scholarly journals Protein Composition and Electron Microscopy Structure of Affinity-Purified Human Spliceosomal B Complexes Isolated under Physiological Conditions

2006 ◽  
Vol 26 (14) ◽  
pp. 5528-5543 ◽  
Author(s):  
Jochen Deckert ◽  
Klaus Hartmuth ◽  
Daniel Boehringer ◽  
Nastaran Behzadnia ◽  
Cindy L. Will ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Potential Role ◽  
Protein Composition ◽  
Nuclear Extract ◽  
Physiological Conditions ◽  
Catalytic Activation ◽  
Composition And Structure ◽  
Spliceosome Activation ◽  
Related Proteins

ABSTRACT The spliceosomal B complex is the substrate that undergoes catalytic activation leading to catalysis of pre-mRNA splicing. Previous characterization of this complex was performed in the presence of heparin, which dissociates less stably associated components. To obtain a more comprehensive inventory of the B complex proteome, we isolated this complex under low-stringency conditions using two independent methods. MS2 affinity-selected B complexes supported splicing when incubated in nuclear extract depleted of snRNPs. Mass spectrometry identified over 110 proteins in both independently purified B complex preparations, including ∼50 non-snRNP proteins not previously found in the spliceosomal A complex. Unexpectedly, the heteromeric hPrp19/CDC5 complex and 10 additional hPrp19/CDC5-related proteins were detected, indicating that they are recruited prior to spliceosome activation. Electron microscopy studies revealed that MS2 affinity-selected B complexes exhibit a rhombic shape with a maximum dimension of 420 Å and are structurally more homogeneous than B complexes treated with heparin. These data provide novel insights into the composition and structure of the spliceosome just prior to its catalytic activation and suggest a potential role in activation for proteins recruited at this stage. Furthermore, the spliceosomal complexes isolated here are well suited for complementation studies with purified proteins to dissect factor requirements for spliceosome activation and splicing catalysis.

scholarly journals Conservation of the Protein Composition and Electron Microscopy Structure of Drosophila melanogaster and Human Spliceosomal Complexes

2008 ◽  
Vol 29 (1) ◽  
pp. 281-301 ◽  
Author(s):  
Nadine Herold ◽  
Cindy L. Will ◽  
Elmar Wolf ◽  
Berthold Kastner ◽  
Henning Urlaub ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Drosophila Melanogaster ◽  
Protein Composition ◽  
Structural Similarity ◽  
Nuclear Extract ◽  
Exon Junction Complex ◽  
Dependent Manner ◽  
Composition And Structure ◽  
C Complex ◽  
Protein Recruitment

ABSTRACT Comprehensive proteomics analyses of spliceosomal complexes are currently limited to those in humans, and thus, it is unclear to what extent the spliceosome's highly complex composition and compositional dynamics are conserved among metazoans. Here we affinity purified Drosophila melanogaster spliceosomal B and C complexes formed in Kc cell nuclear extract. Mass spectrometry revealed that their composition is highly similar to that of human B and C complexes. Nonetheless, a number of Drosophila-specific proteins were identified, suggesting that there may be novel factors contributing specifically to splicing in flies. Protein recruitment and release events during the B-to-C transition were also very similar in both organisms. Electron microscopy of Drosophila B complexes revealed a high degree of structural similarity with human B complexes, indicating that higher-order interactions are also largely conserved. A comparison of Drosophila spliceosomes formed on a short versus long intron revealed only small differences in protein composition but, nonetheless, clear structural differences under the electron microscope. Finally, the characterization of affinity-purified Drosophila mRNPs indicated that exon junction complex proteins are recruited in a splicing-dependent manner during C complex formation. These studies provide insights into the evolutionarily conserved composition and structure of the metazoan spliceosome, as well as its compositional dynamics during catalytic activation.

scholarly journals Preparation and Characterization of Polymer Composite Materials Containing Magnetite

2021 ◽  
Vol 15 ◽  
pp. 1421-1428
Author(s):  
E. R. Sadretdinova ◽  
Thant Zin Hein
Keyword(s):  
Electron Microscopy ◽  
Phase Composition ◽  
Composite Materials ◽  
Ir Spectroscopy ◽  
Polymer Processing ◽  
Flow State ◽  
Standard Methods ◽  
Composition And Structure ◽  
Polyethylene Melt

In this work, magnetite nanoparticles are introduced into a polyethylene melt at the stage of a viscous-flow state by standard methods of polymer processing (extrusion), which makes it possible to obtain a nanocomposite with a uniform nanofiller distribution. The phase composition and structure of the nanocomposite were confirmed by XRF, electron microscopy, and IR spectroscopy. It is shown that the preparation of Fe3O4/LLDPE nanocomposites is not complicated by the appearance of unidentified phases and changes in the structure of the polymer matrix.

scholarly journals Characterization of MXene as a Cancer Photothermal Agent Under Physiological Conditions

2021 ◽  
Vol 3 ◽  
Author(s):  
Samar Shurbaji ◽  
Nimshitha P. Abdul Manaph ◽  
Samia M. Ltaief ◽  
Abeer R. Al-Shammari ◽  
Ahmed Elzatahry ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Shear Stress ◽  
Cancer Cells ◽  
Fluid Shear Stress ◽  
Energy Dispersive Spectrometer ◽  
Physiological Conditions ◽  
Viability Assay ◽  
Transmission Electron ◽  
Power Densities

A growing interest has recently emerged in the use of nanomaterials in medical applications. Nanomaterials, such as MXene, have unique properties due to their 2D ultra-thin structure, which is potentially useful in cancer photothermal therapy. To be most effective, photothermal agents need to be internalized by the cancer cells. In this study, MXene was fabricated using chemical reactions and tested as a photothermal agent on MDA-231 breast cancer cells under static and physiological conditions. Fluid shear stress (∼0.1 Dyn/cm2) was applied using a perfusion system to mimic the physiological tumor microenvironment. The uptake of MXene was analyzed under fluid flow compared to static culture using confocal microscopy, scanning electron microscopy (SEM), energy dispersive spectrometer (EDS), and transmission electron microscopy (TEM). Furthermore, a viability assay was used to assess cell’s survival after exposing the treated cells to photothermal laser at different power densities and durations. We showed that when incubated with cancer cells, 2D MXene nanoparticles were successfully internalized into the cells resulting in increased intracellular temperatures when exposed to NIR laser. Interestingly, dynamic culture alone did not result in a significant increase in uptake suggesting the need for surface modifications for enhanced cellular uptake under shear stress.

scholarly journals Crystallization and biochemical characterization of the human spliceosomal Aar2–Prp8RNaseHcomplex

2015 ◽  
Vol 71 (11) ◽  
pp. 1421-1428
Author(s):  
Karine Santos ◽  
Marco Preussner ◽  
Anna Christina Heroven ◽  
Gert Weber
Keyword(s):  
Biochemical Characterization ◽  
Homology Modelling ◽  
Molecular Replacement ◽  
Noncoding Sequences ◽  
Internal Loop ◽  
Catalytic Activation ◽  
Maturation Factor ◽  
Spliceosome Activation

In eukaryotes, the removal of nuclear noncoding sequences (pre-mRNA splicing) is catalyzed by the spliceosome, which consists of five ribonucleoprotein particles (U1, U2, U4, U5 and U6 snRNPs, each with a respective snRNA) and a plethora of protein factors that aid spliceosomal maturation, assembly, activation and disassembly. Recently, the U5 snRNP maturation factor Aar2p from baker's yeast has been characterized structurally and biochemically. Aar2p binds to the RNaseH (RH) and Jab1/MPN domains of the highly conserved U5-specific Prp8p, which forms a framework for the spliceosomal catalytic centre. Thereby, Aar2p sterically excludes Brr2p, a helicase essential for the catalytic activation of the spliceosome, from Prp8p binding. At the same time, Aar2p blocks U4/U6 di-snRNA binding to Prp8p. Aar2p therefore prevents premature spliceosome activation and its functions are regulated by reversible phosphorylation. To date, little is known about the hypothetical human Aar2 (hsAar2) orthologue C20ORF4. This study identifies C20ORF4 (i) as part of the HeLa proteome by Western blotting and (ii) as a true Aar2 orthologue which binds to the RH domain (hsRH) of Prp8 and corroborates an evolutionary link between yeast and human Aar2 function. An elaborate strategy was devised to crystallize hsAar2 in complex with hsRH. The analysis of initial weakly diffracting crystals obtained byin situproteolysis and homology modelling guided the design of an hsAar2 construct in which an internal loop was replaced by three serines (hsAar2Δloop). A complex of hsAar2Δloopand hsRH crystallized in space groupC2; the crystals diffracted to 2.35 Å resolution and were suitable for structure determination by molecular-replacement approaches. The study presented here suggests a connection between Aar2 and the spliceosome in human cells and paves the way for structural studies of human Aar2.

Synthesis and Characterization of MoO2 Nanoplates Prepared by Thermal Evaporation of MoO3 and S Powders

2016 ◽  
Vol 697 ◽  
pp. 3-6
Author(s):  
Han Qing Li ◽  
Zhi Jian Peng ◽  
Jing Wen Qian ◽  
Zhen Guang Shen ◽  
Xiu Li Fu
Keyword(s):  
Electron Microscopy ◽  
Large Scale ◽  
Thermal Evaporation ◽  
Silicon Substrates ◽  
X Ray Diffraction ◽  
Super Capacitor ◽  
Transmission Electron ◽  
Composition And Structure ◽  
Battery Electrode

MoO2 has been widely used in many fields such as catalyst, gas-senor, super capacitor and Li-ion battery electrode. In this paper, MoO2 nanoplates were synthesized in high density and large scale on silicon substrates via simple thermal evaporation of MoO3 and S powders at 950 °C in a tube furnace. The morphology, composition and structure of the nanoplates were characterized by scanning electron microscopy, X-ray diffraction and transmission electron microscopy. The results indicate that the as-synthesized nanoplates are of well crystalline structure, and the thickness of these nanoplates is in the range of 100-300 nm. The growth mechanism of the nanoparticles was proposed as a vapor-solid process.

scholarly journals Tailoring cryo-electron microscopy grids by photo-micropatterning for in-cell structural studies

2019 ◽  
Author(s):  
Mauricio Toro-Nahuelpan ◽  
Ievgeniia Zagoriy ◽  
Fabrice Senger ◽  
Laurent Blanchoin ◽  
Manuel Théry ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Specimen Preparation ◽  
Cell Architecture ◽  
Molecular Machinery ◽  
Cell Positioning ◽  
Related Proteins

AbstractSpatially-controlled cell adhesion on electron microscopy (EM) supports remains a bottleneck in specimen preparation for cellular cryo-electron tomography. Here, we describe contactless and mask-free photo-micropatterning of EM grids for site-specific deposition of extracellular matrix-related proteins. We attained refined cell positioning for micromachining by cryo-focused ion beam milling. Complex patterns generated predictable intracellular organization, allowing direct correlation between cell architecture and in-cell 3D-structural characterization of the underlying molecular machinery.

scholarly journals Tailoring cryo-electron microscopy grids by photo-micropatterning for in-cell structural studies

2019 ◽  
Author(s):  
Mauricio Toro-Nahuelpan ◽  
Ievgeniia Zagoriy ◽  
Fabrice Senger ◽  
Laurent Blanchoin ◽  
Manuel Théry ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Carbon Films ◽  
Specimen Preparation ◽  
Complex Shapes ◽  
Cell Positioning ◽  
Related Proteins

Abstract Micromachining by cryo-focused ion beam (FIB) milling coupled to cryo-electron tomography (ET) enables visualization of macromolecules directly inside cells. Yet, spatial control of cell adhesion on electron microscopy (EM) grids remains a bottleneck in the specimen preparation pipeline. This protocol describes a contactless and mask-free photo-micropatterning of EM grids for site-specific deposition of extracellular matrix-related proteins. We achieved accurate and reproducible cell positioning, leading to optimized preparations for cryo-FIB milling. We tested HeLa and RPE1 cell lines on various grid types (gold or titanium mesh coated with SiO2, gold or carbon films). Briefly, grids were passivated with an anti-fouling agent, followed by controlled ablation of the passivation layer, and further functionalization with fibronectin. The micropatterning procedure takes ~3 h. Employing micropatterning to produce complex shapes generated a predictable intracellular organization, allowing direct correlation between cellular architecture and in-cell 3D-structural characterization of the underlying machinery at molecular resolution.

scholarly journals Isolation and characterization of extracellular vesicles in Candida albicans

2020 ◽  
pp. 99-108
Author(s):  
Virginia Pérez-Doñate ◽  
Facundo Pérez-Giménez ◽  
Lucas Del Castillo Agudo ◽  
Juan Alberto Castillo-Garit ◽  
Mar Soria-Merino ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Extracellular Vesicles ◽  
Fungal Infections ◽  
Protein Composition ◽  
Yeast Cultures ◽  
Isolation And Characterization ◽  
Fungal Organisms ◽  
Systemic Infections ◽  
Molecular Components

Background: The occurrence of systemic infections due to C. albicans has increased especially in critically ill patients. In fungal infections, secretory mechanisms are key events for disease establishment. Recent findings demonstrate that fungal organisms release many molecular components to the extracellular space in extracellular vesicles.Aims: We develop a method to obtain exosomes from yeast cultures of the Candida albicans.Methods: Yeast strains used in this work were C. albicans SC5314, C. parapsilosis (ATCC 22019) and C. krusei (ATCC 6258). Yeasts were grown at 37.º in liquid YPD medium. The cell cultures were centrifuged and the supernatant filtered through sterile nitrocellulose. Filtrates were concentrated and centrifuged using an ultracentrifuge. The sediment was analyzed by electron microscopy of transmission.Results: The transmission of electron microscopy and nanoparticle tracking analysis confirmed the presence of extracellular vesicles (exosomes) of sizes between 100 and 200 nm and the absence of cellular contaminants. This was ratified by the characterization of proteins performed through the western blot technique, where the absence of cell contamination in the preparations was assessed.Conclusions: The method proves to be highly effective due to the homogeneity and purity of the obtained microvesicles. The protocol developed in this paper proves to be effective for obtaining exosomes of other Candida species, which will allow future studies to determine its protein composition and the role that these vesicles can play.

Characterization of Tamm-Horsfall Urinary Glycoprotein

1973 ◽  
Vol 31 ◽  
pp. 420-421
Author(s):  
John P. Robinson ◽  
J. David Puett
Keyword(s):  
Electron Microscopy ◽  
Circular Dichroism ◽  
Secondary Structure ◽  
Quaternary Structure ◽  
Normal Adult ◽  
Morphological Properties ◽  
Adult Males ◽  
Circular Dichroïsm ◽  
Urinary Glycoprotein

Much work has been reported on the chemical, physical and morphological properties of urinary Tamm-Horsfall glycoprotein (THG). Although it was once reported that cystic fibrotic (CF) individuals had a defective THG, more recent data indicate that THG and CF-THG are similar if not identical.No studies on the conformational aspects have been reported on this glycoprotein using circular dichroism (CD). We examined the secondary structure of THG and derivatives under various conditions and have correlated these results with quaternary structure using electron microscopy.THG was prepared from normal adult males and CF-THG from a 16-year old CF female by the method of Tamm and Horsfall. CF female by the method of Tamm and Horsfall.

Electron Microscopic Detection and Characterization of Nonhuman Primate Enteric Viruses

1982 ◽  
Vol 40 ◽  
pp. 306-307
Author(s):  
G. C. Smith ◽  
R. L. Heberling ◽  
S. S. Kalter
Keyword(s):  
Electron Microscopy ◽  
Animal Model ◽  
Nonhuman Primate ◽  
Clinical Condition ◽  
Intestinal Tract ◽  
Enteric Viruses ◽  
Electron Microscopic ◽  
Microscopic Detection

A number of viral agents are recognized as and suspected of causing the clinical condition “gastroenteritis.” In our attempts to establish an animal model for studies of this entity, we have been examining the nonhuman primate to ascertain what viruses may be found in the intestinal tract of “normal” animals as well as animals with diarrhea. Several virus types including coronavirus, adenovirus, herpesvirus, and picornavirus (Table I) were detected in our colony; however, rotavirus, astrovirus, and calicivirus have not yet been observed. Fecal specimens were prepared for electron microscopy by procedures reported previously.

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