scholarly journals Physical change in cytoplasmic messenger ribonucleoproteins in cells treated with inhibitors of mRNA transcription.

1984 ◽  
Vol 4 (3) ◽  
pp. 415-423 ◽  
Author(s):  
G Dreyfuss ◽  
S A Adam ◽  
Y D Choi
Keyword(s):  
Protein Synthesis ◽  
Actinomycin D ◽  
Rrna Synthesis ◽  
Cross Linking ◽  
Protein Synthesis Inhibitor ◽  
Nuclear Staining ◽  
Mrna Synthesis ◽  
Intact Cells ◽  
In Cells

Exposure of intact cells to UV light brings about cross-linking of polyadenylated mRNA to a set of cytoplasmic proteins which are in direct contact with the mRNA in vivo. Substantial amounts of an additional protein of molecular weight 38,000 (38K) become cross-linked to the mRNA when cells are treated with inhibitors of mRNA synthesis (actinomycin D, camptothecin, and 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole) or after infection with vesicular stomatitis virus. Cordycepin, which inhibits polyadenylation but not mRNA synthesis, has no such effect. Inhibitors of protein synthesis and of rRNA synthesis are also without effect on 38K cross-linking to mRNA. The onset of the effect of inhibitors of mRNA synthesis on the UV cross-linkable interaction between mRNA and 38K is rapid and reaches a maximal level in less than 60 min, and it is completely and rapidly reversible. In cells treated with actinomycin D, the amount of 38K which becomes cross-linked to mRNA is proportional to the extent of inhibition of mRNA synthesis. The association of 38K with mRNA during transcriptional arrest does not require protein synthesis because simultaneous treatment with the protein synthesis inhibitor emetine does not interfere with it. The effectors which promote the interaction of 38K with mRNA do not affect the proteins which are in contact with polyadenylated heterogeneous nuclear RNA and do not markedly affect protein synthesis in the cell. The 38K protein can be isolated with the polyribosomal polyadenylated fraction from which it was purified, and monoclonal antibodies against it were prepared. Immunofluorescence microscopy shows mostly cytoplasmic and some nuclear staining. These observations demonstrate that commonly used inhibitors of transcription affect the physical state of messenger ribonucleoproteins in vivo.

Physical change in cytoplasmic messenger ribonucleoproteins in cells treated with inhibitors of mRNA transcription

1984 ◽  
Vol 4 (3) ◽  
pp. 415-423
Author(s):  
G Dreyfuss ◽  
S A Adam ◽  
Y D Choi
Keyword(s):  
Protein Synthesis ◽  
Actinomycin D ◽  
Rrna Synthesis ◽  
Cross Linking ◽  
Protein Synthesis Inhibitor ◽  
Nuclear Staining ◽  
Mrna Synthesis ◽  
Intact Cells ◽  
In Cells

Exposure of intact cells to UV light brings about cross-linking of polyadenylated mRNA to a set of cytoplasmic proteins which are in direct contact with the mRNA in vivo. Substantial amounts of an additional protein of molecular weight 38,000 (38K) become cross-linked to the mRNA when cells are treated with inhibitors of mRNA synthesis (actinomycin D, camptothecin, and 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole) or after infection with vesicular stomatitis virus. Cordycepin, which inhibits polyadenylation but not mRNA synthesis, has no such effect. Inhibitors of protein synthesis and of rRNA synthesis are also without effect on 38K cross-linking to mRNA. The onset of the effect of inhibitors of mRNA synthesis on the UV cross-linkable interaction between mRNA and 38K is rapid and reaches a maximal level in less than 60 min, and it is completely and rapidly reversible. In cells treated with actinomycin D, the amount of 38K which becomes cross-linked to mRNA is proportional to the extent of inhibition of mRNA synthesis. The association of 38K with mRNA during transcriptional arrest does not require protein synthesis because simultaneous treatment with the protein synthesis inhibitor emetine does not interfere with it. The effectors which promote the interaction of 38K with mRNA do not affect the proteins which are in contact with polyadenylated heterogeneous nuclear RNA and do not markedly affect protein synthesis in the cell. The 38K protein can be isolated with the polyribosomal polyadenylated fraction from which it was purified, and monoclonal antibodies against it were prepared. Immunofluorescence microscopy shows mostly cytoplasmic and some nuclear staining. These observations demonstrate that commonly used inhibitors of transcription affect the physical state of messenger ribonucleoproteins in vivo.

Effect of Some Metabolic Inhibitors on Vinblastine-Induced Ribosomal-Granular Material Complexes

1971 ◽  
Vol 29 ◽  
pp. 548-549
Author(s):  
Awtar Krishan ◽  
Dora Hsu
Keyword(s):  
Granular Material ◽  
Rna Synthesis ◽  
Culture Medium ◽  
Actinomycin D ◽  
Metabolic Inhibitors ◽  
Protein And Rna Synthesis ◽  
Apparent Reduction ◽  
Plant Alkaloids ◽  
In Cells

Cells exposed to antitumor plant alkaloids, vinblastine and vincristine sulfate have large proteinacious crystals and complexes of ribosomes, helical polyribosomes and electron-dense granular material (ribosomal complexes) in their cytoplasm, Binding of H3-colchicine by the in vivo crystals shows that they contain microtubular proteins. Association of ribosomal complexes with the crystals suggests that these structures may be interrelated.In the present study cultured human leukemic lymphoblasts (CCRF-CEM), were incubated with protein and RNA-synthesis inhibitors, p. fluorophenylalanine, puromycin, cycloheximide or actinomycin-D before the addition of crystal-inducing doses of vinblastine to the culture medium. None of these compounds could completely prevent the formation of the ribosomal complexes or the crystals. However, in cells pre-incubated with puromycin, cycloheximide, or actinomycin-D, a reduction in the number and size of the ribosomal complexes was seen. Large helical polyribosomes were absent in the ribosomal complexes of cells treated with puromycin, while in cells exposed to cycloheximide, there was an apparent reduction in the number of ribosomes associated with the ribosomal complexes (Fig. 2).

Abstract 365: Targeted Delivery of IGF-1 Following Acute Myocardial Infarction

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Raffay S Khan ◽  
Jay C Sy ◽  
Milton Brown ◽  
Mario D Martinez ◽  
Niren Murthy ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Acute Myocardial Infarction ◽  
Targeted Delivery ◽  
Coronary Artery Ligation ◽  
Nuclear Staining ◽  
Post Injection ◽  
Artery Ligation ◽  
Intact Cells ◽  
Double Stranded Dna

During acute myocardial infarction (MI) there is excessive necrosis of myocardial cells, leading to the release of large amounts of DNA, representing a potential target for drug delivery. Hoechst, a commonly used molecule for staining nuclei, binds to the minor groove of double-stranded DNA and can be functionalized to contain reactive groups such as free amines, sulfhydryls, and biotin moieties. Insulin-like growth factor-1 (IGF-1), a small molecule with a short half-life is protective immediately following MI, though there is potential for long-term toxicity and off-target effects. Therefore, we hypothesized that conjugating IGF-1 to Hoechst would increase targeting of IGF-1 to the injured myocardium. Hoechst-IGF1 (H-IGF1) was synthesized by binding Hoechst-biotin to biotinylated IGF-1 via a fluorescent streptavidin linker. Intact cells did not show nuclear staining with H-IGF1, while permeabilized cells had a significant increase in blue fluorescent Hoechst staining, indicating H-IGF1 was cell impermeable but could still bind DNA. Activity of H-IGF1 was demonstrated by Akt phosphorylation in cultured cardiac progenitor cells and was similar to native IGF-1. To determine in-vivo targeting of H-IGF1 to MI, mice underwent 30 minutes of coronary artery ligation followed by reperfusion (I/R). Six hours following MI, mice were injected intravenously with 70ng of H-IGF1, S-IGF1 (streptavidin bound IGF-1 only) or PBS followed by in vivo imaging at 30 and 120 minutes post-injection. At 30 minutes post-injection, we found 3.2% (2.2 of 70ng) of the injected dose of H-IGF1 in infarcted hearts compared with 1.8% (1.3 of 70ng) of S-IGF1 (n=5-7; p<0.05). To confirm that targeting of H-IGF1 was dependent on binding DNA, H-IGF1 pre-bound to double-stranded DNA was injected intravenously after I/R. This led to a significant (p<0.05) decrease in targeted IGF-1 levels. IGF-1 levels determined by ELISA 2 hours post-injection demonstrated a similar trend with increased targeting of H-IGF1 compared with S-IGF1 treated mice (4.2±0.6 ng vs. 2.4±0.2 ng; p<0.05). In conclusion, our data demonstrate that intravenous delivery of Hoechst-conjugated IGF-1 increases myocardial targeting. This provides a novel strategy for delivery of growth factors for the treatment of MI.

scholarly journals PPARγ regulates adipose triglyceride lipase in adipocytes in vitro and in vivo

2007 ◽  
Vol 293 (6) ◽  
pp. E1736-E1745 ◽  
Author(s):  
Erin E. Kershaw ◽  
Michael Schupp ◽  
Hong-Ping Guan ◽  
Noah P. Gardner ◽  
Mitchell A. Lazar ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Adipose Tissue ◽  
Lipid Metabolism ◽  
Protein Synthesis Inhibitor ◽  
Adipose Triglyceride Lipase ◽  
Dependent Manner ◽  
Triglyceride Lipase ◽  
Brown Adipose

Peroxisome proliferator-activated receptor-γ (PPARγ) regulates adipocyte genes involved in adipogenesis and lipid metabolism and is the molecular target for thiazolidinedione (TZD) antidiabetic agents. Adipose triglyceride lipase (ATGL) is a recently described triglyceride-specific lipase that is induced during adipogenesis and remains highly expressed in mature adipocytes. This study evaluates the ability of PPARγ to directly regulate ATGL expression in adipocytes in vitro and in vivo. In fully differentiated 3T3-L1 adipocytes, ATGL mRNA and protein are increased by TZD and non-TZD PPARγ agonists in a dose- and time-dependent manner. Rosiglitazone-mediated induction of ATGL mRNA is rapid and is not inhibited by the protein synthesis inhibitor cycloheximide, indicating that intervening protein synthesis is not required for this effect. Rosiglitazone-mediated induction of ATGL mRNA and protein is inhibited by the PPARγ-specific antagonist GW-9662 and is also significantly reduced following siRNA-mediated knockdown of PPARγ, supporting the direct transcriptional regulation of ATGL by PPARγ. In vivo, ATGL mRNA and protein are increased by rosiglitazone treatment in white and brown adipose tissue of mice with and without obesity due to high-fat diet or leptin deficiency. Thus, PPARγ positively regulates ATGL mRNA and protein expression in mature adipocytes in vitro and in adipose tissue in vivo, suggesting a role for ATGL in mediating PPARγ's effects on lipid metabolism.

scholarly journals The effect of inhibitors in vivo on protein synthesis and the amino acid pool in the sheep blowfly, Lucilia cuprina

1975 ◽  
Vol 150 (2) ◽  
pp. 227-234 ◽  
Author(s):  
A J Campbell ◽  
L M Birt
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Actinomycin D ◽  
Pool Size ◽  
Dynamic State ◽  
Amino Acid Pool ◽  
Pronounced Increase ◽  
Acid Pool ◽  
Quantitative Estimates

1. The rates of detoxification of cycloheximide (33 μg/g fresh wt.), puromycin (167 μg/g fresh wt.) and actinomycin D (1 μg/g fresh wt.) were assessed in vivo on the basis of acid-insoluble [14C]leucine incorporation in the sheep blowfly, Lucilla cuprina; these were compared with quantitative estimates which took account not only of incorporation data but also of leucine pool size and turnover. Quantitatively, cycloheximide and puromycin were still at least 50% effective in inhibiting protein synthesis after 6.5 and 24.5h of exposure respectively, whereas values based only on incorporation data suggested that cycloheximide was 83% effective and puromycin completely ineffective after the respective periods. Quantitative estimates also showed that actinomycin D effectiveness increased with increasing exposure over 24.5h, in contrast with values based only on incorporation data, which suggested that it was completely ineffective after 24h.2. All inhibitors affected the dynamic state of the amino acid pool; there was a marked decrease in the rate of leucine-pool turnover as well as an increase in the half-life of leucine in the pool. 3. Inhibition of protein synthesis resulted in changes in leucine-pool size; the most pronounced increase occurred with cycloheximide and puromycin and the most pronounced decreases with actinomycin D. 4. Evidence is presented which suggests that proteolysis is functionally linked to protein synthesis, which determines its rate indirectly.

scholarly journals In vivo evidence that TATA-binding protein/SL1 colocalizes with UBF and RNA polymerase I when rRNA synthesis is either active or inactive.

1996 ◽  
Vol 133 (2) ◽  
pp. 225-234 ◽  
Author(s):  
P Jordan ◽  
M Mannervik ◽  
L Tora ◽  
M Carmo-Fonseca
Keyword(s):  
Rna Polymerase ◽  
Actinomycin D ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Rna Polymerase I ◽  
Rrna Genes ◽  
Rrna Synthesis ◽  
Rrna Transcription ◽  
Polymerase I

Here we show that the TATA-binding protein (TBP) is localized in the nucleoplasm and in the nucleolus of mammalian cells, consistent with its known involvement in transcription by RNA polymerase I, II, and III. In the nucleolus of actively growing cells, TBP colocalizes with upstream binding factor (UBF) and RNA polymerase I at the sites of rRNA transcription. During mitosis, when rRNA synthesis is down-regulated, TBP colocalizes with TBP-associated factors for RNA polymerase I (TAF(I)s), UBF, and RNA polymerase I on the chromosomal regions containing the rRNA genes. Treatment of cells with a low concentration of actinomycin D inhibits rRNA synthesis and causes a redistribution of the rRNA genes that become concentrated in clusters at the periphery of the nucleolus. A similar redistribution was observed for the major components of the rRNA transcription machinery (i.e., TBP, TAF(I)s, UBF, and RNA polymerase I), which still colocalized with each other. Furthermore, anti-TBP antibodies are shown to coimmunoprecipitate TBP and TAF(I)63 in extracts prepared from untreated and actinomycin D-treated cells. Collectively, the data indicate that in vivo TBP/promoter selectivity factor, UBF, and RNA polymerase I remain associated with both active and inactive rRNA genes.

scholarly journals Composition of the λ plasmid heritable replicationcomplex

2002 ◽  
Vol 364 (3) ◽  
pp. 857-862 ◽  
Author(s):  
Katarzyna POTRYKUS ◽  
Sylwia BARAŃSKA ◽  
Alicja WĘGRZYN ◽  
Grzegorz WĘGRZYN
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Protein Synthesis ◽  
Plasmid Dna ◽  
Pcr Analysis ◽  
Cross Linking ◽  
Bacteriophage Λ ◽  
Yeast Saccharomyces Cerevisiae ◽  
Replication Complex

Previous studies indicated during replication of plasmids derived from bacteriophage λ (the so-called λ plasmids), that, once assembled, replication complex can be inherited by one of the two daughter plasmid copies after each replication round, and may function in subsequent replication rounds. It seems that similar processes occur during replication of other DNA molecules, including chromosomes of the yeast Saccharomyces cerevisiae. However, apart from some suggestions based on genetic experiments, composition of the λ heritable replication complex remains unknown. In amino acid-starved Escherichia coli relA mutants, replication of λ plasmid DNA is carried out exclusively by the heritable replication complex as assembly of new complexes is impaired due to inhibition of protein synthesis. Here, using a procedure based on in vivo cross-linking, cell lysis, immunoprecipitation with specific sera, de-cross-linking and PCR analysis, we demonstrate that the λ heritable replication complex consists of O, P, DnaB and, perhaps surprisingly, DnaK proteins.

Corticotropin-Releasing Factor Produces a Protein Synthesis–Dependent Long-Lasting Potentiation in Dentate Gyrus Neurons

2000 ◽  
Vol 83 (1) ◽  
pp. 343-349 ◽  
Author(s):  
Hai L. Wang ◽  
Li Y. Tsai ◽  
Eminy H. Y. Lee
Keyword(s):  
Protein Synthesis ◽  
Synaptic Transmission ◽  
Dentate Gyrus ◽  
Late Phase ◽  
Long Term Potentiation ◽  
Corticotropin Releasing Factor ◽  
Rat Hippocampus ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor

Corticotropin-releasing factor (CRF) was shown to produce a long-lasting potentiation of synaptic efficacy in dentate gyrus neurons of the rat hippocampus in vivo. This potentiation was shown to share some similarities with tetanization-induced long-term potentiation (LTP). In the present study, we further examined the mechanism underlying CRF-induced long-lasting potentiation in rat hippocampus in vivo. Results indicated that the RNA synthesis inhibitor actinomycin-D, at a concentration that did not change basal synaptic transmission alone (5 μg), significantly decreased CRF-induced potentiation. Similarly, the protein synthesis inhibitor emetine, at a concentration that did not affect hippocampal synaptic transmission alone (5 μg), also markedly inhibited CRF-induced potentiation. These results suggest that like the late phase of LTP, CRF-induced long-lasting potentiation also critically depend on protein synthesis. Further, prior maximum excitation of dentate gyrus neurons with tetanization occluded further potentiation of these neurons produced by CRF and vise versa. Moreover, quantitative reverse transcription-polymerase chain reaction analysis revealed that CRF mRNA level in the dentate gyrus was significantly increased 1 h after LTP recording. Together with our previous findings that CRF antagonist dose-dependently diminishes tetanization-induced LTP, these results suggest that both CRF-induced long-lasting potentiation and tetanization-induced LTP require protein synthesis and that CRF neurons are possibly involved in the neural circuits underlying LTP.

scholarly journals Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies.

1984 ◽  
Vol 4 (6) ◽  
pp. 1104-1114 ◽  
Author(s):  
G Dreyfuss ◽  
Y D Choi ◽  
S A Adam
Keyword(s):  
Monoclonal Antibodies ◽  
Uv Light ◽  
Protein Complexes ◽  
Cross Linking ◽  
Intact Cells ◽  
Molecular Weights ◽  
Nuclear Rna ◽  
Nonionic Detergent ◽  
Sufficient Intensity

Exposure of cells to UV light of sufficient intensity brings about cross-linking of RNA to proteins which are in direct contact with it in vivo. The major [35S]methionine-labeled proteins which become cross-linked to polyadenylated heterogeneous nuclear RNA in HeLa cells have molecular weights of 120,000 (120K), 68K, 53K, 43K, 41K, 38K, and 36K. Purified complexes of polyadenylated RNA with proteins obtained by UV cross-linking in intact cells were used to immunize mice and generate monoclonal antibodies to several of these proteins. Some properties of three of the proteins, 41K, 43K, and 120K, were characterized with these antibodies. The 41K and 43K polypeptides are highly related. They were recognized by the same antibody (2B12) and have identical isoelectric points (pl = 6.0 +/- 0.2) but different partial peptide maps. The 41K and 43K polypeptides were part of the 40S heterogeneous nuclear ribonucleoprotein particle and appear to correspond to the previously described C proteins (Beyer et al., Cell II:127-138, 1977). A different monoclonal antibody (3G6) defined a new major heterogeneous ribonucleoprotein of 120K. The 41K, 43K, and 120K polypeptides were associated in vivo with both polyadenylated and non-polyadenylated nuclear RNA, and all three proteins were phosphorylated. The monoclonal antibodies recognized similar proteins in human and monkey cells but not in several other vertebrates. Immunofluorescence microscopy demonstrated that these proteins are segregated to the nucleus, where they are part of a fine particulate nonnucleolar structure. In cells extracted in situ with nonionic detergent, all of the 41K and 43K polypeptides were associated with the nucleus at salt concentrations up to 0.5 M NaCl, whereas the 120K polypeptide was completely extracted at this NaCl concentration. A substantial fraction of the 41K and 43K polypeptides (up to 40%) was retained with a nuclear matrix--a structure which is resistant to digestion with DNase I and to extraction by 2 M NaCl, but the 41K and 43K polypeptides were quantitatively removed at 0.5 M NaCl after digestion with RNase.

scholarly journals Cross-linking peptide and repurposed drugs inhibit both entry pathways of SARS-CoV-2

2020 ◽  
Author(s):  
Hanjun Zhao ◽  
Hoiyan Lam ◽  
Xinxin Zhou ◽  
Kelvin To ◽  
Jasper Chan ◽  
...  
Keyword(s):  
Drug Combination ◽  
Endocytic Pathway ◽  
In Vivo Studies ◽  
Cross Linking ◽  
Fusion Inhibitor ◽  
Dual Functional ◽  
Endosomal Acidification ◽  
Repurposed Drugs ◽  
In Cells

Abstract So far, effective antivirals have not been widely available for treating COVID-19. In this study, we identify a dual-functional cross-linking peptide 8P9R which can inhibit the two entry pathways (endocytic pathway and TMPRSS2-mediated surface pathway) of SARS-CoV-2 in cells. The endosomal acidification inhibitors (8P9R and chloroquine) can synergistically enhance the activity of arbidol, a spike-ACE2 fusion inhibitor, against SARS-CoV-2 and SARS-CoV in cells. In vivo studies indicate that 8P9R or the combination of repurposed drugs (arbidol, chloroquine and camostat which is a TMPRSS2 inhibitor), simultaneously interfering with the two entry pathways of coronavirus, can significantly suppress SARS-CoV-2 replication in hamsters and SARS-CoV in mice. Here, we use drug combination (arbidol, chloroquine, and camostat) and a dual-functional 8P9R to demonstrate that blocking the two entry pathways of coronavirus can be a promising and achievable approach for inhibiting SARS-CoV-2 replication in vivo. Cocktail therapy of these drug combinations should be considered in treatment trials for COVID-19.

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