G-protein-coupled receptors have a major role in transmembrane signalling in most eukaryotes and many are important drug targets. Here we report the 2.7 A resolution crystal structure of a beta(1)-adrenergic receptor in complex with the high-affinity antagonist cyanopindolol. The modified turkey (Meleagris gallopavo) receptor was selected to be in its antagonist conformation and its thermostability improved by earlier limited mutagenesis. The ligand-binding pocket comprises 15 side chains from amino acid residues in 4 transmembrane alpha-helices and extracellular loop 2. This loop defines the entrance of the ligand-binding pocket and is stabilized by two disulphide bonds and a sodium ion. Binding of cyanopindolol to the beta(1)-adrenergic receptor and binding of carazolol to the beta(2)-adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the beta(2)-adrenergic receptor, directly interacts by means of a tyrosine with the highly conserved DRY motif at the end of helix 3 that is essential for receptor activation. 相似文献
Glutamate ionotropic alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (AMPARs) mediate most fast excitatory synaptic transmission in the central nervous system. The content and composition of AMPARs in postsynaptic membranes (which determine synaptic strength) are dependent on the regulated trafficking of AMPAR subunits in and out of the membranes. AMPAR trafficking is a key mechanism that drives nascent synapse development, and is the main determinant of both Hebbian and homeostatic plasticity in mature synapses. Hebbian plasticity seems to be the biological substrate of at least some forms of learning and memory; while homeostatic plasticity (also known as synaptic scaling) keeps neuronal circuits stable by maintaining changes within a physiological range. In this review, we examine recent findings that provide further understanding of the role of AMPAR trafficking in synapse maturation, Hebbian plasticity, and homeostatic plasticity. 相似文献
Cell growth and proliferation require coordinated ribosomal biogenesis and translation. Eukaryotic initiation factors (eIFs) control translation at the rate-limiting step of initiation. So far, only two eIFs connect extracellular stimuli to global translation rates: eIF4E acts in the eIF4F complex and regulates binding of capped messenger RNA to 40S subunits, downstream of growth factors, and eIF2 controls loading of the ternary complex on the 40S subunit and is inhibited on stress stimuli. No eIFs have been found to link extracellular stimuli to the activity of the large 60S ribosomal subunit. eIF6 binds 60S ribosomes precluding ribosome joining in vitro. However, studies in yeasts showed that eIF6 is required for ribosome biogenesis rather than translation. Here we show that mammalian eIF6 is required for efficient initiation of translation, in vivo. eIF6 null embryos are lethal at preimplantation. Heterozygous mice have 50% reduction of eIF6 levels in all tissues, and show reduced mass of hepatic and adipose tissues due to a lower number of cells and to impaired G1/S cell cycle progression. eIF6(+/-) cells retain sufficient nucleolar eIF6 and normal ribosome biogenesis. The liver of eIF6(+/-) mice displays an increase of 80S in polysomal profiles, indicating a defect in initiation of translation. Consistently, isolated hepatocytes have impaired insulin-stimulated translation. Heterozygous mouse embryonic fibroblasts recapitulate the organism phenotype and have normal ribosome biogenesis, reduced insulin-stimulated translation, and delayed G1/S phase progression. Furthermore, eIF6(+/-) cells are resistant to oncogene-induced transformation. Thus, eIF6 is the first eIF associated with the large 60S subunit that regulates translation in response to extracellular signals. 相似文献
RNA interference (RNAi) is an evolutionarily conserved defence mechanism whereby genes are specifically silenced through degradation of messenger RNAs; this process is mediated by homologous double-stranded (ds)RNA molecules. In invertebrates, long dsRNAs have been used for genome-wide screens and have provided insights into gene functions. Because long dsRNA triggers a nonspecific interferon response in many vertebrates, short interfering (si)RNA or short hairpin (sh)RNAs must be used for these organisms to ensure specific gene silencing. Here we report the generation of a genome-scale library of endoribonuclease-prepared short interfering (esi)RNAs from a sequence-verified complementary DNA collection representing 15,497 human genes. We used 5,305 esiRNAs from this library to screen for genes required for cell division in HeLa cells. Using a primary high-throughput cell viability screen followed by a secondary high content videomicroscopy assay, we identified 37 genes required for cell division. These include several splicing factors for which knockdown generates mitotic spindle defects. In addition, a putative nuclear-export terminator was found to speed up cell proliferation and mitotic progression after knockdown. Thus, our study uncovers new aspects of cell division and establishes esiRNA as a versatile approach for genomic RNAi screens in mammalian cells. 相似文献
The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine. 相似文献
Human carbonic anhydrase IX (hCA IX) is a tumour-associated enzyme present in a limited number of normal tissues, but overexpressed in several malignant human tumours. It is a transmembrane protein, where the extracellular region consists of a greatly investigated catalytic CA domain and a much less investigated proteoglycan-like (PG) domain. Considering its important role in tumour biology, here, we report for the first time the full characterization of the PG domain, providing insights into its structural and functional features. In particular, this domain has been produced at high yields in bacterial cells and characterized by means of biochemical, biophysical and molecular dynamics studies. Results show that it belongs to the family of intrinsically disordered proteins, being globally unfolded with only some local residual polyproline II secondary structure. The observed conformational flexibility may have several important roles in tumour progression, facilitating interactions of hCA IX with partner proteins assisting tumour spreading and progression. 相似文献
The rational ruled surface is a typical modeling surface in computer aided geometric design. A rational ruled surface may have different representations with respective advantages and disadvantages. In this paper, the authors revisit the representations of ruled surfaces including the parametric form, algebraic form, homogenous form and Plücker form. Moreover, the transformations between these representations are proposed such as parametrization for an algebraic form, implicitization for a parametric form, proper reparametrization of an improper one and standardized reparametrization for a general parametrization. Based on these transformation algorithms, one can give a complete interchange graph for the different representations of a rational ruled surface. For rational surfaces given in algebraic form or parametric form not in the standard form of ruled surfaces, the characterization methods are recalled to identify the ruled surfaces from them.
Summary Previtellogenic oocytes ofOdontophrynus americanus display hundreds of chromatin circles. Electron microscopy of spread preparations of isolated nuclei shows that the circles originate from the chromatin. The circles change their morphology and form new copies. The length of the DNA packed in the nucleosomal circles is about 2.5–3.5 m or multiples of this value. Assuming that histones need not be removed from chromatin before DNA replication3 we suggest that the circles might belong to the process of rDNA amplification.This work was supported by grants from the Brazilian CNPq, FAPESP and FEDIB.We thank Dr A. Brunner Jr for the use of the electron microscope and Dr N. Leon for his valuable help. 相似文献