A possible structure of the rabbit reticulocyte ribosome. An exercise in model building.
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ABSTRACT:
SUBMITTER: Cox RA
PROVIDER: S-EPMC1184963 | biostudies-other | 1969 Oct
REPOSITORIES: biostudies-other
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Project description:1. Parts of the 16s and 30s RNA species of reticulocytes are readily hydrolysed by pancreatic ribonuclease. The biological activity of the ribosomes is diminished after treatment with low concentrations of the enzyme (e.g. 1ng. of ribonuclease/2.5mg. of polyribosome fraction/ml.). A high proportion of the chain scissions are ;hidden' owing to the secondary structure of the RNA moiety. 2. As the concentration of ribonuclease is increased RNA is lost from the ribosome. About 20-30% of the RNA may be removed from the ribosome without altering appreciably its sedimentation coefficient or its appearance in the electron microscope. 3. The amount of RNA removed from the ribosome is not increased by raising the concentration of enzyme from about 1mug. to 2.5mg. of ribonuclease/2.5mg. of polyribosome fraction/ml., or by increasing the temperature from 0 degrees to 30 degrees , or by first converting the RNA moiety into a single-stranded form before exposure to ribonuclease. 4. Untreated polyribosomes aggregate at about 75 degrees , whereas ribosomes treated with ribonuclease aggregate at about 45 degrees . The aggregates that are found on heating ribosomes after enzymic hydrolysis contain about 40-50% of the complement of RNA of intact ribosomes. 5. From the size of the fragments of RNA isolated from RNA-depleted ribosomes it is inferred that there is one site/60-100 nucleotides that is sensitive to ribonuclease. 6. The RNA moiety of RNA-depleted ribosomes has some double-helical character as shown by the optical properties and X-ray-diffraction pattern of ribonuclease-treated ribosomes and by the ;melting' properties of the isolated RNA. 7. Subparticles prepared by titration with an excess of EDTA are readily hydrolysed by ribonuclease to fragments of S(20,w) less than 4s, in contrast with the intact particle.
| S-EPMC1184962 | biostudies-other
Project description:In the present study, the effect of long-term exercise training was investigated on myocardial morphological and functional remodeling and on proarrhythmic sensitivity in a rabbit athlete's heart model. New-Zealand white rabbits were trained during a 12-week long treadmill running protocol and compared with their sedentary controls. At the end of the training protocol, echocardiography, in vivo and in vitro ECG recordings, proarrhythmic sensitivity with dofetilide (nM) were performed in isolated hearts, and action potential duration (APD) measurements at different potassium concentrations (4.5 and 2 mM) were made in the isolated papillary muscles. Expression levels of the slow component of delayed rectifier potassium current and fibrosis synthesis and degradation biomarkers were quantified. Echocardiography showed a significantly dilated left ventricle in the running rabbits. ECG PQ and RR intervals were significantly longer in the exercised group (79 ± 2 vs. 69 ± 2 ms and 325 ± 11 vs. 265 ± 6 ms, p < 0.05, respectively). The in vivo heart rate variability (HRV) (SD of root mean square: 5.2 ± 1.4 ms vs. 1.4 ± 0.2 ms, p < 0.05) and Tpeak-Tend variability were higher in the running rabbits. Bradycardia disappeared in the exercised group in vitro. Dofetilide tended to increase the QTc interval in a greater extent, and significantly increased the number of arrhythmic beats in the trained animals in vitro. APD was longer in the exercised group at a low potassium level. Real-time quantitative PCR (RT-qPCR) showed significantly greater messenger RNA expression of fibrotic biomarkers in the exercised group. Increased repolarization variability and higher arrhythmia incidences, lengthened APD at a low potassium level, increased fibrotic biomarker gene expressions may indicate higher sensitivity of the rabbit "athlete's heart" to life-threatening arrhythmias.
| S-EPMC8882986 | biostudies-literature
Project description:Turnover of mRNA is a critical step that allows cells to control gene expression. Endoribonucleases, enzymes cleaving RNA molecules internally, are some of the key components of the degradation process. Here we provide a detailed characterization of novel endoribonuclease SLFN14 purified from rabbit reticulocyte lysate. Schlafen genes encode a family of proteins limited to mammals. Their cellular function is unknown or incompletely understood. In reticulocytes, SLFN14 is strongly overexpressed, represented exclusively by the short form, all tethered to ribosomes, and appears to be one of the major ribosome-associated proteins. SLFN14 binds to ribosomes and ribosomal subunits in the low part of the body and cleaves RNA but preferentially rRNA and ribosome-associated mRNA. This results in the degradation of ribosomal subunits. This process is strictly Mg(2+)- and Mn(2+)-dependent, NTP-independent, and sequence nonspecific. However, in other cell types, SLFN14 is a full-length solely nuclear protein, which lacks ribosomal binding and nuclease activities. Mutational analysis revealed the ribosomal binding site and the aspartate essential for the endonucleolytic activity of protein. Only few endoribonucleases participating in ribosome-mediated processes have been characterized to date. Moreover, none of them are shown to be directly associated with the ribosome. Therefore, our findings expand the general knowledge of endoribonucleases involved in mammalian translation control.
| S-EPMC4461289 | biostudies-literature
Project description:MAIN is software that has been designed to interactively perform the complex tasks of macromolecular crystal structure determination and validation. Using MAIN, it is possible to perform density modification, manual and semi-automated or automated model building and rebuilding, real- and reciprocal-space structure optimization and refinement, map calculations and various types of molecular structure validation. The prompt availability of various analytical tools and the immediate visualization of molecular and map objects allow a user to efficiently progress towards the completed refined structure. The extraordinary depth perception of molecular objects in three dimensions that is provided by MAIN is achieved by the clarity and contrast of colours and the smooth rotation of the displayed objects. MAIN allows simultaneous work on several molecular models and various crystal forms. The strength of MAIN lies in its manipulation of averaged density maps and molecular models when noncrystallographic symmetry (NCS) is present. Using MAIN, it is possible to optimize NCS parameters and envelopes and to refine the structure in single or multiple crystal forms.
| S-EPMC3727325 | biostudies-literature
Project description:Establishing an in vitro "red blood cell matrix" that would allow uninterrupted access to a stable, homogeneous reticulocyte population would facilitate the establishment of continuous, long-term in vitro Plasmodium vivax blood stage cultures. In this study, we have explored the suitability of the erythroleukemia K562 cell line as a continuous source of such reticulocytes and have investigated regulatory factors behind the terminal differentiation (and enucleation, in particular) of this cell line that can be used to drive the reticulocyte production process. The Duffy blood group antigen receptor (Fy), essential for P. vivax invasion, was stably introduced into K562 cells by lentiviral gene transfer. miRNA-26a-5p and miRNA-30a-5p were downregulated to promote erythroid differentiation and enucleation, resulting in a tenfold increase in the production of reticulocytes after stimulation with an induction cocktail compared with controls. Our results suggest an interplay in the mechanisms of action of miRNA-26a-5p and miRNA-30a-5p, which makes it necessary to downregulate both miRNAs to achieve a stable enucleation rate and Fy receptor expression. In the context of establishing P. vivax-permissive, stable, and reproducible reticulocytes, a higher enucleation rate may be desirable, which may be achieved by the targeting of further regulatory mechanisms in Fy-K562 cells; promoting the shift in hemoglobin production from fetal to adult may also be necessary. Despite the fact that K562 erythroleukemia cell lines are of neoplastic origin, this cell line offers a versatile model system to research the regulatory mechanisms underlying erythropoiesis.
| S-EPMC7097847 | biostudies-literature
Project description:Translation termination is an essential cellular process that is also of therapeutic interest for diseases that manifest from premature stop codons. In eukaryotes, translation termination requires eRF1, which recognizes stop codons, catalyzes the release of nascent proteins fom ribosomes, and facilitates ribosome recycling. The small molecule SRI-41315 triggers eRF1 degradation and enhances translational readthrough of premature stop codons. SRI-41315 promotes retention of eRF1 on ribosomes and leads to a higher frequency of translation termination at near-cognate stop codons. In this study, the systematic effect of SRI-41315 on translation termination at cognate and near-cognate stop codons is evaluated using a rabbit reticulocyte lysate-based in vitro translation system with endogenous transcript as well as reporter transcripts containing defined near-cognate stop codon.
2023-12-31 | GSE248946 | GEO
Project description:The highly divergent ribosomes of human mitochondria (mitoribosomes) synthesize 13 essential proteins of oxidative phosphorylation complexes. We have determined the structure of the intact mitoribosome to 3.5 angstrom resolution by means of single-particle electron cryogenic microscopy. It reveals 80 extensively interconnected proteins, 36 of which are specific to mitochondria, and three ribosomal RNA molecules. The head domain of the small subunit, particularly the messenger (mRNA) channel, is highly remodeled. Many intersubunit bridges are specific to the mitoribosome, which adopts conformations involving ratcheting or rolling of the small subunit that are distinct from those seen in bacteria or eukaryotes. An intrinsic guanosine triphosphatase mediates a contact between the head and central protuberance. The structure provides a reference for analysis of mutations that cause severe pathologies and for future drug design.
| S-EPMC4501431 | biostudies-literature
Project description:THE EFFECT OF EXPOSING RABBIT RETICULOCYTE RIBOSOMES TO CONCENTRATED SOLUTIONS OF POTASSIUM CHLORIDE WAS EXAMINED BY: (a) dialysis against 0.5m-potassium chloride; (b) zone centrifugation through a sucrose gradient in 0.5m-potassium chloride; (c) differential centrifugation of a solution made 0.5m with respect to potassium chloride. The products of each treatment and their ability to support protein synthesis in a reticulocyte cell-free system, in the presence and in the absence of polyuridylic acid, were examined. The following results were found. (1) Exposing the polysomes to 0.5m-potassium chloride was not a sufficient condition for the complete dissociation of ribosomes into subparticles; the reaction showed a concentration-dependence, implying the existence of an equilibrium between the various ribosomal species. Disturbance of the equilibrium by removing certain products, as in zone centrifuging, can lead to complete dissociation. (2) The subparticles produced by dialysis or sucrose-gradient fractionation were biologically inactive and unstable. (3) The pellet obtained by differential centrifuging consisted of subparticles, if suspended in a Mg(2+)-free buffer; addition of Mg(2+) converted about 30% of the material into heavier sedimenting species, and the preparation had 20-40% of the activity of the untreated control polysomes in the cell-free system. Addition of the 0.5m-potassium chloride supernatant fraction resulted in further apparent reconstitution of sub-particles into ribosomes and polysomes and in a 50-100% restoration of biological activity. When both polyuridylic acid and supernatant factors were present incorporations similar to or higher than those of the control were attained.
| S-EPMC1187112 | biostudies-other
Project description:ObjectivesThe objective of this study was to perform a quantitative analysis of the structural and functional alterations in the intervertebral disc during in vivo degeneration, using emerging tools that enable rigorous assessment from the microscale to the macroscale, as well as to correlate these outcomes with noninvasive, clinically relevant imaging parameters.DesignDegeneration was induced in a rabbit model by puncturing the annulus fibrosus (AF) with a 16-gauge needle. 2, 4, 8, and 12 weeks following puncture, degenerative changes in the discs were evaluated via magnetic resonance imaging (MRI), whole motion segment biomechanics, atomic force microscopy, histology and polarized light microscopy, immunohistochemistry, biochemical content, and second harmonic generation imaging.ResultsFollowing puncture, degeneration was evident through marked changes in whole disc structure and mechanics. Puncture acutely compromised disc macro and microscale mechanics, followed by progressive stiffening and remodeling. Histological analysis showed substantial anterior fibrotic remodeling and osteophyte formation, as well as an overall reduction in disc height, and disorganization and infolding of the AF lamellae into the NP space. Increases in NP collagen content and aggrecan breakdown products were also noted within 4 weeks. On MRI, NP T2 was reduced at all post-puncture time points and correlated significantly with microscale indentation modulus.ConclusionThis study defined the time dependent changes in disc structure-function relationships during IVD degeneration in a rabbit annular injury model and correlated degeneration severity with clinical imaging parameters. Our findings identified AF infolding and occupancy of the space as a principle mechanism of disc degeneration in response to needle puncture, and provide new insights to direct the development of novel therapeutics.
| S-EPMC6875634 | biostudies-literature
Project description:The PHENIX AutoBuild wizard is a highly automated tool for iterative model building, structure refinement and density modification using RESOLVE model building, RESOLVE statistical density modification and phenix.refine structure refinement. Recent advances in the AutoBuild wizard and phenix.refine include automated detection and application of NCS from models as they are built, extensive model-completion algorithms and automated solvent-molecule picking. Model-completion algorithms in the AutoBuild wizard include loop building, crossovers between chains in different models of a structure and side-chain optimization. The AutoBuild wizard has been applied to a set of 48 structures at resolutions ranging from 1.1 to 3.2 A, resulting in a mean R factor of 0.24 and a mean free R factor of 0.29. The R factor of the final model is dependent on the quality of the starting electron density and is relatively independent of resolution.
| S-EPMC2394820 | biostudies-literature