Inhibition of nucleoplasmic transcription and the translation of rapidly labeled nuclear proteins by low concentrations of actinomycin D in vivo. Proposed role of messenger RNA in ribosomal RNA transcription

Biochemistry ◽  
1978 ◽  
Vol 17 (7) ◽  
pp. 1154-1160 ◽  
Author(s):  
Thomas J. Lindell ◽  
Anthony F. O'Malley ◽  
Brian Puglisi
Keyword(s):  
Ribosomal Rna ◽  
Messenger Rna ◽  
Actinomycin D ◽  
Nuclear Proteins ◽  
Rna Transcription ◽  
Low Concentrations

Intranuclear Accumulation of Rna Resembling the Smaller Ribosomal Rna Component

1970 ◽  
Vol 6 (1) ◽  
pp. 53-72
Author(s):  
M. E. BRAMWELL
Keyword(s):  
Ribosomal Rna ◽  
Base Composition ◽  
Actinomycin D ◽  
Total Radioactivity ◽  
16S Ribosomal Rna ◽  
Low Concentrations ◽  
Rapid Accumulation ◽  
Nuclear Rna ◽  
Step Down ◽  
Nucleolar Rna

A study was made of the nuclear RNA in HeLa cells with particular reference to the rapidly labelled fractions. It was found that if cells were incubated at a high density, that is, under ‘step-down’ conditions, there was a rapid accumulation of RNA in the nucleus. The fraction of the nuclear RNA which includes rapidly labelled RNA and which binds tightly to columns of methylated albumin on kieselguhr increased in amount and reached levels which permitted enough of the material to be isolated for direct measurement of its base composition. This was found to be very similar to that of 16s ribosomal RNA. When cells growing logarithmically were treated with low concentrations of actinomycin D and then incubated in the presence of [3H]uridine it was found that an RNA fraction which bound tightly to methylated albumin on kieselguhr again accumulated in the nucleus. This fraction resembled that which accumulated under ‘step-down’ conditions. It contained over 85% of the total radioactivity in the nuclear RNA and again had a base composition very similar to 16s ribosomal RNA. Since nucleolar RNA synthesis was inhibited by the concentrations of actinomycin D used, it appeared that an RNA closely resembling 16s ribosomal RNA was synthesized outside the nucleolus. Sedimentation patterns on sucrose density gradients and thermal denaturation profiles lent support to the view that the RNA which binds tightly to columns of methylated albumin on kieselguhr probably represents ‘nascent’ 16s ribosomal RNA.

scholarly journals Impact of Deoxycholic Acid on Oesophageal Adenocarcinoma Invasion: Effect on Matrix Metalloproteinases

2020 ◽  
Vol 21 (21) ◽  
pp. 8042
Author(s):  
Fran Quilty ◽  
Anne-Marie Byrne ◽  
John Aird ◽  
Sheeren El Mashad ◽  
Adolfo Parra-Blanco ◽  
...  
Keyword(s):  
Matrix Metalloproteinases ◽  
Deoxycholic Acid ◽  
Therapeutic Strategies ◽  
Oesophageal Adenocarcinoma ◽  
Low Concentrations ◽  
Cancer Invasiveness ◽  
Mrna And Protein Expression

Bile acids (BAs) have been implicated in the development of oesophagitis, Barrett’s oesophagus and oesophageal adenocarcinoma (OAC). However, whether BAs promote cancer invasiveness has not been elucidated. We evaluated the role of BAs, in particular deoxycholic acid (DCA), in OAC invasion. Migration and invasiveness in untreated and BA-treated oesophageal SKGT-4 cancer cells were evaluated. Activity and expression of different matrix metalloproteinases (MMPs) were determined by zymography, ELISA, PCR and Western blot. Finally, human OAC tissues were stained for MMP-10 by immunohistochemistry. It was found that SKGT-4 cells incubated with low concentrations of DCA had a significant increase in invasion. In addition, MMP-10 mRNA and protein expression were also increased in the presence of DCA. MMP-10 was found to be highly expressed both in-vitro and in-vivo in neoplastic OAC cells relative to non-neoplastic squamous epithelial cells. Our results show that DCA promotes OAC invasion and MMP-10 overexpression. This study will advance our understanding of the pathophysiological mechanisms involved in human OAC and shows promise for the development of new therapeutic strategies.

scholarly journals The role of divalent cations in the regulation of microtubule assembly. In vivo studies on microtubules of the heliozoan axopodium using the ionophore A23187.

1976 ◽  
Vol 70 (3) ◽  
pp. 527-540 ◽  
Author(s):  
M Schliwa
Keyword(s):  
Divalent Cations ◽  
Light And Electron Microscopy ◽  
Microtubule Assembly ◽  
In Vivo Studies ◽  
Ionophore A23187 ◽  
Low Concentrations ◽  
Microtubule Formation

Low concentrations of calcium and magnesium ions have been shown to influence microtubule assembly in vitro. To test whether these cations also have an effect on microtubules in vivo, specimens of Actinosphaerium eichhorni were exposed to different concentrations of Ca++ and Mg++ and the divalent cation ionophore A23187. Experimental degradation and reformation of axopodia were studied by light and electron microscopy. In the presence of Ca++ and the ionophore axopodia gradually shorten, the rate of shortening depending on the concentrations of Ca++ and the ionophore used. Retraction of axopodia was observed with a concentration of Ca++ as low as 0.01 mM. After transfer to a Ca++-free solution containing EGTA, axopodia re-extend; the initial length is reached after about 2 h. Likewise, reformation of axopodia of cold-treated organisms is observed only in solutions of EGTA or Mg++, whereas it is completely inhibited in a Ca++ solution. Electron microscope studies demonstrate degradation of the axonemal microtubular array in organisms treated with Ca++ and A23187. No alteration was observed in organisms treated with Mg++ or EGTA plus ionophore. The results suggest that, in the presence of the ionophore, formation of axonemal microtubules can be regulated by varying the Ca++ concentration in the medium. Since A23187 tends to equilibrate the concentrations of divalent cations between external medium and cell interior, it is likely that microtubule formation invivo is influenced by micromolar concentrations of Ca++. These concentrations are low enough to be of physiological significance for a role in the regulation of microtubule assembly in vivo.

scholarly journals Up-regulation of MicroRNA-21 Mediates Isoflurane-induced Protection of Cardiomyocytes

2015 ◽  
Vol 122 (4) ◽  
pp. 795-805 ◽  
Author(s):  
Jessica M. Olson ◽  
Yasheng Yan ◽  
Xiaowen Bai ◽  
Zhi-Dong Ge ◽  
Mingyu Liang ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Messenger Rna ◽  
Functional Role ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Cell Death Protein

Abstract Background: Anesthetic cardioprotection reduces myocardial infarct size after ischemia–reperfusion injury. Currently, the role of microRNA in this process remains unknown. MicroRNAs are short, noncoding nucleotide sequences that negatively regulate gene expression through degradation or suppression of messenger RNA. In this study, the authors uncovered the functional role of microRNA-21 (miR-21) up-regulation after anesthetic exposure. Methods: MicroRNA and messenger RNA expression changes were analyzed by quantitative real-time polymerase chain reaction in cardiomyocytes after exposure to isoflurane. Lactate dehydrogenase release assay and propidium iodide staining were conducted after inhibition of miR-21. miR-21 target expression was analyzed by Western blot. The functional role of miR-21 was confirmed in vivo in both wild-type and miR-21 knockout mice. Results: Isoflurane induces an acute up-regulation of miR-21 in both in vivo and in vitro rat models (n = 6, 247.8 ± 27.5% and 258.5 ± 9.0%), which mediates protection to cardiomyocytes through down-regulation of programmed cell death protein 4 messenger RNA (n = 3, 82.0 ± 4.9% of control group). This protective effect was confirmed by knockdown of miR-21 and programmed cell death protein 4 in vitro. In addition, the protective effect of isoflurane was abolished in miR-21 knockout mice in vivo, with no significant decrease in infarct size compared with nonexposed controls (n = 8, 62.3 ± 4.6% and 56.2 ± 3.2%). Conclusions: The authors demonstrate for the first time that isoflurane mediates protection of cardiomyocytes against oxidative stress via an miR-21/programmed cell death protein 4 pathway. These results reveal a novel mechanism by which the damage done by ischemia/reperfusion injury may be decreased.

scholarly journals EFFECT OF ACTINOMYCIN D ON RNA SYNTHESIS AND ANTIBODY FORMATION IN THE ANAMNESTIC RESPONSE IN VITRO

1964 ◽  
Vol 119 (6) ◽  
pp. 881-893 ◽  
Author(s):  
J. Donald Smiley ◽  
John G. Heard ◽  
Morris Ziff
Keyword(s):  
Messenger Rna ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Lymphoid Cells ◽  
Limiting Factor ◽  
High Concentration ◽  
Antibody Synthesis ◽  
Anamnestic Response ◽  
Low Concentrations

Antibody synthesis in anamnestic lymphoid cells, measured by incorporation of leucine-C14 into specific antibody, was inhibited at moderate concentrations of actinomycin D. This was accompanied by marked inhibition of synthesis of RNA as measured by incorporation of H3-cytidine monophosphate. However, at low concentrations of actinomycin D, antibody synthesis was unaffected or even increased while RNA synthesis continued to be inhibited. The results obtained suggest that messenger RNA for antibody synthesis, either because it is relatively stable or present in excess, does not become a limiting factor until its synthesis is maximally inhibited. Puromycin, an inhibitor of amino acid coupling, abolished antibody synthesis in low concentration. 6-Mercaptopurine had no effect on the synthesis of antibody or RNA even at high concentration. The data obtained support the view that antibody synthesis follows pathways similar to those utilized for the formation of other types of proteins.

Role of exosite binding modulators in the inhibition of Fxa by TFPI

2016 ◽  
Vol 115 (03) ◽  
pp. 580-590 ◽  
Author(s):  
Alexandra Heinzmann ◽  
Tilman M. Hackeng ◽  
Rudolf Hartmann ◽  
Friedrich Scheiflinger ◽  
Michael Dockal ◽  
...  
Keyword(s):  
Plasma Proteins ◽  
Tissue Factor Pathway Inhibitor ◽  
Anticoagulant Activity ◽  
Protein S ◽  
Low Concentrations ◽  
Tissue Factor Pathway ◽  
Coagulation Pathway ◽  
Prothrombin Activation

SummaryTissue factor pathway inhibitor (TFPI) down-regulates the extrinsic coagulation pathway by inhibiting FXa and FVIIa. Both TFPI and FXa interact with several plasma proteins (e. g. prothrombin, FV/FVa, protein S) and non-proteinaceous compounds (e. g. phospholipids, heparin). It was our aim to investigate effects of ligands that bind to FXa and TFPI on FXa inhibition by full-length TFPI (designated TFPI) and truncated TFPI (TFPI1-150). Inhibition of FXa by TFPI and TFPI1-150 and effects of phospholipids, heparin, prothrombin, FV, FVa, and protein S thereon was quantified from progress curves of conversion of the FXa-specific chromogenic substrate CS11-(65). Low concentrations negatively charged phospholipids (~10 μM) already maximally stimulated (up to 5- to 6-fold) FXa inhibition by TFPI. Unfractionated heparin at concentrations (0.2–1 U/ml) enhanced FXa inhibition by TFPI ~8-fold, but impaired inhibition at concentrations > 1 U/ml. Physiological protein S and FV concentrations both enhanced FXa inhibition by TFPI 2- to 3-fold. In contrast, thrombin-activated FV (FVa) impaired the ability of TFPI to inhibit FXa. FXa inhibition by TFPI1–150 was not affected by FV, FVa, protein S, phospholipids and heparin. TFPI potently inhibited FXa-catalysed prothrombin activation in the absence of FVa, but hardly inhibited prothrombin activation in the presence of thrombin-activated FVa. In conclusion, physiological concentrations TFPI (0.25–0.5 nM TFPI) inhibit FXa with a t1/2 between 3–15 minutes. Direct FXa inhibition by TFPI is modulated by physiological concentrations prothrombin, FV, FVa, protein S, phospholipids and heparin indicating the importance of these modulators for the in vivo anticoagulant activity of TFPI.

scholarly journals LncRNA UCA1 facilitated cell growth and invasion through the miR-206/CLOCK axis in glioma

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Zhi Huang ◽  
Xuya Zhao ◽  
Xiaowen Wu ◽  
Lei Xiang ◽  
Yingnan Yuan ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Epithelial Mesenchymal Transition ◽  
Mesenchymal Transition ◽  
Brain Functions ◽  
Promising Therapeutic Target ◽  
Glioma Growth ◽  
Life Threatening ◽  
The Brain

Abstract Background Glioma is a lethal malignant brain tumor, which affects the brain functions and is life-threatening. LncRNA UCA1 was identified as a pivotal regulator for tumorigenesis of glioma. MiR-206 was discovered to promote tumorigenesis and is critical in the regulation of cell proliferation in glioma. This study will discuss the expression of UCA1 regarding miR-206 and CLOCK, and their integrative effects in the proliferation and cell cycle of glioma cells. Methods qRT-PCR was conducted to measure the mRNA expressions of IgG and Ago2 in cells co-transfected with UCA1, and miR-216 in U251. Bioinformation was analyzed for the prediction of association between UCA1 and miR-206. Transwell migrations assays and invasion assays were utilized to observe the cell invasive ability. Western blot and immunofluorescence imaging were used to examine the protein expressions. In vivo comparisons and observations were also performed to investigate the role of UCA1 in glioma growth. Results LncRNA UCA1 was up-regulated in glioma cell lines and tissues. It elevated cell invasion via the inducing of epithelial-mesenchymal transition. We found that UCA1 can modulate miR-206 expression and serve as an endogenous sponge of miR-206. The EMT-inducer CLOCK was validated as a messenger RNA target of miR-206. At last, we demonstrated that UCA1 exerted the biology function through regulating miR-206 and CLOCK in vivo. Conclusions Overall, the results demonstrated that UCA1/miR-206/CLOCK axis participated in the progressing of glioma and could act as a promising therapeutic target.

Mechanisms responsible for increasing immunoreactivity of apolipoprotein A-I with storage: the role of oxidation.

1989 ◽  
Vol 35 (10) ◽  
pp. 2082-2086 ◽  
Author(s):  
X L Wang ◽  
N P Dudman ◽  
J Wang ◽  
D E Wilcken
Keyword(s):  
Density Lipoprotein ◽  
Polyclonal Antiserum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Carrier Protein ◽  
Nonspecific Binding ◽  
Apolipoprotein A ◽  
Low Concentrations ◽  
Peroxidase Conjugate

Abstract Atherogenesis has been linked to low concentrations of high-density lipoprotein and its principal carrier protein, apolipoprotein (apo) A-I, in serum. We measured apo A-I by enzyme-linked immunosorbent assay, using polyclonal antiserum raised against purified apo A-I and conjugated to peroxidase. During storage at 4 degrees C and above, the immunoreactivity of apo A-I increased so that measured values in stored serum were difficult to interpret. The immunoreactivity of serum apo A-I also increased substantially (up to 50-fold) during exposure to NaIO4. This increase was apo A-I concentration dependent, was linearly dependent on the concentration of NaIO4 up to 200 mmol/L at pH 7.4 and 25 degrees C, and was not ascribable to nonspecific binding of antibody-peroxidase conjugate to the reaction wells. Treatment with NaIO4 caused apo A-I immunoreactivity to increase to a peak or plateau, then gradually decline. In 100 mmol/L NaIO4 (pH 7.4, 25 degrees C), this plateau occurred 30-60 min after oxidation commenced. When frozen serum and stored nonfrozen serum were both oxidized with NaIO4 under the same conditions, the apo A-I from the frozen serum had a significantly (P = 0.0004) greater increase in immunoreactivity. Sera from different patients with different apo A-I concentrations did not have proportionately increased immunoreactivity after treatment with NaIO4. Our findings suggest that the increase in apo A-I immunoreactivity on storage at 4 degrees C or higher involves atmospheric oxidation, and they raise the possibility of in vivo oxidized and nonoxidized forms of apo A-I.

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