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Stem Cell Research & Therapy Mar 2022Spinal interneurons (INs) relay sensory and motor control information between the brain and body. When this relay circuitry is disrupted from injury or disease, it is...
BACKGROUND
Spinal interneurons (INs) relay sensory and motor control information between the brain and body. When this relay circuitry is disrupted from injury or disease, it is devastating to patients due to the lack of native recovery in central nervous system (CNS) tissues. Obtaining a purified population of INs is necessary to better understand their role in normal function and as potential therapies in CNS. The ventral V0 (V0) INs are excitatory neurons involved in locomotor circuits and are thus of interest for understanding normal and pathological spinal cord function. To achieve scalable amounts of V0 INs, they can be derived from pluripotent sources, such as mouse embryonic stem cells (mESCs), but the resultant culture is heterogenous, obscuring the specific role of V0 INs. This study generated a transgenic mESC line to enrich V0 INs from induced cultures to allow for a scalable, enriched population for future in vitro and in vivo studies.
METHODS
The transgenic Evx1-PAC mESC line was created by CRISPR-Cas9-mediated insertion of puromycin-N-acetyltransferase (PAC) into the locus of V0 IN marker Evx1. Evx1 and PAC mRNA expression were measured by qPCR. Viability staining helped establish the selection protocol for V0 INs derived from Evx1-PAC mESCs inductions. Immunostaining was used to examine composition of selected inductions. Cultures were maintained up to 30 days to examine maturation by expression of mature/synaptic markers, determined by immunostaining, and functional activity in co-cultures with selected motor neurons (MNs) and V2a INs on microelectrode arrays (MEAs).
RESULTS
V0 IN inductions were best selected with 4 µg/mL puromycin on day 10 to 11 and showed reduction of other IN populations and elimination of proliferative cells. Long-term selected cultures were highly neuronal, expressing neuronal nuclear marker NeuN, dendritic marker MAP2, pre-synaptic marker Bassoon, and glutamatergic marker VGLUT2, with some cholinergic VAChT-expressing cells. Functional studies on MEAs showed that co-cultures with MNs or MNs plus V2a INs created neuronal networks with synchronized bursting.
CONCLUSIONS
Evx1-PAC mESCs can be used to purify V0 IN cultures for largely glutamatergic neurons that can be used in network formation studies or for rodent models requiring transplanted V0 INs.
Topics: Animals; Homeodomain Proteins; Humans; Interneurons; Mice; Mice, Transgenic; Motor Neurons; Mouse Embryonic Stem Cells; Puromycin
PubMed: 35346349
DOI: 10.1186/s13287-022-02801-7 -
Journal of Pineal Research May 2011Melatonin, a naturally occurring molecule, is produced by the pineal gland in a circadian manner to regulate biologic rhythms in humans. Recent studies report that...
Melatonin, a naturally occurring molecule, is produced by the pineal gland in a circadian manner to regulate biologic rhythms in humans. Recent studies report that melatonin may be an attractive candidate as an anticancer agent or for combined therapy because of its antioxidant, oncostatic and immunoregulatory activities. In this study, the potentiating effect of melatonin was evaluated on the apoptosis induced by puromycin as an anticancer drug in acute promyelocytic leukemia HL-60 cells. Melatonin did not show significant cytotoxicity against HL-60 cells compared to puromycin. However, melatonin significantly augmented the cytotoxicity of puromycin. Consistently, combined treatment of melatonin and puromycin reduced the expression of anti-apoptotic proteins, such as bcl-2 and bcl-x(L) , and also induced caspase-3 activation and poly (ADP-ribose) polymerase (PARP) cleavage compared to puromycin treatment alone. Furthermore, cell cycle analysis revealed that melatonin promoted puromycin-induced apoptosis by increasing the sub-G1 population, but suppressing G2/M arrest in HL-60 cells. Interestingly, melatonin activated the phosphorylation of 5'-adenosine monophosphate-activated kinase (AMPK) in combination with puromycin. Taken together, our results suggest that melatonin potentiates puromycin-induced apoptosis with caspase-3 and AMPK activation in HL-60 cells, and thus, melatonin treatment can be effectively applied to leukemia treatment as a potential sensitizer for chemotherapeutic agents.
Topics: AMP-Activated Protein Kinases; Apoptosis; Blotting, Western; Cell Cycle; HL-60 Cells; Humans; Melatonin; Puromycin
PubMed: 21244482
DOI: 10.1111/j.1600-079X.2010.00852.x -
Journal of Biochemistry Mar 2021Monitoring translational regulation in response to environmental signals is crucial for understanding cellular proteostasis. However, only limited approaches are...
Monitoring translational regulation in response to environmental signals is crucial for understanding cellular proteostasis. However, only limited approaches are currently available for quantifying acute changes in protein synthesis induced by stimuli. Recently, a clickable puromycin analogue, O-propargyl-puromycin (OPP), was developed and applied to label the C-termini of nascent polypeptide chains (NPCs). Following affinity purification via a click reaction, OPP allows for a proteomic analysis of NPCs. Despite its advantage, the affinity purification of NPCs using magnetic beads or resins inherently suffers from significant non-specific protein binding, which hinders accurate quantification of the nascent proteins. To address this issue, we employed dual-pulse labelling of NPCs with both OPP and stable isotope-labelled amino acids to distinguish bona fide NPCs from non-specific proteins, thereby enabling the accurate quantitative profiling of NPCs. We applied this method to dissecting translation responses upon transcriptional inhibition and quantified ∼3,000 nascent proteins. We found that the translation of a subset of ribosomal proteins (e.g. RPSA, RPLP0) as well as signalling proteins (e.g. BCAR3, EFNA1, DUSP1) was significantly repressed by transcription inhibition. Together, the present method provides an accurate and broadly applicable nascent proteome profiling for many biological applications at the level of translation.
Topics: Amino Acids; HeLa Cells; Humans; Isotope Labeling; Mass Spectrometry; Protein Biosynthesis; Proteome; Proteomics; Puromycin
PubMed: 32926143
DOI: 10.1093/jb/mvaa104 -
Molecular Imaging and Biology Feb 2013The purpose of this study was to investigate whether (44)Sc-labeled puromycin can be utilized for imaging of protein synthesis in vivo.
PURPOSE
The purpose of this study was to investigate whether (44)Sc-labeled puromycin can be utilized for imaging of protein synthesis in vivo.
METHODS
For micro-positron emission tomographic (μPET) studies, 20-25 MBq of [(44)Sc]-DOTA-puromycin was administered to tumor-bearing rats, and animals were scanned for 1 h dynamically. Results were further validated by dissecting organs and tissues of the animals after the measurement and in vitro blocking experiments using puromycin or cycloheximide to block protein synthesis.
RESULTS
μPET images of tumor-bearing rats showed significant tumor uptake of [(44)Sc]-DOTA-puromycin and a clear-cut tumor visualization. In both blocking experiments, cellular uptake of [(44)Sc]-DOTA-puromycin ([(44)Sc]-DOTA-Pur) could be suppressed by blocking protein synthesis.
CONCLUSIONS
We report for the first time successful μPET imaging with (44)Sc obtained from a (44)Ti/(44)Sc generator, as well as noninvasive μPET imaging of ribosomal activity, respectively protein synthesis, with a puromycin-based radiopharmaceutical and the direct correlation between cellular uptake of [(44)Sc]-DOTA-Pur and protein synthesis.
Topics: Animals; Cell Line, Tumor; Heterocyclic Compounds, 1-Ring; Humans; Kinetics; Male; Molecular Imaging; Positron-Emission Tomography; Protein Biosynthesis; Proteins; Puromycin; Rats; Scandium; Tissue Distribution
PubMed: 22565849
DOI: 10.1007/s11307-012-0561-3 -
Bioorganic & Medicinal Chemistry Mar 2009We have synthesized a series of 5'-phosphorylated and 5'-cytidylyl-(3'-5')-cytidylyl-(3'-5')-puromycin derivatives that have backbone-elongated substrates. All the...
We have synthesized a series of 5'-phosphorylated and 5'-cytidylyl-(3'-5')-cytidylyl-(3'-5')-puromycin derivatives that have backbone-elongated substrates. All the synthesized puromycin derivatives showed good solubility in water and were applied to translation inhibitory assay in a reconstituted Escherichia coli translation system.
Topics: Chromatography, High Pressure Liquid; Escherichia coli; Phosphorylation; Protein Biosynthesis; Puromycin; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 19261481
DOI: 10.1016/j.bmc.2009.02.006 -
Life Sciences Feb 1976
Topics: Animals; Escherichia coli; Kidney; Male; Peptidyl Transferases; Proteinuria; Puromycin; Puromycin Aminonucleoside; Rats; Ribosomes; Structure-Activity Relationship
PubMed: 768682
DOI: 10.1016/0024-3205(76)90063-1 -
Biochemical and Biophysical Research... May 2016The amino-nucleoside antibiotic, puromycin, acts by covalently linking to elongating polypeptide chains on ribosomes to generate prematurely terminated immature...
The amino-nucleoside antibiotic, puromycin, acts by covalently linking to elongating polypeptide chains on ribosomes to generate prematurely terminated immature polypeptides. The trafficking of puromycin-conjugated (puromycylated) immature polypeptides within cell has, however, remained elusive. In this study, using O-propargyl-puromycin (OP-Puro), the distribution of puromycylated polypeptides was assessed in HeLa cells by click chemistry. Under standard culture conditions, OP-Puro signals were detected in the cytoplasm and nucleus with the highest concentrations in the nucleolus. Intriguingly, when proteasome activities were aborted using MG132, OP-Puro signals began to accumulate at promyelocytic leukemia nuclear bodies (PML-NBs) in addition to the nucleolus. We also found promiscuous association of OP-Puro signals with SUMO-2/3 and ubiquitin at PML-NBs, but not at the nucleolus, during abortive proteasome activities. This study reveals a previously unknown distribution of OP-Puro that argues for a nuclear function in regulating immature protein homeostasis.
Topics: Cell Nucleus; Click Chemistry; Intranuclear Inclusion Bodies; Promyelocytic Leukemia Protein; Proteasome Endopeptidase Complex; Puromycin; SUMO-1 Protein; Ubiquitin
PubMed: 27125456
DOI: 10.1016/j.bbrc.2016.03.155 -
The Journal of Organic Chemistry Dec 2008Conformationally locked North and South versions of puromycin analogues built on a bicyclo[3.1.0]hexane pseudosugar template were synthesized. The final assembly of the...
Conformationally locked North and South versions of puromycin analogues built on a bicyclo[3.1.0]hexane pseudosugar template were synthesized. The final assembly of the products was accomplished by the Staudinger-Vilarrasa coupling of the corresponding North (2 and 3) and South (6 and 7) 3'-azidopurine carbanucleosides with the Fmoc-protected 1-hydroxybenzotriazole ester of 4-methoxy-L-tyrosine. North azides 2 and 3 were reported earlier. The 3'-azido intermediates 6 and 7 that are necessary for the synthesis of the South puromycin analogues are described herein for the first time.
Topics: Antimetabolites, Antineoplastic; Chemistry, Organic; Chemistry, Pharmaceutical; Drug Design; Models, Chemical; Molecular Conformation; Nucleosides; Peptides; Puromycin; RNA, Transfer; Ribosomes
PubMed: 18991379
DOI: 10.1021/jo8016132 -
Journal of Medicinal Chemistry Mar 1981A series of ortho- and para-substituted L-phenylalanylpuromycin analogues were synthesized and evaluated as substrates for the peptidyltransferase reaction of...
A series of ortho- and para-substituted L-phenylalanylpuromycin analogues were synthesized and evaluated as substrates for the peptidyltransferase reaction of Escherichia coli ribosomes. Kinetic results reveal that substitution of the p-methoxy group of the puromycin molecule alters the peptidyltransferase activity of the molecule with the following decreasing order of substrate efficiencies: p-NH2 greater than p-NHCOCH3 greater than p-NO2 = p-NHCO(CH2)2CH3 greater than p-NHCOCH2Br. However, the inability of the ribosome to tolerate a nitro group at the ortho position of the phenylalanine ring precluded the use of the photosensitive puromycin analogue, 2-nitro-4-azidophenylalanylpuromycin aminonucleoside (7a), as a photoaffinity label for the peptidyltransferase site.
Topics: Acyltransferases; Binding Sites; Kinetics; Peptidyl Transferases; Puromycin; Ribosomes; Structure-Activity Relationship
PubMed: 7265117
DOI: 10.1021/jm00135a013 -
Antimicrobial Agents and Chemotherapy Jul 1973The incorporation of [(3)H]puromycin into nascent polypeptide chains of polyribosomes has proved to be a sensitive method of evaluating effects of inhibitors on peptide...
The incorporation of [(3)H]puromycin into nascent polypeptide chains of polyribosomes has proved to be a sensitive method of evaluating effects of inhibitors on peptide bond synthesis. Several analogues of puromycin were found to react with polyribosomes from both bacteria and rat liver. The K(m) for puromycin is 4 muM with bacterial polyribosomes; under the same conditions, the K(i) for psi-hydroxy-puromycin (6-dimethylamino-9-[3-(l-beta-phenyllactylamino)-3-deoxy-beta- d-ribofuranosyl] purine) is 240 muM and for a carbocyclic analogue of puromycin (6-dimethylamino-9- {R- [2R-hydroxy-3R- (p-methoxyphenyl-l-alanylamino)]-cyclopentyl}purine) is 1 muM. Both were found to be competitive inhibitors of puromycin. The K(m) for C-A-C-C-A(Phe) is 250 muM. In addition, the dissociation constant for C-A-C-C-A(Phe) binding to washed ribosomes was found to be 1 and 0.03 muM in the absence and presence, respectively, of 20% (vol/vol) ethanol. The results with these analogues lead to the following conclusions. Substitution of a hydroxyl group for the alpha-amino group of puromycin results in an active analogue with about one-sixtieth the affinity of puromycin in the reaction. Omission of the 5'-hydroxymethyl group or substitution of the furanosyl ring oxygen by a carbon atom in the carbocyclic analogue reduces its activity compared with puromycin only slightly. Additionally, the relatively high K(m) for C-A-C-C-A(Phe) as an acceptor compared with puromycin suggests the existence of a protective mechanism on polyribosomes, which prevents aminoacyl-transfer ribonucleic acid (tRNA) free in solution from stripping nascent chains from polyribosomes so that only aminoacyl-tRNA bound to ribosomes through the appropriate coding mechanism can form a peptide bond.
Topics: Animals; Binding, Competitive; Escherichia coli; In Vitro Techniques; Kinetics; Liver; Peptide Biosynthesis; Polyribosomes; Puromycin; Rats; Structure-Activity Relationship; Tritium
PubMed: 4598844
DOI: 10.1128/AAC.4.1.37