-
Cell Reports Oct 2018Ribosome profiling, or Ribo-seq, is based on large-scale sequencing of RNA fragments protected from nuclease digestion by ribosomes. Thanks to its unique ability to...
Ribosome profiling, or Ribo-seq, is based on large-scale sequencing of RNA fragments protected from nuclease digestion by ribosomes. Thanks to its unique ability to provide positional information about ribosomes flowing along transcripts, this method can be used to shed light on mechanistic aspects of translation. However, current Ribo-seq approaches lack the ability to distinguish between fragments protected by either ribosomes in active translation or inactive ribosomes. To overcome this possible limitation, we developed RiboLace, a method based on an original puromycin-containing molecule capable of isolating active ribosomes by means of an antibody-free and tag-free pull-down approach. RiboLace is fast, works reliably with low amounts of input material, and can be easily and rapidly applied both in vitro and in vivo, thereby generating a global snapshot of active ribosome footprints at single nucleotide resolution.
Topics: Animals; Cell Line; High-Throughput Nucleotide Sequencing; Humans; Mice; Microspheres; Puromycin; RNA, Messenger; Ribosomes
PubMed: 30355487
DOI: 10.1016/j.celrep.2018.09.084 -
Scientific Reports Jul 2022Podocytes are highly specialized cells playing a key role in the filtration function of the kidney. A damaged podocyte ultrastructure is associated with a reorganization...
Podocytes are highly specialized cells playing a key role in the filtration function of the kidney. A damaged podocyte ultrastructure is associated with a reorganization of the actin cytoskeleton and accompanied with a loss of adhesion to the glomerular basement membrane leading to proteinuria in many forms of glomerular diseases, e.g. nephrotic syndrome. If the first-line therapy with glucocorticoids fails, alternative immunosuppressive agents are used, which are known to have the potential to stabilize the actin cytoskeleton. A new option for preventing relapses in steroid dependent nephrotic syndrome is the monoclonal antibody rituximab, which, in addition to its B-cell depleting effect, is assumed to have direct effects on podocytes. We here provide data on the non-immunological off-target effects of the immunosuppressant rituximab on podocyte structure and dynamics in an in vitro puromycin aminonucleoside model of podocyte injury. A conditionally immortalized human podocyte cell line was used. Differentiated podocytes were treated with puromycin aminonucleoside and rituximab. Our studies focussed on analyzing the structure of the actin cytoskeleton, cellular adhesion and apoptosis using immunofluorescence staining and protein biochemistry methods. Treatment with rituximab resulted in a stabilization of podocyte actin stress fibers in the puromycin aminonucleoside model, leading to an improvement in cell adhesion. A lower apoptosis rate was observed after parallel treatment with puromycin aminonucleoside and rituximab visualized by reduced nuclear fragmentation. Consistent with this data, Western-blot analyses demonstrated that rituximab directly affects the caspase pathways by inhibiting the activation of Caspases-8, -9 and -3, suggesting that rituximab may inhibit apoptosis. In conclusion, our results indicate an important role of the immunosuppressant rituximab in terms of stability and morphogenesis of podocytes, involving apoptosis pathways. This could help to improve therapeutical concepts for patients with proteinuria mediated by diseased podocytes.
Topics: Apoptosis; Cells, Cultured; Humans; Immunosuppressive Agents; Nephrotic Syndrome; Podocytes; Proteinuria; Puromycin; Puromycin Aminonucleoside; Rituximab
PubMed: 35853959
DOI: 10.1038/s41598-022-16333-w -
Cell Reports Methods Apr 2022The regulation of gene expression via protein translation is critical for growth, development, and stress response. While puromycin-based techniques have been used to...
The regulation of gene expression via protein translation is critical for growth, development, and stress response. While puromycin-based techniques have been used to quantify protein translation in , they have been limited to using lysate from whole worms. To achieve tissue-specific quantification of ribosome activity in intact , we report the application of O-propargyl-puromycin in a cuticle defective mutant followed by conjugation of an azide fluorophore for detection using fluorescent confocal microscopy. We apply this technique to quantify translation in response to heat shock, cycloheximide, or knockdown of translation factors Furthermore, we demonstrate that O-propargyl-puromycin can be used to quantify translation between tissues or within a tissue like the germline. This technique is expected to have a broad range of applications in determining how protein translation is altered in different tissues in response to stress or gene knockdowns or with age.
Topics: Animals; Caenorhabditis elegans; Protein Biosynthesis; Puromycin; Microscopy, Fluorescence
PubMed: 35497499
DOI: 10.1016/j.crmeth.2022.100203 -
Asian Pacific Journal of Cancer... May 2022Cancer is life-threatening disease and being global health problems. Chemotherapy is one of the most used therapy for cancer since many years ago. Chemotherapy is also...
UNLABELLED
Cancer is life-threatening disease and being global health problems. Chemotherapy is one of the most used therapy for cancer since many years ago. Chemotherapy is also toxic for normal cell, not specific to the target cells. Consequently, chemotherapy has various side effects. Monoclonal antibody (MAb) has been developed for specific therapy which only has killing effect in cancer cells, but the survival rate of most MAbs around 20%. Therefore, in clinical practice, MAbs administration should combine with chemotherapeutic agents. For effectiveness of therapy and to minimalize adverse effects, anticancer agent with selective cytotoxic effect on target cells is needed, the immunotoxin.
OBJECTIVE
This study introduces a novel approach to conjugate monoclonal antibody (Cetuximab) and toxin (Puromycin), in order to selectively inhibit proliferation of triple negative breast cancer (TNBC) and to enhance the efficacy of MAb in target cells killing.
METHODS
Cetuximab was conjugated with Puromycin using a linker, i.e SATP (Succinimidyl-acetylthiopropionate) and tested on triple negative breast cancer cell lines (MDA-MB-231) which expressed EGFR (epidermal growth factor receptor). Cetuximab is MAb which targets EGFR. MCF-7 was used as control cells since it has low or no EGFR expression. Cell counting were conducted as viability assay at 24 hours, 48 hours, and 72 hours after treatment.
RESULTS
The results showed significant reduction of live cells number in Conjugate 20 µg/mL cultured in MDA-MB-231 compared to MCF-7 after 24 hours, 48 hours, and 72 hours incubation. In all time period of incubation, significant reduction of MDA-MB-231 live cells number was also observed in Conjugate 20 µg/mL compared to Cetuximab 20 µg/mL.
CONCLUSION
Synthesized conjugate showed its target-specific effect in TNBC and improved the efficacy of Cetuximab on TNBC. In the future, this conjugate can be a potential anticancer therapy in treating triple-negative breast cancer.
Topics: Antibodies, Monoclonal; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cetuximab; ErbB Receptors; Humans; Puromycin; Triple Negative Breast Neoplasms
PubMed: 35633567
DOI: 10.31557/APJCP.2022.23.5.1803 -
International Journal of Medical... 2022Podocytes are specialized cells of the glomerulus that play important structural and functional roles in maintaining the filtration barrier. Loss and injury of podocytes...
Podocytes are specialized cells of the glomerulus that play important structural and functional roles in maintaining the filtration barrier. Loss and injury of podocytes are leading factors of glomerular disease and kidney failure. Recent studies found that phosphatase and tensin homolog (PTEN) may play a critical role in maintaining the normal structure and function in podocytes. However, we still understand very little about how PTEN is regulated under podocyte injury conditions. In this study, We therefore investigated whether PTEN could play a role in podocyte injury induced by puromycin aminonucleoside (PAN), and whether dexamethasone (DEX) alleviates podocyte injury by PTEN/PI3K/Akt signaling. Our results showed that PI3K/Akt pathway was activated in podocytes exposed to PAN conditions, accompanied by down-regulation of the PTEN and microtubule-associated light chain 3 (LC3) expression.podocyte-specific knockout of PTEN significantly promoted podocyte injury, The potential renoprotection of overexpressed PTEN in podocytes was partly attributed with an improvement in autophagy and the inhibition of apoptosis.These novel findings also suggest that targeting PTEN might be a novel and promising therapeutic strategy against podocyte injury.
Topics: Autophagy; Phosphatidylinositol 3-Kinases; Podocytes; Proto-Oncogene Proteins c-akt; Puromycin Aminonucleoside
PubMed: 36035365
DOI: 10.7150/ijms.72988 -
STAR Protocols Jun 2022Gene functions can be assessed in mouse embryonic stem (ES) cells and in mutant mice derived from mutant ES cells. Here, we describe an approach for efficient isolation...
Gene functions can be assessed in mouse embryonic stem (ES) cells and in mutant mice derived from mutant ES cells. Here, we describe an approach for efficient isolation of the ES clones carrying deletion mutations at the target genes by CRISPR-Cas9. Two sgRNAs against a target gene are co-expressed with puromycin-resistant gene in ES cells through co-transfection followed by transient puromycin selection. Deletion mutations are identified by PCR from individual ES clones that are picked from puromycin-selected ES cells.
Topics: Animals; CRISPR-Cas Systems; Embryonic Stem Cells; Mice; Mouse Embryonic Stem Cells; Puromycin; Transfection
PubMed: 35693210
DOI: 10.1016/j.xpro.2022.101436 -
Applied Biochemistry and Biotechnology Dec 2023Minimal change disease (MCD) is the most common cause of idiopathic nephrotic syndrome in children. The current major therapy is hormones for most steroid-sensitive...
Minimal change disease (MCD) is the most common cause of idiopathic nephrotic syndrome in children. The current major therapy is hormones for most steroid-sensitive patients. However, many patients have recurrent relapses of the disease and require long-term immunosuppression, leading to significant morbidity due to the side effects of the drugs. Therefore, better drugs need to be urgently explored to treat nephrotic syndrome while avoiding the side effects of drugs. Minnelide, a water-soluble prodrug of triptolide, has been proved to be effective in treating cancers in many clinical trials. This study aimed to investigate the therapeutic effect of minnelide in mice with adriamycin (ADR) nephropathy, its underlying protection mechanisms, and its reproductive toxicity. Minnelide was administered intraperitoneally to 6-8-week female mice with adriamycin nephropathy for 2 weeks, and the urine, blood, and kidney tissues were taken to analyze the therapeutic effect. In addition, we evaluated reproductive toxicity by measuring the levels of gonadal hormones and observing the histological changes in ovaries and testes. Primary mouse podocytes were exposed to puromycin (PAN) to damage the cytoskeleton and induce apoptosis, and then, triptolide was used to evaluate the therapeutic effect and underlying protection mechanisms in vitro. It was observed that minnelide dramatically alleviated proteinuria and apoptosis in mice with adriamycin nephropathy. In vitro, triptolide ameliorated puromycin-induced cytoskeletal rearrangement and apoptosis via reactive oxygen species-mediated mitochondrial pathway. In addition, minnelide caused no reproductive toxicity to male and female mice. The results suggested that minnelide might be a promising drug for nephrotic syndrome.
Topics: Humans; Child; Mice; Male; Female; Animals; Doxorubicin; Nephrotic Syndrome; Podocytes; Kidney Diseases; Proteinuria; Puromycin
PubMed: 37000351
DOI: 10.1007/s12010-023-04333-z -
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 -
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 -
ELife Aug 2020Puromycin is a tyrosyl-tRNA mimic that blocks translation by labeling and releasing elongating polypeptide chains from translating ribosomes. Puromycin has been used in...
Puromycin is a tyrosyl-tRNA mimic that blocks translation by labeling and releasing elongating polypeptide chains from translating ribosomes. Puromycin has been used in molecular biology research for decades as a translation inhibitor. The development of puromycin antibodies and derivatized puromycin analogs has enabled the quantification of active translation in bulk and single-cell assays. More recently, in vivo puromycylation assays have become popular tools for localizing translating ribosomes in cells. These assays often use elongation inhibitors to purportedly inhibit the release of puromycin-labeled nascent peptides from ribosomes. Using in vitro and in vivo experiments in various eukaryotic systems, we demonstrate that, even in the presence of elongation inhibitors, puromycylated peptides are released and diffuse away from ribosomes. Puromycylation assays reveal subcellular sites, such as nuclei, where puromycylated peptides accumulate post-release and which do not necessarily coincide with sites of active translation. Our findings urge caution when interpreting puromycylation assays in vivo.
Topics: Animals; Caenorhabditis elegans; Cell Nucleus; Emetine; Peptides; Protein Biosynthesis; Protein Synthesis Inhibitors; Puromycin; RNA, Transfer; Rabbits; Ribosomes; Single-Cell Analysis
PubMed: 32844748
DOI: 10.7554/eLife.60303