Human embryonic kidney HEK293T cells (CRL-11268) and human cervical cancer HeLa cells (CCL-2) from ATCC were grown in Dulbecco's modified Eagle's medium supplemented with 10% dialyzed fetal bovine serum (US origin, Life Technologies), and 1% penicillin–streptomycin. Cells were passaged at the confluency of 70%–90% for ∼10 passages to generate Q-depleted cell lines (designated as 0Q cells). Total RNA was isolated using the TRIzol method, and the tRNA queuosine modification level was determined by northern blot analysis with specific tRNA probes as described below. 0Q cell lines proliferated normally under standard culture conditions, and were stocked for subsequent experiments. To fully restore tRNA Q modification, Q-depleted cells were supplemented with queuine (Toronto Research Chemicals) at 1 µM for 24 h (designated as 100Q cells).
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Queuine
Queuine
Queuine is a naturally occurring modified nucleoside found in the anticodon of certain transfer RNAs.
It plays a role in regulating gene expression and has been linked to various biological processes, including cellular differentiation and metabolic control.
Queuine is synthesized by certain microorganisms and must be obtained from dietary sources in humans and other mammals.
Its presence in tRNAs is believed to enhance translational fidelity and influence cellular function.
Reserchers are investigating the potential therapeutic applications of queuine and its analogues in areas such as cancer, neurodegeneration, and metabolic disorders.
It plays a role in regulating gene expression and has been linked to various biological processes, including cellular differentiation and metabolic control.
Queuine is synthesized by certain microorganisms and must be obtained from dietary sources in humans and other mammals.
Its presence in tRNAs is believed to enhance translational fidelity and influence cellular function.
Reserchers are investigating the potential therapeutic applications of queuine and its analogues in areas such as cancer, neurodegeneration, and metabolic disorders.
Most cited protocols related to «Queuine»
Cell Lines
Cells
Cervical Cancer
Embryo
Fetal Bovine Serum
HeLa Cells
Homo sapiens
Kidney
Northern Blotting
Nucleoside Q
Penicillins
queuine
Streptomycin
Transfer RNA
trizol
Bisulfite sequencing of tRNAs was performed as described previously (11 (link),14 (link),28 (link)). Briefly, PCR amplicons of tRNAs were generated from bisulfite-treated total RNA of S. pombe or D. discoideum by reverse transcription using a tRNA 3′-specific stem-loop primer, followed by amplification with primers binding only to the deaminated sequences at the 5’ end. Subsequently, standard cloning of the PCR amplicons and sequencing of several independent subclones was performed. Primer sequences are listed in Supplementary Tables S3 and S4. For high-throughput RNA bisulfite sequencing of individual tRNAs from S. pombe, PCR products obtained after bisulfite treatment of total RNA, reverse transcription and PCR amplification were subjected to Illumina sequencing (28 (link)). Library preparation was performed by ligation of Illumina TrueSeq LT indexes to PCR products using T4 ligase (NEB). Post-ligation amplification was carried out with Herculase II Fusion DNA Polymerase (Agilent) for 8–12 cycles. A total of 150 bp paired-end sequencing was done using the Illumina MiSeq v3 platform. Reads were processed using in-house R scripting and the Bioconductor package ShortRead (29 (link)). Processing included trimming of PCR primers, selection of high quality reads and sorting of the reads based on the sequence in the degenerate region of the RT-primer. Bisulfite conversion in 1024 unique reads per sample were plotted.
Base Sequence
DNA Library
DNA Polymerase II
hydrogen sulfite
Ligase
Ligation
Oligonucleotide Primers
Reverse Transcription
Schizosaccharomyces pombe
Stem, Plant
The S. pombe and D. discoideum strains used in this study are shown in Supplementary Table S2. S. pombe strains were cultured in standard full medium (YES) (5 g/l yeast extract, 30 g/l glucose, 250 mg/l adenine, 250 mg/l histidine, 250 mg/l leucine, 250 mg/l uracil, 250 mg/l lysine). Gene knockouts in S. pombe and D. discoideum were obtained by homologous recombination, and correct knockout was verified by polymerase chain reaction (PCR) analysis (24 (link)–26 (link)). Primer sequences are listed in Supplementary Tables S3 and S4. All Dictyostelium strains used in this study are derived from strain Ax2–214. Cells were cultured in HL5 medium (Formedium) containing ampicillin (50 μg/ml), amphotericin-B (0.25 μg/ml), penicillin/ streptomycin (100 μg/ml) and chloramphenicol (34 μg/ml). Where required, antibiotics for selection (blasticidin S and/or G418 sulfate, 10 μg/ml each) were added. For RNA preparation, cells were grown shaking under continuous light at 22°C up to a cell density of 2 × 106 cells/ml. Queuine isolated from bovine amniotic fluid was added to S. pombe cultures in YES medium at 0.03 μM. D. discoideum cultures were grown for at least four days with 0.1 μM queuine, kindly provided by Klaus Reuter, Marburg (27 (link)).
Adenine
Amniotic Fluid
Amphotericin B
Ampicillin
antibiotic G 418
Antibiotics, Antitubercular
blasticidin S
Bos taurus
Cells
Chloramphenicol
Dictyostelium
Gene Knockout Techniques
Glucose
Histidine
Homologous Recombination
Leucine
Light
Lysine
Oligonucleotide Primers
Penicillins
Polymerase Chain Reaction
queuine
Schizosaccharomyces pombe
Strains
Streptomycin
Sulfates, Inorganic
Uracil
Yeast, Dried
Trophozoites (2 × 106/ml) were grown with and without queuine (0.1 μM for 3 days at 37°C) and were exposed or not to 2.5 mM H2O2 for 20 min at 37°C. After treatment with H2O2, the trophozoites were incubated with 10 μg/ml puromycin (Sigma) for 20 min. For pretreatment of the trophozoites with cycloheximide (Sigma), the trophozoites were incubated with 100 μg/ml cycloheximide for 5 min before the addition of puromycin. The trophozoites were lysed with 1% IGEPAL (Sigma) in phosphate-buffered saline (PBS) with protease inhibitors. puromycin was detected by immunoblotting as described above with a 12D10 clone monoclonal puromycin antibody (Millipore). The amount of total protein in each lane was determined by using the No-Stain protein labeling reagent (Thermo Fisher Scientific). Imaging for puromycin and total protein signals was performed on a FUSION FX7 EDGE imaging system (Witec AG). Quantification of signal density was performed using ImageJ.
Aftercare
Clone Cells
Cycloheximide
Monoclonal Antibodies
Peroxide, Hydrogen
Phosphates
Protease Inhibitors
Proteins
Puromycin
queuine
Saline Solution
Stains
Trophozoite
The queuine base was synthesized either as described previously26 (link), or with a modified synthesis strategy for the (1R,2S,3S)-1-bromo-2,3-O-isopropylidene-cyclopent-4-ene building block starting from inexpensive methyl α-D-galactopyranoside. This synthesis is described in detail in the supplementary information.
Anabolism
Cyclopent
Galactose
propylene
queuine
Most recents protocols related to «Queuine»
Queuine standard was dissolved in ultrapure water to prepare 1 mM stock solution.
Phosphate buffered saline (PBS) with 4% bovine serum albumin (BSA) was sued as surrogate matrix. Both q and Q stock solutions were aliquoted and stored at -20 ֯ C. Calibration standards (1, 0.3, 0.1, 0.03, 0.01, 0.003, 0.001 and 0.0003 µM), and 3 quality controls (QCs; 0.8, 0.02 and 0.0008 µM) were prepared freshly either in ultrapure water; or spiked in matrix surrogate and then extracted using a PBA cartridge on the day of analysis.
Phosphate buffered saline (PBS) with 4% bovine serum albumin (BSA) was sued as surrogate matrix. Both q and Q stock solutions were aliquoted and stored at -20 ֯ C. Calibration standards (1, 0.3, 0.1, 0.03, 0.01, 0.003, 0.001 and 0.0003 µM), and 3 quality controls (QCs; 0.8, 0.02 and 0.0008 µM) were prepared freshly either in ultrapure water; or spiked in matrix surrogate and then extracted using a PBA cartridge on the day of analysis.
The clinical scores for the mice were graphed by days post treatment. Day 0 was when the mouse reaches a clinical score of approximately 2 (identified upon inspection of the animals each morning). At this point the animal receives its first injection of control or queuine analogue. Each mouse was tracked individually, scored and weighed until the day after the last injection. Data was plotted using Prism Software (GraphPad, USA) and analysed using two-way ANOVA (i.e. variables of time and treatment) with multiple comparisons.
RASFC cultures from different patients (n = 6 compounds 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23; n = 3 compounds 21, 24, 25, 26) were treated with TNFα (10 ng mL−1) in the presence of controls or queuine analogues at a concentration of 200 μm. Culture media was replaced after 24 h and fresh media and compound were added. Cell supernatants were collected at the 72 h time point and tested for IL-6 secretion by ELISA. DMSO (4% in PBS), Tofacitinib (Xeljanz – Pfizer, Janus kinase inhibitor, 1 μM), IgG isotype antibody (1 μM) and Adalimumab (Humira – Abbvie, anti-TNF monoclonal antibody, 1 μM) served as experimental controls.
HEK293T cells were cultured in high-glucose (4.5g/l) DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% sodium pyruvate. Stress experiments were conducted in 96 well plates for cell viability assay by MTT or in 6 well plates or 10cm dishes when samples were collected for western blotting or sequencing. Cells were seeded 24 hours before the stress experiment and cultured either in regular media, or in serum free media with or without 1μM Queuine supplementation (Toronto research, Cat# Q525000) Cells were stressed by Arsenite (sodium metaArsenite), Rotenone (Respiratory complex I inhibitor), Thenoyltrifluoroacetone (TTFA; Respiratory complex II inhibitor), Antimycin A (Respiratory complex III inhibitor), potassium cyanide (KCN; Respiratory complex IV inhibitor), or Oligomycin (Respiratory complex V inhibitor) for the indicated doses and duration. Cell proliferation was conducted by passaging the cells at low concentrations into 96 wells or 6 wells plates (10,000 cells per well) and analyzing the cells over time using MTT or by cell counting by Trypan blue respectively.
The Enzyme Function Initiative (EFI) suite of web tools was used to generate the SSN (45 ). Visualization of SSNs was carried out using Cytoscape 3.10.1ape (46 (link)). 7,625 PF02592 family sequences were retrieved from UniProt using the family option with fraction of 3 and submitted to EFI. The initial SSN was generated with an alignment score cutoff set such that each connection (edge) represents a sequence identity of above approximately 40%. The obtained SSN was first colored according to the configurations for salvaging preQ1, preQ0, queuine, and Queuosine de novo synthesis. Then more stringent SSNs were created by increasing the alignment score cutoff in small increments (usually by 5). This process was repeated until most clusters were homogeneous in their colors. The UniProt IDs were associated with the genome ID including GenBank/EMBL, RefSeq nucleotide, BV-BRC genome ID, Ensembl genome ID, using homemade scripts (scripts available upon request). The UniProt IDs of PF02592 family sequences in the SSN are listed in Table S7 as well as corresponding presence of Q pathway genes. The connection between UniProt IDs and genome information was performed by querying UniProt ID mapping file using homemade scripts (scripts available upon request).
Top products related to «Queuine»
Sourced in United States
Queuine Hydrochloride is a chemical compound used in various laboratory applications. It serves as a precursor for the synthesis of the modified nucleoside queuine, which is found in certain transfer RNAs. The core function of Queuine Hydrochloride is to facilitate research and experiments involving the detection, quantification, or manipulation of queuine and its related biological processes.
Sourced in United States
Etoperidone Hydrochloride is a chemical compound used in research applications. It is a white to off-white crystalline powder. Etoperidone Hydrochloride is often used as a reference standard or intermediate in the development and analysis of pharmaceutical products.
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Ultra Culture Serum-free Medium is a cell culture medium designed for the growth and maintenance of a variety of cell lines. It is a chemically-defined, protein-free formulation that supports cell proliferation and viability without the need for animal-derived components.
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Penicillin/streptomycin is a commonly used antibiotic solution for cell culture applications. It contains a combination of penicillin and streptomycin, which are broad-spectrum antibiotics that inhibit the growth of both Gram-positive and Gram-negative bacteria.
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Thiamine hydrochloride is a water-soluble chemical compound that serves as a source of vitamin B1 (thiamine). It is commonly used in various laboratory applications, including as a nutrient supplement in cell culture media and as a component in biochemical assays and analyses.
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The pGEM-T Easy Vector is a high-copy-number plasmid designed for cloning and sequencing of PCR products. It provides a simple, efficient method for the insertion and analysis of PCR amplified DNA fragments.
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TRIzol reagent is a monophasic solution of phenol, guanidine isothiocyanate, and other proprietary components designed for the isolation of total RNA, DNA, and proteins from a variety of biological samples. The reagent maintains the integrity of the RNA while disrupting cells and dissolving cell components.
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TRIzol is a monophasic solution of phenol and guanidine isothiocyanate that is used for the isolation of total RNA from various biological samples. It is a reagent designed to facilitate the disruption of cells and the subsequent isolation of RNA.
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Protran BA83 is a laboratory filter membrane product. It is made of cellulose nitrate and has a pore size of 0.2 microns.
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Lipofectamine LTX is a transfection reagent used for the delivery of nucleic acids, such as plasmid DNA or RNA, into mammalian cells. It is designed to efficiently and gently introduce these molecules into the cells, enabling their expression or functional studies.
More about "Queuine"
Queuine is a naturally occurring modified nucleoside that plays a crucial role in regulating gene expression and various biological processes.
This unique compound is found in the anticodon of certain transfer RNAs (tRNAs) and is synthesized by specific microorganisms.
Humans and other mammals must obtain queuine from dietary sources.
The presence of queuine in tRNAs is believed to enhance translational fidelity and influence cellular function.
Researchers are actively investigating the potential therapeutic applications of queuine and its analogues, such as Queuine Hydrochloride, in areas like cancer, neurodegeneration, and metabolic disorders.
Queuine is closely related to other compounds like Etoperidone Hydrochloride, which have their own important roles in biological systems.
Additionally, specialized cell culture media like Ultra Culture Serum-free Medium and the use of antibiotics like Penicillin/Streptomycin can play a crucial role in maintaining cellular environments for queuine-related research.
To study queuine and its effects, researchers may employ various techniques and tools, including the use of the PGEM-T Easy vector, TRIzol reagent (or TRIzol) for RNA extraction, and Protran BA83 membranes for blotting.
Furthermore, transfection reagents like Lipofectamine LTX can facilitate the introduction of queuine-related genetic material into cells.
By understanding the intricacies of queuine and its associated compounds, researchers can uncover new insights, develop innovative therapies, and advance our knowledge of this fascinating modified nucleoside and its impact on human health and disease.
This unique compound is found in the anticodon of certain transfer RNAs (tRNAs) and is synthesized by specific microorganisms.
Humans and other mammals must obtain queuine from dietary sources.
The presence of queuine in tRNAs is believed to enhance translational fidelity and influence cellular function.
Researchers are actively investigating the potential therapeutic applications of queuine and its analogues, such as Queuine Hydrochloride, in areas like cancer, neurodegeneration, and metabolic disorders.
Queuine is closely related to other compounds like Etoperidone Hydrochloride, which have their own important roles in biological systems.
Additionally, specialized cell culture media like Ultra Culture Serum-free Medium and the use of antibiotics like Penicillin/Streptomycin can play a crucial role in maintaining cellular environments for queuine-related research.
To study queuine and its effects, researchers may employ various techniques and tools, including the use of the PGEM-T Easy vector, TRIzol reagent (or TRIzol) for RNA extraction, and Protran BA83 membranes for blotting.
Furthermore, transfection reagents like Lipofectamine LTX can facilitate the introduction of queuine-related genetic material into cells.
By understanding the intricacies of queuine and its associated compounds, researchers can uncover new insights, develop innovative therapies, and advance our knowledge of this fascinating modified nucleoside and its impact on human health and disease.