Projecte de condicionament del camí rural de l’Aiguabarreig al terme municipal de Massalcoreig (Lleida)

El tema d’aquest projecte és el condicionament del camí rural, situat al llarg del riu Cinca, al terme municipal de Massalcoreig (Lleida). Aquest projecte està format per quatre documents, classificats en dos volums. Al primer volum trobem el document número 1, format per la memòria amb els seus annexes on es defineixen les característiques bàsiques a tenir en compte per a realitzar el condicionament del camí, així com el desenvolupament de les activitats. Al segon volum, trobem la resta de documents. En primer lloc, trobem el segon document, els plànols necessaris per a dur a terme l’obra. A continuació, el tercer document és el plec de condicions en el que s’estipulen les normes que s’han de tenir en compte durant l’execució del projecte. Finalment, el quart document, és el pressupost, on hi ha el cost de l’obra

Characterisierung vom proteinen mit ENTH domänen und ihre Interaktion mit SNARE proteinen in S.cerevisiae

SNAREs spielen eine wichtige Rolle bei der Membranfusion als fusiogene Proteine. Folglich muß die Lokalisation und Funktion von SNAREs streng gesteuert werden. Regulatorische Proteine binden an N-terminale Domänen einiger SNAREs. Vti1b ist ein Säugetier- SNARE, das in späten endosomalen Fusionen eine Rolle spielt. Um die Rolle des N-Terminus von vti1b zu charakterisieren, wurde eine yeast two hybrid Analyse durchgeführt. Der N-Terminus von vti1b wirkte spezifisch auf die ENTH-Domänen von Enthoprotin/CLINT/EpsinR ein. Diese Abhängigkeit wurde durch in-vitro Bindungsstudien bestätigt. Die komplexe Anordnung zwischen einem SNARE und einer ENTH-Domäne ist zwischen Säugetieren und Hefe konserviert. Hefe Vti1p wechsel auf die ENTH- Domäne von, Ent3p ein. ENTH-Proteine sind an der Bildung der clathrin coated vesicles beteiligt. Beide, EpsinR und Ent3p binden Adapterproteine am TGN. Vti1p wird für mehrere Transportschritte im endosomalen System benötigt. Genetische Interaktionen zwischen VTI1 und ENT3 wurden untersucht. Künstliche Defekte lassen vermuten, daß Vti1p und Ent3p beim Transport vom TGN zum prevacuolären Endosom kooperieren. Zusätzlich wirkt VTI1 genetisch auf ENT5 ein. Ent3p und Ent5p haben teilweise reduntante Funktionen. Weitere Analysen zeigten, daß Ent3p auch auf den N-Terminus anderer SNAREs einwirkt

PIWI-like protein, HIWI2 is aberrantly expressed in retinoblastoma cells and affects cell-cycle potentially through OTX2

Abstract Retinoblastoma (RB), a childhood cancer, is caused by biallelic mutation of the RB1 gene, but its development is not clearly understood. Furthermore, the presence of a cancer stem cell subpopulation in RB might impact its treatment. PIWI protein, known for its role in stem cell self-renewal, is aberrantly expressed in cancers. We examined the role of the PIWI-like protein HIWI2 in RB and its effect on the stem cell markers in cells of the RB line, Y79. The expression of HIWI2 is significantly increased in Y79 compared with its level in HeLa and ARPE19 cells. The stem cell markers Oct-3/4, Nanog and Sox-2 were not altered upon HIWI2 knockdown in Y79 cells. Interestingly, OTX2 was significantly downregulated in the absence of HIWI2. Otx2 transcripts also decreased in HIWI2-silenced Y79 and ARPE19 cells. Moreover, silencing HIWI2 in Y79 accumulated the cells at G2–M phase and reduced the levels of proliferating cell nuclear antigen (PCNA) and the tumor suppressor, p16. Our results demonstrate that HIWI2 is aberrantly expressed in Y79 cells and silencing of HIWI2 downregulates OTX2, suggesting that HIWI2 might play a role in the progression of RB

Ent3p-a yeast ENTH domain protein acts in forward and retrograde endosomal transport with cargo adaptor functions for three SNAREs

Zimmermann J, Chidambaram S, Fischer von Mollard G. Ent3p-a yeast ENTH domain protein acts in forward and retrograde endosomal transport with cargo adaptor functions for three SNAREs. In: European Journal of Cell Biology. EUROPEAN JOURNAL OF CELL BIOLOGY. Vol 89. ELSEVIER GMBH, URBAN & FISCHER VERLAG; 2010: 25-25

ENTH domain proteins are cargo adaptors for multiple SNARE proteins at the TGN endosome

Chidambaram S, Zimmermann J, Fischer von Mollard G. ENTH domain proteins are cargo adaptors for multiple SNARE proteins at the TGN endosome. JOURNAL OF CELL SCIENCE. 2008;121(3):329-338

Dissecting Ent3p: The ENTH domain binds different SNAREs via distinct amino acid residues while the C-terminus is sufficient for retrograde transport from endosomes.

Zimmermann J, Chidambaram S, Fischer von Mollard G. Dissecting Ent3p: The ENTH domain binds different SNAREs via distinct amino acid residues while the C-terminus is sufficient for retrograde transport from endosomes. Biochemical Journal. 2010;431(1):123-134

Defective CPY sorting in <i>vti1-3</i> cells.

<p>CPY (carboxypeptidase Y) sorting was analyzed by pulse-chase labeling and CPY immunoprecipitations from cellular extracts (I, intracellular) and medium (E, extracellular). The numbers are percentage of vacuolar mCPY in the experiment shown. (A) The N-terminal double mutant Vti1p Q29R W79R secreted Golgi-modified p2CPY at 37°C but not at 24°C. The corresponding single mutants Q29R and W79R displayed no sorting phenotype at 37°C. Strains used: FvMY6 pBK120 (<i>CEN</i> Q29R W79R), FvMY6 pBK123 (<i>CEN</i> Q29R), FvMY6 pBK128 (<i>CEN</i> W79R). (B) Genomic integration of the <i>vti1-3</i> mutant resulted in an aggravated CPY missorting. Some Golgi-modified p2CPY was secreted at 24°C. At 37°C, ER-modified p1CPY and Golgi-modified p2CPY accumulated intracellularly, Golgi-modified p2CPY was secreted and very little vacuolar mCPY was detected. Overproduction of <i>vti1-3p</i> reduced the CPY sorting defect at 24°C. Strains used: SCY14 (<i>vti1-3</i>), SCY14 pSC14 (<i>vti1-3</i>+2µ <i>vti1-3</i>). (C) Vti1p or <i>vti1-3p</i> protein levels in different strains at 24°C or after 2 h at 37°C detected by western blotting using antiserum against Vti1p.</p

Levels and stability of late endosomal SNAREs.

<p>(A) Protein level of Pep12p was drastically increased in <i>vti1-3</i> cells after 2 h at 37°C. Protein extracts were analyzed by SDS-PAGE and immunoblotting. (B) Western blots were quantified and normalized to the amount in wild type cells at 24°C. Three independent experiments were quantified. mean ±standard deviation, **P<0.01, unpaired Student’s t test (C) Pep12p was more stable in <i>vti1-3</i> cells at 37°C. Cells were labeled with [³⁵S]methionine and [³⁵S]cysteine for 25 min and chased for 10 min, 3 h or 5 h. Pep12p was immunoprecipitated from the cellular extracts and the percentage of Pep12p remaining after 3 and 5 hours was calculated. Strains used: SEY6210 (WT), SCY14 (<i>vti1-3</i>). (D) GFP-Pep12p accumulated in intracelullar spots that did not colocalize with DsRed-FYVE in <i>vti1-3</i> cells after 2 h at 37°C. Strains used: SEY6210 pTK16 (WT GFP-Pep12p), SCY14 pTK16 (<i>vti1-3 </i>GFP-Pep12p).</p

Plasmids used in this study.

<p>Plasmids used in this study.</p