemalca in s tem znižati stroške priprave izobraževalnega materiala ter posledično cene storitve izobraževanja. To smo storili z razvojem programske rešitve za avtomatiziran nizkocenovni zajem videa in sinhronizacijo le-tega s ppt prosojnicami. V delu je predstavljena implementacija te rešitve v obliki vtičnika za e-izobraževalni sistem ECHO, ki ga je razvil Laboratorij za telekomunikacije Fakultete za elektrotehniko v Ljubljani.New technologies enabled education systems which allow distance learning online, also called e-learning. This kind of learning is not time- or space-related and is more and more widespread. It features short videos, lectures, written notes, powerpoint (ppt) presentations and audio recordings. To be student- or study-focused, instead of technology-focused, e-learning tries to involve main styles of learning and perception styles: auditory (hearing), visual (sight) and kinesthetic (movement). All three learning types together are excellently covered with video lectures in combination with ppt slides. Preparation of such material usually requires expensive technology and additional personnel (cameraman). Because of the high competition on the market, we wanted to enable lector to record video without cameraman and thus lower the cost of learning material preparation, ultimately lowering the costs of education. We did this by developing software solution for automatic low-cost video recording and synchronization with ppt presentation. This thesis describes implementation of the software solution in form of plug-in for e-learning system ECHO, which is developed by Laboratory for Telecommunications at the Faculty of Electrical Engineering, University of Ljubljana

Etude pilote (faisabilité de la pose écho-guidée du cathéter paravertébral dans la prise en charge analgésique de thoracotomie)

La chirurgie thoracique est pourvoyeuse de douleur post opératoire sévère. L'analgésie paravertébrale occupe une place importante dans cette indication. Plusieurs techniques de cathétérisation de l'espace paravertébral ont été décrites. Parmi celles-ci l'utilisation conjointe de l'échographie semble intéressante. L'objectif de notre étude était d'évaluer la faisabilité de l'insertion du cathéter en paravertébral sous contrôle échographique en période pré opératoire. Cohorte prospective observationnelle. Le critère principal était le taux d'échec global (de pose ou d'efficacité). La technique échoguidée évaluée, "in plane" avec ponction de latéral à médial correspondait à celle proposée par Shibata. La procédure fut volontairement limitée à 2 opérateurs. Tous les délais de la procédure échoguidée juqu'au contrôle des dermatomes anesthésiés étaient relevés. Un contrôle radiographique du cathéter après injection de produit de contraste avait lieu en post opératoire immédiat. Un dosage plasmatique du taux de ropivacaïne était réalisé à j0, j1 et j2. Une surveillance analgésique, hémodynamique et respiratoire était poursuivie pendant 48 heures. Dans l'Analyse intermédiaire après 26 patients inclus, le taux d'échec global était de 31%. 2 poses n'ont pas eu lieu pour défaut de visualisation échographique des structures concernant des patients d'IMC >30.2 cathéters ont été retrouvés en position intra pleurale ainsi qu'un autre en position intramusculaire. 3 patients ont nécessité une analgésie de recours conséquence de l'inefficacité du bloc paravertébral. 1 cas d'anesthésie intercostale a également été observé. La médiane du délai de procédure échoguidée était de 56 minutes. Une période d'apprentissage prolongée de la technique a perturbé les premières procédures. La plèvre était toujours visualisée, 5 cas de non visualisation du ligament costo-transverse ont été relevés au cours des ponctions. Le contrôle radiographique postopératoire mettait en évidence une ponction trop latérale. La pose échoguidée du cathéter paravertébral pour thoracotomie doit être reprécisée. Cette technique nécessite un apprentissage préalable difficile, du matériel performant et une organisation structurelle adaptée. Pour autant, la faisabilité de cette technique peut parfaitement s'intégrer en pratique courante en chirurgie thoracique.CLERMONT FD-BCIU-Santé (631132104) / SudocSudocFranceF

Thermostability of recombinant pernisine^co.

<p>Time courses of the residual activity of recombinant pernisine at the indicated temperatures, at pH 8.0.</p

Residual protease activities of recombinant pernisine in the presence of the reductants, denaturants and detergent.

<p>Residual protease activities of recombinant pernisine in the presence of the reductants, denaturants and detergent.</p

Residual protease activities of recombinant pernisine in the presence of the protease inhibitors.

<p>ND, no data.</p><p>Residual protease activities of recombinant pernisine in the presence of the protease inhibitors.</p

SDS-PAGE analysis and zymography of purified pernisine^co and pernisine^S355Aco.

<p>Representative gels of the purified pernisines, following electrophoresis on standard 12% SDS-PAGE (A) and on 12% SDS-PAGE with casein as substrate (B) for the zymography activity (4 h at 80°C). Staining was with Coomassie blue dye. Lanes 0, protein MW markers (indicated left); lanes 1, recombinant pernisine^co; lanes 2, recombinant pernisine^S355Aco; lanes 3 and 4, heat-activated pernisine^co and pernisine^S355Aco. Selected protein bands of pernisine^co that were analysed by MS/MS are marked as I and II, (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123288#pone.0123288.s003" target="_blank">S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123288#pone.0123288.s004" target="_blank">S4</a> Figs). *protein load of pernisine^{<b>S355Aco</b>} is three times higher than pernisine^co.</p

Dependence of the activity of recombinant pernisine on temperature and pH.

<p>Relative activity of recombinant pernisine according to temperature and pH (as corrected based on temperature change). Assays were carried out in triplicates, and means of the relative activity dependence are presented as a function of temperature and corrected pH. Colour legend on the left indicates the relative activities.</p

Experimental flowchart and pernisine expression and analysis.

<p>(A) Flowchart of the experimental procedures. (B, C) Gel-exchange chromatography of pernisine^co in pMCSG7 using BL21(DE3) <i>E</i>. <i>coli</i> cells (B), and the corresponding SDS-PAGE analysis (C). The red line represents the selected fractions. (D) Time expression analysis after induction (1, 2, 3, 4 h) of <i>pernisine</i>^<i>wt</i> and <i>pernisine</i>^<i>co</i> for total cell lysates. Proteins were transferred (dot blot) onto nitrocellulose membranes and His₆-tagged pernisine was detected with anti-His₅-tag antibodies. Quantification was done using the ImageJ software (right panel). (E) SDS-PAGE analysis of pernisine^co and pernisine^S355Aco for total cell lysates of BL21(DE3) <i>E</i>. <i>coli</i> containing the pMCSGx series of vectors. 1, 4, Pernisine^co/wt-pMCSG7; 2, 5, Pernisine^co/wt-pMCSG9; 3, 6, Pernisine^co/wt-pMCSG10. (F) Immunodetection of pernisine^wt and pernisine^co for cell lysates containing the pMCSGx series of vectors. Proteins were transferred onto nitrocellulose membranes and His₆-tagged pernisine was detected using anti-His₆-tag antibodies. (G, H) Azocasein assays of the purified pernisine^co, showing effects of CaCl₂ (G) and NaCl (H). Relative proteolytic activities of activated pernisine^co are shown according to the CaCl₂ concentrations, and to the NaCl concentrations in the presence (grey, dot-dash line) and absence (black line) of 1 mM CaCl₂. Non-activated pernisine^co in the absence of 1 mM CaCl₂ is also shown (black, dot line). Abbreviations: co-codon-optimised, wt-wild-type.</p