331 research outputs found
The Ribosome Cooperates with the Assembly Chaperone pICln to Initiate Formation of snRNPs
No full textSummaryThe formation of macromolecular complexes within the crowded environment of cells often requires aid from assembly chaperones. PRMT5 and SMN complexes mediate this task for the assembly of the common core of pre-mRNA processing small nuclear ribonucleoprotein particles (snRNPs). Core formation is initiated by the PRMT5-complex subunit pICln, which pre-arranges the core proteins into spatial positions occupied in the assembled snRNP. The SMN complex then accepts these pICln-bound proteins and unites them with small nuclear RNA (snRNA). Here, we have analyzed how newly synthesized snRNP proteins are channeled into the assembly pathway to evade mis-assembly. We show that they initially remain bound to the ribosome near the polypeptide exit tunnel and dissociate upon association with pICln. Coincident with its release activity, pICln ensures the formation of cognate heterooligomers and their chaperoned guidance into the assembly pathway. Our study identifies the ribosomal quality control hub as a site where chaperone-mediated assembly of macromolecular complexes can be initiated
Assembly of RNPs: help needed
No full textThis extract was created in the absence of an abstract: In 1968 the Nomura lab in Madison made an astonishing observation: when ribosomal proteins were mixed under appropriate conditions with 16S rRNA, fully functional ribosomal 30S subunits were formed. Along with the subsequent total reconstitution of 50S subunits, it was established that even entire ribosomes could be assembled in vitro from their constituents. Neither energy in the form of nucleoside triphosphates, nor other “helping factors,” were required for this reaction. It was thus concluded that the structural information required for the formation of even complex macromolecules lies within its individual components itself and hence allows “self assembly.” This notion was soon supported by many other labs, which managed to assemble macromolecular RNPs in spontaneous reactions, including the signal recognition particle (SRP), spliceosomal subcomplexes (U snRNPs) and nucleolar RNPs (snoRNPs), to name just a few. These findings were in full accordance with the observations of the Anfinsen lab in the 1950s
Introduction
No full textSchedewie F, Grüner F, Utz R. Introduction. In: Fischer von Weikersthal F, Grüner F, Hohler S, Schedewie F, Utz R, eds. The Russian Revolution of 1905 in Transcultural Perspective. Identities, Peripheries, and the Flow of Ideas. Allan K. Wildman Group historical series. Vol 6. Bloomington: Slavica Publishers; 2013: 1-9
The Russian Revolution of 1905 in Transcultural Perspective. Identities, Peripheries, and the Flow of Ideas
No full textFischer von Weikersthal F, Grüner F, Hohler S, Schedewie F, Utz R, eds. The Russian Revolution of 1905 in Transcultural Perspective. Identities, Peripheries, and the Flow of Ideas. Allan K. Wildman Group historical series. Vol 6. Bloomington: Slavica Publishers; 2013
The Russian Revolution of 1905 at the Periphery: The Case of the Manchurian City of Harbin
No full textGrüner F. The Russian Revolution of 1905 at the Periphery: The Case of the Manchurian City of Harbin. In: Fischer von Weikersthal F, Grüner F, Hohler S, Schedewie F, Utz R, eds. The Russian Revolution of 1905 in Transcultural Perspective. Identities, Peripheries, and the Flow of Ideas. Allan K. Wildman Group historical series. Vol 6. Bloomington: Slavica Publishers; 2013: 175-196
Molecular cloning and targeted deletion of PEP2 which encodes a novel aspartic proteinase from Aspergillus fumigatus
No full textAn aspartic proteinase PEP2 [EC 3.4.23.25] was purified from a cell wall fraction of Aspergillus fumigatus. The enzyme, which showed a broad range of activity from pH 2.0 to 7.0 and migrated as a single band of 39 kDa in SDS-PAGE, was not detected in the culture supernatant. A specific gene probe was designed on the basis of the N-terminal sequence of the native protein, and the PEP2 genomic and cDNA were isolated from corresponding libraries. The deduced amino acid sequence of PEP2 consists of 398 amino acids. A signal sequence of 18 amino acids and a proregion of another 52 amino acids were identified. The mature protein consists of 328 amino acids which include the two DTG-motifs of the active site common to almost all pepsin-like enzymes; PEP2 showed a 64% identity with the vacuolar proteinase A (PrA), of Saccharomyces cerevisiae, and an 88% identity with PEPE, an aspartic proteinase of Aspergillus niger. Recombinant PEP2 was overexpressed in Pichia pastoris and the active enzyme was secreted into the culture supernatant. Targeted deletion of PEP2 did not affect vegetative growth or cell and colony morphology. Identification of proteinases, such as PEP2, which are apparently associated with the Aspergillus cell wall raises new interest in these molecules with respect to their possible function in the pathogenesis of invasive aspergillosis
Molecular cloning and targeted deletion of PEP2 which encodes a novel aspartic proteinase from Aspergillus fumigatus
No full textAn aspartic proteinase PEP2 [EC 3.4.23.25] was purified from a cell wall fraction of Aspergillus fumigatus. The enzyme, which showed a broad range of activity from pH 2.0 to 7.0 and migrated as a single band of 39 kDa in SDS-PAGE, was not detected in the culture supernatant. A specific gene probe was designed on the basis of the N-terminal sequence of the native protein, and the PEP2 genomic and cDNA were isolated from corresponding libraries. The deduced amino acid sequence of PEP2 consists of 398 amino acids. A signal sequence of 18 amino acids and a proregion of another 52 amino acids were identified. The mature protein consists of 328 amino acids which include the two DTG-motifs of the active site common to almost all pepsin-like enzymes; PEP2 showed a 64% identity with the vacuolar proteinase A (PrA), of Saccharomyces cerevisiae, and an 88% identity with PEPE, an aspartic proteinase of Aspergillus niger. Recombinant PEP2 was overexpressed in Pichia pastoris and the active enzyme was secreted into the culture supernatant. Targeted deletion of PEP2 did not affect vegetative growth or cell and colony morphology. Identification of proteinases, such as PEP2, which are apparently associated with the Aspergillus cell wall raises new interest in these molecules with respect to their possible function in the pathogenesis of invasive aspergillosis
Bruce Chatwin: Utz, su novela sobre el mundo del arte
No full textBritish writer Bruce Chatwin (1940-1989) makes a compulsive collector the central axis of his novel Utz. Through that character, he reflects on the art world remembering his work at the auction house Sotheby´s. His experience there gave him the knowledge and look that would condition his life. He learnt to analyze a work of art, to describe it in just a few words and to value it in the market. All of it can be noticed in his writing style, in which he pursues the kind of the precision observed in cataloguers. He developed his own technique. Besides, he acquired a strong contact network around the world. He put his eyes on peculiar personalities that led to the creation of Utz, personification of all those art dealers and collectors that he met during his time as the head of the firm's Impressionist and Modern Art department. Moreover, his work at Sotheby´s awakened his eternal dilemma between possession, search for immortality and creation. This article examines how the author treats the above topics in his novel.El escritor británico Bruce Chatwin (1940-1989) convierte a un coleccionista compulsivo en eje central de su novela Utz. A través de su protagonista, reflexiona acerca del mundo del arte, rememorando su pasado cuando trabajaba en la casa de subastas Sotheby´s. Su experiencia allí le proporcionó unos conocimientos y una mirada que determinarían su vida, aprendió a estudiar una obra de arte, a describirla en pocas palabras y valorar su precio en el mercado. Todo ello se percibe en su estilo narrativo, donde persigue la precisión de las descripciones típicas de los catalogadores. Desarrolló un estilo propio. Además, adquirió una red de contactos trascendental alrededor del mundo. Fijó su interés en personalidades peculiares que desembocaron en la creación de Utz, personificación de ese entramado de marchantes y coleccionistas que le facilitó su puesto de director en el departamento de impresionistas y arte moderno. Al mismo tiempo, su época en Sotheby´s despertó en él la eterna disyuntiva entre la posesión, la búsqueda de la inmortalidad y la creación artística. El presente artículo examina cómo el autor aborda todos estos temas en la novela
Studies on the posttranscriptional regulation of the CDK inhibitors p27Kip1 and p21Cip1
No full textDie intrazelluläre Konzentration des CDK-Inhibitors p27Kip1 spielt eine kritische Rolle bei der Entscheidung zwischen Proliferation und Wachstumsarrest in der G1-Phase des Zellzyklus.
Zur Analyse der translationalen Regulation von p27 wurde eine 2516 nt lange humane p27-cDNA kloniert. Transiente Transfektionen von HeLa-Zellen zeigten, daß die 575 nt lange 5'UTR an ihrem 5'-Ende ein Element von etwa 100 nt enthält, das die Translation eines Reportergens in G1-Zellen im Vergleich zu Zellen der S-Phase um den Faktor 2 bis 2,5 verstärkt. Dieses Element gewährleistet demnach eine translationale Regulation, wie sie die endogene p27-mRNA am Übergang von der G1- zur S-Phase erfährt. Es wurde daher als 'cell cycle responsive element' (CCRE) bezeichnet. Stromabwärts des CCRE wurde eine 'internal ribosomal entry site' (IRES) identifiziert, die es Ribosomen ermöglicht, die Translation von p27 unabhängig von der 5'-Cap-Struktur zu initiieren und vermutlich zur Aufrechterhaltung der p27-Expression in quieszenten Zellen beiträgt.
Es wurde weiter gezeigt, daß RNA-Bindeproteine PTB (polypyrimidine tract binding protein) und Nucleolin in vitro spezifisch und unter stringenten Bedingungen an das CCRE binden. Die Überexpression von PTB reprimiert spezifisch die Reportergenexpression von Transkripten mit p27-5'UTR. Diese Beobachtungen stützen die Hypothese, daß PTB in vivo an der translationalen Regulation von p27 beteiligt sein könnte.
Der CDK-Inhibitor p21Cip1 ist mit p27Kip1 nahe verwandt und wird unter anderem durch den Tumorsuppressor p53 als Reaktion auf DNA-Schäden induziert.
In dieser Arbeit wurde gezeigt, daß p21 durch Cyclin A/CDK2 in vitro zu drei Phosphoisoformen unterschiedlicher elektrophoretischer Beweglichkeit umgesetzt werden kann. Es konnte weiter gezeigt werden, daß diese Phosphoisoformen als Monomere Cyclin A/CDK2 inhibieren und sich darin von unmodifiziertem p21 nicht unterscheiden.The intracellular level of CDK inhibitor p27Kip1 plays an important role in controlling the decision between proliferation and growth arrest during G1 phase. In addition to altered protein stability, translational control of p27 synthesis contributes to this regulation.
In order to investigate the mechanisms of regulated translation of p27, an extended human p27 cDNA of 2516 nt was isolated. Using transient transfections of HeLa cells, an element of about 100 nt was identified at the 5' end of the 5'UTR, that causes a 2 to 2.5 fold increase of reporter mRNA translation in G1 cells when compared to S phase cells. This regulation is similar to the translational control of endogenous p27 mRNA at the G1/S transition. Therefore the 100 nt 5'UTR region was designated the cell cycle responsive element (CCRE). An internal ribosomal entry site (IRES) was identified downstream of the CCRE allowing ribosomes to initiate translation independent of the 5' cap structure. The IRES is likely to contribute to the maintenance of p27 expression in quiescent cells.
Furthermore it was shown, that the RNA binding proteins PTB (polypyrimidine tract binding protein) und Nucleolin specifically bind to the CCRE in vitro under stringent conditions. Overexpression of PTB specifically represses reporter gene expression from transcripts with p27 5'UTR. These observations suggest that PTB participates in p27 translational control in vivo.
The CDK inhibitor p21Cip1 is a close relative of p27Kip1 and is induced for example by the tumorsuppressor p53 in response to DNA damage.
Here it was shown, that p21 is phosphorylated in vitro by its target kinase Cyclin A/CDK2. Three phosphoisoforms were observed with different electrophoretic mobilities. The isoforms with the lowest electrophoretic mobilities are likely to contain two or three phosphate groups respectively. It was demonstrated that these phosphoisoforms inhibit Cyclin A/CDK2 in a monomeric form as had been shown for unmodified p21
Molecular characterization and influence on fungal development of ALP2, a novel serine proteinase from Aspergillus fumigatus
No full textA novel subtilisin-related serine proteinase (ALP2) [EC 3.4.21.48] with a broad range of activity between pH 4.5 and 11.0 was released from a cell wall fraction of Aspergillus fumigatus by an alkaline pH shift. The enzyme which was not detected in the culture supernatant was partially purified by phenylbutylamine agarose chromatography. The N-terminal sequence revealed that ALP2 is the same protein identified as the major allergen of A. fumigatus in patients suffering from extrinsic bronchial asthma (Shen et al. 1999, Int. Arch. Allergy Immunol. 119, 259-264). Based on this N-terminal sequence and on a conserved region of fungal subtilisins, a specific PCR probe was generated and the ALP2 genomic and cDNA were isolated from corresponding phage libraries. ALP2 shares a 49 % identity with the vacuolar proteinase B (PrB) of Saccharomyces cerevisiae. In addition there is a 78 % identity with PEPC, a serine proteinase which has been described in Aspergillus niger. Targeted disruption of the ALP2-encoding gene resulted in a slightly decreased speed of vegetative growth and in a more than 80% reduction of sporulation in the alp2-negative mutants, correlated with an approximately 50 % reduction of the median diameter of conidiophore vesicles. The requirement of ALP2 for regular sporulation, in addition to its role in allergic asthma, raises further interest in cellular proteinases in respect to morphogenesis and pathogenesis in A. fumigatus
- …
