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Journal of Virology Oct 1967The attachment and eclipse of adenovirus have been studied with the aid of highly purified (14)C-threonine and (32)P-labeled adenovirus type 2 in KB cells in suspension...
The attachment and eclipse of adenovirus have been studied with the aid of highly purified (14)C-threonine and (32)P-labeled adenovirus type 2 in KB cells in suspension cultures. Adenovirus particles and infectivity appear to attach at the same rate. The attachment rate appears to be highly dependent on the cell concentration and less dependent on virus concentration within the multiplicity range from 0.15 to 3 plaque-forming units per cell, probably corresponding to 4.5 to 90 particles per cell. Subsequent to attachment, 5 to 8% of the (14)C label is eluted from the cell at a structure level, corresponding to free hexon. The (32)P activity is rapidly associated with the cells and is converted within 20 to 30 min to 65 to 85% deoxyribonuclease-susceptible material. This process is unaffected by actinomycin and puromycin. The deoxyribonuclease-sensitive material is, however, associated with (14)C label for an extended period after infection, and does not sediment as free deoxyribonucleic acid in sucrose gradients. The implications of these findings on the penetration mechanism of animal viruses are discussed.
Topics: Adenoviridae; Carbon Isotopes; Carcinoma; Cell Line; Cell Transformation, Neoplastic; Centrifugation, Density Gradient; Culture Techniques; Cytopathogenic Effect, Viral; Dactinomycin; Humans; Mouth Neoplasms; Phosphorus Isotopes; Puromycin; Threonine; Viral Proteins; Virus Replication
PubMed: 5621483
DOI: 10.1128/JVI.1.5.868-875.1967 -
BMC Microbiology Apr 2021Translation is an important point of regulation in protein synthesis. However, there is a limited number of methods available to measure global translation activity in...
BACKGROUND
Translation is an important point of regulation in protein synthesis. However, there is a limited number of methods available to measure global translation activity in yeast. Recently, O-propargyl-puromycin (OPP) labelling has been established for mammalian cells, but unmodified yeasts are unsusceptible to puromycin.
RESULTS
We could increase susceptibility by using a Komagataella phaffii strain with an impaired ergosterol pathway (erg6Δ), but translation measurements are restricted to this strain background, which displayed growth deficits. Using surfactants, specifically Imipramine, instead, proved to be more advantageous and circumvents previous restrictions. Imipramine-supplemented OPP-labelling with subsequent flow cytometry analysis, enabled us to distinguish actively translating cells from negative controls, and to clearly quantify differences in translation activities in different strains and growth conditions. Specifically, we investigated K. phaffii at different growth rates, verified that methanol feeding alters translation activity, and analysed global translation in strains with genetically modified stress response pathways.
CONCLUSIONS
We set up a simple protocol to measure global translation activity in yeast on a single cell basis. The use of surfactants poses a practical and non-invasive alternative to the commonly used ergosterol pathway impaired strains and thus impacts a wide range of applications where increased drug and dye uptake is needed.
Topics: Imipramine; Protein Biosynthesis; Puromycin; Saccharomycetales; Surface-Active Agents
PubMed: 33879049
DOI: 10.1186/s12866-021-02185-3 -
Science Advances Jun 2019An effective transformation of the cell culture dishes that biologists use every day into microfluidic devices would open many avenues for miniaturizing cell-based...
An effective transformation of the cell culture dishes that biologists use every day into microfluidic devices would open many avenues for miniaturizing cell-based workflows. In this article, we report a simple method for creating microfluidic arrangements around cells already growing on the surface of standard petri dishes, using the interface between immiscible fluids as a "building material." Conventional dishes are repurposed into sophisticated microfluidic devices by reshaping, on demand, the fluid structures around living cells. Moreover, these microfluidic arrangements can be further reconfigured during experiments, which is impossible with most existing microfluidic platforms. The method is demonstrated using workflows involving cell cloning, the selection of a particular clone from among others in a dish, drug treatments, and wound healing. The versatility of the approach and its biologically friendly aspects may hasten uptake by biologists of microfluidics, so the technology finally fulfills its potential.
Topics: Cell Line; Cell Movement; Cell Survival; Humans; Microfluidics; Puromycin
PubMed: 31183401
DOI: 10.1126/sciadv.aav8002 -
Stem Cell Research & Therapy Jan 2022Many drugs have the potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in hepatocytes. Hepatocytes can be...
BACKGROUND
Many drugs have the potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in hepatocytes. Hepatocytes can be accurately evaluated for drug-mediated CYP3A4 induction; this is the gold standard for in vitro hepatic toxicology testing. However, the variation from lot to lot is an issue that needs to be addressed. Only a limited number of immortalized hepatocyte cell lines have been reported. In this study, immortalized cells expressing CYP3A4 were generated from a patient with drug-induced liver injury (DILI).
METHODS
To generate DILI-derived cells with high expression of CYP3A4, a three-step approach was employed: (1) Differentiation of DILI-induced pluripotent stem cells (DILI-iPSCs); (2) Immortalization of the differentiated cells; (3) Selection of the cells by puromycin. It was hypothesized that cells with high cytochrome P450 gene expression would be able to survive exposure to cytotoxic antibiotics because of their increased drug-metabolizing activity. Puromycin, a cytotoxic antibiotic, was used in this study because of its rapid cytocidal effect at low concentrations.
RESULTS
The hepatocyte-like cells differentiated from DILI-iPSCs were purified by exposure to puromycin. The puromycin-selected cells (HepaSM or SI cells) constitutively expressed the CYP3A4 gene at extremely high levels and exhibited hepatocytic features over time. However, unlike primary hepatocytes, the established cells did not produce bile or accumulate glycogen.
CONCLUSIONS
iPSC-derived hepatocyte-like cells with intrinsic drug-metabolizing enzymes can be purified from non-hepatocytes and undifferentiated iPSCs using the cytocidal antibiotic puromycin. The puromycin-selected hepatocyte-like cells exhibited characteristics of hepatocytes after immortalization and may serve as another useful source for in vitro hepatotoxicity testing of low molecular weight drugs.
Topics: Cells, Cultured; Chemical and Drug Induced Liver Injury; Cytochrome P-450 CYP3A; Hepatocytes; Humans; Liver; Puromycin
PubMed: 35012658
DOI: 10.1186/s13287-021-02680-4 -
PloS One 2013Development of Nourseothricin N-acetyl transferase (NAT) as a selection marker for mammalian cells is described. Mammalian cells are acutely susceptible to...
Development of Nourseothricin N-acetyl transferase (NAT) as a selection marker for mammalian cells is described. Mammalian cells are acutely susceptible to Nourseothricin, similar to the widely used drug Puromycin, and NAT allows for quick and robust selection of transfected/transduced cells in the presence of Nourseothricin. NAT is compatible with other selection markers puromycin, hygromycin, neomycin, blasticidin, and is a valuable addition to the repertoire of mammalian selection markers.
Topics: Acetyltransferases; Animals; Anti-Bacterial Agents; Biomarkers; Cell Line; Cinnamates; Humans; Hygromycin B; Neomycin; Puromycin; Pyrrolidinones; Streptothricins; Transfection
PubMed: 23861913
DOI: 10.1371/journal.pone.0068509 -
Journal of Biotechnology Oct 2015cDNA display is a powerful in vitro display technology used to explore functional peptides and proteins from a huge library by in vitro selection. In addition to...
cDNA display is a powerful in vitro display technology used to explore functional peptides and proteins from a huge library by in vitro selection. In addition to expediting the in vitro selection cycle by using cDNA display, easy and rapid functional analysis of selected candidate clones is crucial for high-throughput screening of functional peptides and proteins. In this report, a versatile puromycin-linker employing an ultrafast photo-cross-linker, 3-cyanovinylcarbazole nucleoside, is introduced. Its utility for both in vitro selection using cDNA display and protein-protein interaction analysis using a surface plasmon resonance (SPR) system is described. Using this versatile puromycin-linker, we demonstrated the model in vitro selection of the FLAG epitope and a SPR-based assay to measure the dissociation constant between the B domain of protein A and immunoglobulin G. Improvement of the puromycin-linker as described herein should make the cDNA display method easier to utilize for design of protein or peptide based affinity reagents.
Topics: Carbazoles; Cross-Linking Reagents; DNA, Complementary; Epitopes; Immunoglobulin G; Nucleosides; Oligopeptides; Protein Structure, Tertiary; Puromycin; RNA, Messenger; Staphylococcal Protein A; Surface Plasmon Resonance; Ultraviolet Rays
PubMed: 26321074
DOI: 10.1016/j.jbiotec.2015.08.020 -
Nucleic Acids Research Aug 2023Extremely diverse libraries are essential for effectively selecting functional peptides or proteins, and mRNA display technology is a powerful tool for generating such...
Extremely diverse libraries are essential for effectively selecting functional peptides or proteins, and mRNA display technology is a powerful tool for generating such libraries with over 1012-1013 diversity. Particularly, the protein-puromycin linker (PuL)/mRNA complex formation yield is determining for preparing the libraries. However, how mRNA sequences affect the complex formation yield remains unclear. To study the effects of N-terminal and C-terminal coding sequences on the complex formation yield, puromycin-attached mRNAs containing three random codons after the start codon (32768 sequences) or seven random bases next to the amber codon (6480 sequences) were translated. Enrichment scores were calculated by dividing the appearance rate of every sequence in protein-PuL/mRNA complexes by that in total mRNAs. The wide range of enrichment scores (0.09-2.10 for N-terminal and 0.30-4.23 for C-terminal coding sequences) indicated that the N-terminal and C-terminal coding sequences strongly affected the complex formation yield. Using C-terminal GGC-CGA-UAG-U sequences, which resulted in the highest enrichment scores, we constructed highly diverse libraries of monobodies and macrocyclic peptides. The present study provides insights into how mRNA sequences affect the protein/mRNA complex formation yield and will accelerate the identification of functional peptides and proteins involved in various biological processes and having therapeutic applications.
Topics: Codon, Terminator; Peptides; Proteins; Puromycin; RNA, Messenger; Peptide Library
PubMed: 37395404
DOI: 10.1093/nar/gkad555 -
Biochemical and Biophysical Research... Dec 2017The precise roles that the major proteolytic pathways play in the regulation of skeletal muscle mass remain incompletely understood, in part due to technical limitations...
The precise roles that the major proteolytic pathways play in the regulation of skeletal muscle mass remain incompletely understood, in part due to technical limitations associated with current techniques used to quantify muscle protein breakdown (MPB). We aimed to develop a method to assess MPB in cells, based on loss of puromycin labelling of translated polypeptide chains. Following an initial 24 h incubation period with puromycin (1 μM), loss of puromycin labelling from murine C2C12 myotubes was assessed over 48 h, both in the presence or absence of protein synthesis inhibitor cycloheximide (CHX). To validate the method, loss of puromycin labelling was determined from cells treated with selected compounds known to influence MPB (e.g. serum starvation, Dexamethasone (Dex), tumour necrosis factor alpha (TNF-α) and MG-132)). Reported established (static) markers of MPB were measured following each treatment. Loss of puromycin labelling from cells pre-incubated with puromycin was evident over a 48 h period, both with and without CHX. Treatment with Dex (-14 ± 2% vs. Ctl; P < 0.01), TNF-α (-20 ± 4% vs. Ctl; P < 0.001) and serum starvation (-14 ± 4% vs. Ctl; P < 0.01) caused a greater loss of puromycin labelling than untreated controls, while the proteasome inhibitor MG-132 caused a relatively lower loss of puromycin labelling (+15 ± 8% vs. Ctl; P < 0.05). Thus, we have developed a novel decorporation method for measuring global changes in MPB, validated in vitro using an established muscle cell line. It is anticipated this non isotopic-tracer alternative to measuring MPB will facilitate insight into the mechanisms that regulate muscle mass/MPB both in vitro, and perhaps, in vivo.
Topics: Animals; Cell Line; Isotope Labeling; Metabolic Clearance Rate; Mice; Muscle Fibers, Skeletal; Muscle Proteins; Pilot Projects; Puromycin; Radioligand Assay
PubMed: 29054406
DOI: 10.1016/j.bbrc.2017.10.085 -
Journal of Biochemistry Feb 1998Blasticidin S-producing Streptomyces morookaensis JCM4673 produces an enzyme which inactivates puromycin (PM) by hydrolyzing an amide linkage between its aminonucleoside...
Blasticidin S-producing Streptomyces morookaensis JCM4673 produces an enzyme which inactivates puromycin (PM) by hydrolyzing an amide linkage between its aminonucleoside and O-methyl-L-tyrosine moieties [Nishimura et al. (1995) FEMS Microbiol. Lett. 132, 95-100]. In this study, we purified to homogeneity the enzyme from the cell-free extracts of S. morookaensis. The molecular weight of PM-hydrolyzing enzyme, estimated by SDS-PAGE and gel filtration, was 68 and 66 kDa, respectively, suggesting that this protein is monomeric. The PM-hydrolyzing activity was strongly inhibited by Zn2+, Fe2+, Cu2+, Hg2+, and N-bromosuccinimide, but was stimulated by DTT. The optimum pH and temperature for PM-hydrolyzing activity were 8.0 and 45 degrees C, respectively. Several L-aminoacyl-beta-naphthylamides were good substrates for the enzyme, suggesting that the PM-inactivating enzyme has an aminopeptidase activity. The N-terminal sequence of the first 14 amino acids (Val-Ser-Thr-Ala-Pro-Tyr-Gly-Ala-Trp-Gln-Ser-Pro-Ile-Asp) of the enzyme showed no significant homology with any published hydrolase sequences.
Topics: Aminopeptidases; Enzyme Inhibitors; Hydrolysis; Metals, Heavy; Molecular Sequence Data; Nucleosides; Puromycin; Streptomyces; Substrate Specificity
PubMed: 9538199
DOI: 10.1093/oxfordjournals.jbchem.a021929 -
The EMBO Journal Aug 1991Bryophyllum fedtschenkoi is a Crassulacean acid metabolism plant whose phosphoenolpyruvate carboxylase is regulated by reversible phosphorylation in response to a...
Bryophyllum fedtschenkoi is a Crassulacean acid metabolism plant whose phosphoenolpyruvate carboxylase is regulated by reversible phosphorylation in response to a circadian rhythm. A partially purified protein kinase phosphorylated phosphoenolpyruvate carboxylase in vitro with a stoichiometry approaching one per subunit and caused a concomitant 5- to 10-fold decrease in the sensitivity of the carboxylase to inhibition by malate. The sites phosphorylated in vitro were identical to those phosphorylated in intact tissue. The activity of the protein kinase was controlled in a circadian fashion. During normal diurnal cycles, kinase activity appeared between 4 and 5 h after the onset of darkness and disappeared 2----3 h before the end of darkness. Kinase activity displayed circadian oscillations in constant environmental conditions. The activity of protein phosphatase 2A, which dephosphorylates phosphoenolpyruvate carboxylase, did not oscillate. Treatment of detached leaves with the protein synthesis inhibitors puromycin and cycloheximide blocked the nocturnal appearance of the protein kinase activity, maintained phosphoenolypyruvate carboxylase in the dephosphorylated state and blocked the circadian rhythms of CO2 output that is observed in constant darkness and CO2-free air. The simplest explanation of the data is that there is a circadian rhythm in the synthesis of phosphoenolpyruvate carboxylase kinase.
Topics: Circadian Rhythm; Cycloheximide; Electrophoresis, Polyacrylamide Gel; Peptide Mapping; Phosphoenolpyruvate Carboxylase; Phosphorylation; Plants; Protein Kinase Inhibitors; Protein Kinases; Protein Serine-Threonine Kinases; Protein Synthesis Inhibitors; Puromycin
PubMed: 2065654
DOI: 10.1002/j.1460-2075.1991.tb07737.x