RESUMEN
This paper presents a new approach to monitoring ultrasonic systems using structurally integrated piezoceramics. These are integrated into the sonotrode at different points and with different orientations. The procedure for integrating the piezoceramics into the sonotrode and their performance is experimentally investigated. We examine whether the measured signal can be used to determine the optimal operating frequency of the ultrasonic system, if integrating several piezoceramics enables discernment of the current vibration shape, and if the piezoceramics can withstand the high strains caused by the vibrations in a frequency range of approximately 20-25 kHz. The signals from the piezoceramic sensors are compared to the real-time displacement at different points of the sonotrode using a 3D laser scanning vibrometer. To evaluate the performance of the sensors, different kinds of excitation of the ultrasonic system are chosen.
RESUMEN
The tumor suppressor p53 has been implicated in the regulation of ribosome biogenesis based on its inhibitory effect on RNA polymerase I (pol I)-dependent transcription. Consistent with this, p53 has been described in nucleoli, albeit under specific experimental conditions. Since data on the intranucleolar localization of p53 are controversial, we have analyzed in detail its subnucleolar distribution. Our results show that p53 does not localize to one of the well-known structural components of the nucleolus involved in ribosome biogenesis, but rather occupies distinct intranucleolar regions that constitute nucleolar cavities. When cells were treated with the proteasome inhibitor MG132, the size and frequency of p53-containing nucleolar cavities increased, and the protein partially colocalized with inactivated proteasomes. Importantly, p53 did not colocalize with pol I at the transcription sites in fibrillar centers (FCs) as has previously been reported. The observed intranucleolar distribution and accumulation of p53 raises the question of how the protein influences rDNA transcription in vivo.
Asunto(s)
Compartimento Celular , Nucléolo Celular/metabolismo , Ribosomas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Compartimento Celular/efectos de los fármacos , Línea Celular Tumoral , Nucléolo Celular/efectos de los fármacos , Nucléolo Celular/ultraestructura , Supervivencia Celular/efectos de los fármacos , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Leupeptinas/farmacología , Inhibidores de Proteasoma , Transporte de Proteínas/efectos de los fármacos , ARN Polimerasa I/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Ribosomas/efectos de los fármacos , Ribosomas/ultraestructuraRESUMEN
Considerable efforts are being undertaken to elucidate the processes of ribosome biogenesis. Although various preribosomal RNP complexes have been isolated and molecularly characterized, the order of ribosomal protein (r-protein) addition to the emerging ribosome subunits is largely unknown. Furthermore, the correlation between the ribosome assembly pathway and the structural organization of the dedicated ribosome factory, the nucleolus, is not well established. We have analyzed the nucleolar localization of several early binding r-proteins in human cells, applying various methods, including live-cell imaging and electron microscopy. We have located all examined r-proteins (S4, S6, S7, S9, S14, and L4) in the granular component (GC), which is the nucleolar region where later pre-ribosomal RNA (rRNA) processing steps take place. These results imply that early binding r-proteins do not assemble with nascent pre-rRNA transcripts in the dense fibrillar component (DFC), as is generally believed, and provide a link between r-protein assembly and the emergence of distinct granules at the DFC-GC interface.
Asunto(s)
Nucléolo Celular/metabolismo , Proteínas Ribosómicas/biosíntesis , Ribosomas/metabolismo , Línea Celular Tumoral , Nucléolo Celular/ultraestructura , Humanos , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/ultraestructura , Ribosomas/ultraestructuraRESUMEN
The cellular response to hypoxia is crucial to organismal survival, and hypoxia-inducible factors (HIF) are the key mediators of this response. HIF-signaling is central to many human diseases and mediates longevity in the nematode. Despite the rapidly increasing knowledge on RNA-binding proteins (RBPs), little is known about their contribution to hypoxia-induced cellular adaptation. We used RNA interactome capture (RIC) in wild-type Caenorhabditis elegans and vhl-1 loss-of-function mutants to fill this gap. This approach identifies more than 1,300 nematode RBPs, 270 of which can be considered novel RBPs. Interestingly, loss of vhl-1 modulates the RBPome. This difference is not primarily explained by protein abundance suggesting differential RNA-binding. Taken together, our study provides a global view on the nematode RBPome and proteome as well as their modulation by HIF-signaling. The resulting RBP atlas is also provided as an interactive online data mining tool (http://shiny.cecad.uni-koeln.de:3838/celegans_rbpome).
RESUMEN
A study of the influence of the local environment on the light-induced luminescence enhancement of CdSe/ZnS quantum dots (QD) embedded in silica colloids that are dispersed in various solvents is presented. The photoluminescence of the embedded QD is enhanced up to a factor of ten upon photoactivation by ultraviolet or visible light. This enhancement is strongly dependent on the local environment. The thickness-dependent permeability of the silica shell covering the QD controls the influence of the solvent on the QD. If foreign ions are present the activation state is stabilized after termination of the activation, whereas in their absence the process is partially reversible. A new qualitative model for the photoactivation of QD in various environments is developed. It comprises light-induced passivation and subsequent oxidation processes. The embedded QD also retain their fluorescence quantum yield inside living cells. Moreover, they can be activated for many hours in living cells by laser radiation in the visible regime.
Asunto(s)
Compuestos de Cadmio/química , Puntos Cuánticos , Compuestos de Selenio/química , Dióxido de Silicio/química , Sulfuros/química , Compuestos de Zinc/química , Coloides , Microscopía Electrónica de Transmisión , FotoquímicaRESUMEN
Phosphoinositides comprise a group of essential phospholipids that control a variety of cellular functions. In the case of the human malaria parasite Plasmodium falciparum, phosphoinositides have been shown to trigger exflagellation and to affect haemoglobin endocytosis and maturation of the parasite's digestive vacuole. A central enzyme in the formation of phosphoinositides is the phosphatidylinositol 4-kinase that catalyzes the production of phosphatidylinositol 4-phosphate from phosphatidylinositol. Here we have identified and characterized a phosphatidylinositol 4-kinase from P. falciparum. Our data show that the corresponding P. falciparum gene, termed PFE0485w, can functionally complement a yeast temperature-sensitive pik1 mutation. Our data add to the concept that P. falciparum maintains its own phospholipids biosynthesis pathway.
Asunto(s)
1-Fosfatidilinositol 4-Quinasa/deficiencia , 1-Fosfatidilinositol 4-Quinasa/genética , Plasmodium falciparum/enzimología , Proteínas Protozoarias/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimología , Prueba de Complementación Genética , Calor , Mutación Missense , Plasmodium falciparum/genética , Saccharomyces cerevisiae/genéticaRESUMEN
In the ciliate Paramecium tetraurelia, 3',5'-cyclic GMP (cGMP) is one of the second messengers involved in several signal transduction pathways. The enzymes for its production and degradation are well established for these cells, whereas less is known about the potential effector proteins. On the basis of a current Paramecium genome project, we have identified a multigene family with at least 35 members, all of which encode cGMP-dependent protein kinases (PKGs). They can be classified into 16 subfamilies with several members each. Two of the genes, PKG1-1 and PKG2-1, were analyzed in more detail after molecular cloning. They encode monomeric enzymes of 770 and 819 amino acids, respectively, whose overall domain organization resembles that in higher eukaryotes. The enzymes contain a regulatory domain of two tandem cyclic nucleotide-binding sites flanked by an amino-terminal region for intracellular localization and a catalytic domain with highly conserved regions for ATP binding and catalysis. However, some Paramecium PKGs show a different structure. In Western blots, PKGs are detected both as cytosolic and as structure-bound forms. Immunofluorescence labeling shows enrichment in the cell cortex, notably around the dense-core secretory vesicles (trichocysts), as well as in cilia. Immunogold electron microscopy analysis reveals consistent labeling of ciliary membranes, of the membrane complex composed of cell membrane and cortical Ca2+ stores, and of regions adjacent to ciliary basal bodies, trichocysts, and trafficking vesicles. Since PKGs (re)phosphorylate the exocytosis-sensitive phosphoprotein pp63/pf upon stimulation, the role of PKGs during stimulated exocytosis is discussed, in addition to a role in ciliary beat regulation.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/genética , Familia de Multigenes/genética , Paramecium/enzimología , Paramecium/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Clonación Molecular , Secuencia de Consenso , Proteínas Quinasas Dependientes de GMP Cíclico/química , Exones/genética , Intrones/genética , Datos de Secuencia Molecular , Paramecium/citología , Paramecium/ultraestructura , Fosforilación , Filogenia , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Protozoarias/genética , Proteínas Recombinantes , Especificidad por SustratoRESUMEN
Nuclear actin and myosin 1 (NM1) are key regulators of gene transcription. Here, we show by biochemical fractionation of nuclear extracts, protein-protein interaction studies and chromatin immunoprecipitation assays that NM1 is part of a multiprotein complex that contains WICH, a chromatin remodelling complex containing WSTF (Williams syndrome transcription factor) and SNF2h. NM1, WSTF and SNF2h were found to be associated with RNA polymerase I (Pol I) and ribosomal RNA genes (rDNA). RNA interference-mediated knockdown of NM1 and WSTF reduced pre-rRNA synthesis in vivo, and antibodies to WSTF inhibited Pol I transcription on pre-assembled chromatin templates but not on naked DNA. The results indicate that NM1 cooperates with WICH to facilitate transcription on chromatin.