We describe a cell-free translation system for evaluating the activity of ribosomes stringently purified from human cells. This system is based on in vitro reconstitution of the cellular translation machinery, in which a ribosome-free rabbit reticulocyte lysate (RRL) is reassembled with human ribosomes and in vitro-transcribed reporter mRNAs. The protocol describes the preparation of the RRL-derived fractions, purification of ribosomes devoid of detectable nonribosomal-associated factors, and assembly of the reactions to evaluate ribosomal translational efficiency and fidelity using appropriate reporter transcripts. The whole procedure can be completed in ∼2.5 d (plus 2 weeks for RRL preparation and cell expansion time). This protocol can be applied to study intrinsic functional properties (cis-acting element-mediated translation initiation or translational fidelity) of ribosome populations from different sources (including nonhuman origin). It is therefore useful for the characterization of ribosomal function in ribosomopathies and cancer, and it will be applicable in the emerging fields of ribosome diversity and specialized ribosomes.
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http://dx.doi.org/10.1038/nprot.2016.072 | DOI Listing |
J Virol
December 2024
College of Animal Science and Technology, Northwest A&F University College of Animal Science and Technology, Yangling, China.
The occurrence of viral diseases poses a huge threat and impact on human public health safety and the development of the animal and fishery industry. Here, a strain of single-chain antibody fragment, scFv-1, was isolated from the phage antibody display library construct by immunizing New Zealand white rabbits with rhabdovirus. analysis showed that the single-chain antibody could inhibit the infection of the virus in multiple pathways, including adsorption, fusion, and release.
View Article and Find Full Text PDFWorld J Gastroenterol
December 2024
Department of Gastroenterology, The First Hospital of Lanzhou University, Lanzhou 730000, Gansu Province, China.
Background: () infection exhibits a familial clustering phenomenon.
Aim: To investigate the prevalence of infection, identify associated factors, and analyze patterns of transmission within families residing in the community.
Methods: From July 2021 to September 2021, a total of 191 families (519 people) in two randomly chosen community health service centers in the Chengguan District of Lanzhou in Gansu Province, were recruited to fill out questionnaires and tested for infection.
Maintaining a balance of inorganic phosphate (Pi) is vital for cellular functionality due to Pi's essential role in numerous biological processes. Proper phosphate levels are managed through Pi import and export, facilitated by specific Pi transport proteins. Although the mechanisms of Pi import have been extensively studied, the processes governing Pi export remain less understood.
View Article and Find Full Text PDFZhonghua Yu Fang Yi Xue Za Zhi
December 2024
Clinical Research Center, The Affiliated Wuxi People's Hospital of Nanjing Medical University, Wuxi214023, China.
The present study was aimed to produce the recombinant protein of allergen component 32 (Tyr p 32) and to identify its immunoreactivity. The cDNA encoding Tyr p 32 was amplified from total RNA of and inserted into pET-28a (+) vector. The constructed plasmid pET-28a (+)-Tyr p 32 was transformed into BL21 (DE3) receptor cells.
View Article and Find Full Text PDFExp Cell Res
December 2024
Department of Maternal-Fetal Medicine Pregnancy Research Centre, Royal Women's Hospital, Parkville, VIC 3052, Australia; University of Melbourne Department of Obstetrics and Gynaecology and Newborn Health, Royal Women's Hospital, Parkville, VIC 3052, Australia. Electronic address:
Increasing evidence shows extracellular vesicles (EVs) are primarily responsible for the beneficial effects of cell-based therapies. EVs derived from mesenchymal stromal cells (MSCs) show promise as a source of EVs for cell-free therapies. The human placental fetal-maternal interface is a rich and abundant source of MSCs from which EVs can be isolated.
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