Synthesis and evaluation of stability of m3G-CAP analogues in serum-supplemented medium and cytosolic extract

Although the entry of DNA into the nucleus is a crucial step of non-viral gene delivery, fundamental features of this transport process have remained unexplored. This study analyzed the effect of linear double stranded DNA size on its passive diffusion, its active transport and its NLS-assisted transport. The size limit for passive diffusion was found to be between 200 and 310 bp. DNA of 310–1500 bp entered the nuclei of digitonin treated cells in the absence of cytosolic extract by an active transport process. Both the size limit and the intensity of DNA nuclear transport could be increased by the attachment of strong nuclear localization signals. Conjugation of a 900 bp expression cassette to nuclear localization signals increased both its nuclear entry and expression in microinjected, living cells.

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Modulation of fibroblast motility by a cytosolic extract of cyanobacteria

Archives of Biochemistry and Biophysics ◽

10.1016/0003-9861(91)90011-7 ◽

1991 ◽

Vol 286 (1) ◽

pp. 85-93 ◽

Cited By ~ 2

Author(s):

Marie-Laure Hodara ◽

Marie-Hélène Bré ◽

Haniaa Bouzinba ◽

Monique Pouphile ◽

Marcelle Lefort-Tran

Keyword(s):

Cytosolic Extract

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Inhibition of DNA replication by fish oil-treated cytoplasm is counteracted by fish oil-treated nuclear extract

AJP Cell Physiology ◽

10.1152/ajpcell.00121.2002 ◽

2002 ◽

Vol 283 (5) ◽

pp. C1365-C1375 ◽

Cited By ~ 10

Author(s):

Sybille Rex ◽

Maria A. Kukuruzinska ◽

Nawfal W. Istfan

Keyword(s):

Fatty Acids ◽

Dna Replication ◽

Dna Synthesis ◽

Fish Oil ◽

Corn Oil ◽

Nuclear Extract ◽

Inhibitory Effect ◽

Free System ◽

Nuclear Factors ◽

Cytosolic Extract

We have recently noted that cells treated with fish oil and n–3-fatty acids show slower DNA replication rates than cells treated with a control emulsion or corn oil only. However, it is not clearly understood how such an effect is induced. Fish oil and its metabolites are known to have several modulating effects on signal transduction pathways. Alternatively, they may influence DNA replication by interacting directly with nuclear components. To investigate this problem in greater detail, we have studied the kinetics of DNA synthesis in a cell-free system derived from HeLa cells. Nuclei and cytosolic extract were isolated from cells synchronized in early S phase after treatment with control emulsion, corn oil, or fish oil, respectively. The nuclei were reconstituted with cytosolic extract and a reaction mixture containing bromodeoxyuridine (BrdU) triphosphate to label newly synthesized DNA. The rate of DNA synthesis was measured by bivariate DNA/BrdU analysis and flow cytometry. We show that fish oil-treated cytosol inhibits the elongation of newly synthesized DNA by ∼80% in control nuclei. However, nuclei treated with fish oil escape this inhibitory effect. We also show that addition of nuclear extract from fish oil-treated cells reverses the inhibitory effect seen in the reconstitution system of control nuclei and fish oil-treated cytosol. These results indicate that polyunsaturated fatty acids can modulate DNA synthesis through cytosolic as well as soluble nuclear factors.

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Binding of ADP to rat liver cytosolic proteins and its influence on the ratio of free ATP/free ADP

Biochemical Journal ◽

10.1042/bj2590117 ◽

1989 ◽

Vol 259 (1) ◽

pp. 117-124 ◽

Cited By ~ 17

Author(s):

S Mörikofer-Zwez ◽

P Walter

Keyword(s):

Ethylene Glycol ◽

Rat Liver ◽

Binding Sites ◽

Dissociation Constants ◽

Cellular Protein ◽

Poly Ethylene Glycol ◽

Cytosolic Proteins ◽

Kd Values ◽

Cytosolic Extract ◽

Poly Ethylene

In a cytosolic extract from rat liver, the number and the concentration of ADP-binding sites as well as their dissociation constants were determined by using the rate-of-dialysis technique. Interfering cytosolic adenylate kinase was extracted from the cytosol by affinity chromatography on Ap5A-agarose, and remaining traces of enzyme activity were inhibited with (+)-catechin. Binding of ADP to cytosolic proteins was increased by poly(ethylene glycol) and decreased by EDTA. The effect of 0.1 mM-EDTA could be reversed by addition of equimolar concentrations of Mn2+ or Mg2+. In presence of 5% poly(ethylene glycol), added to increase local protein concentration, two binding sites for ADP were observed, with KD values of 1.9 microM (site I) and 10.8 microM (site II). The concentration of these binding sites, when extrapolated to cellular protein concentrations, were 30 microM (site I) and 114 microM (site II). It is concluded that a minimum of about 50% of total cytosolic ADP is bound to proteins, and that the ratio of free ATP/free ADP is at least twice that of total ATP/total ADP.

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Downregulation of the Ah receptor in mouse hepatoma cells treated in culture with 2,3,7,8-tetrachlorodibenzo-p-dioxin

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y91-176 ◽

1991 ◽

Vol 69 (8) ◽

pp. 1204-1210 ◽

Cited By ~ 44

Author(s):

Rebecca D. Prokipcak ◽

Allan B. Okey

Keyword(s):

Nuclear Receptor ◽

Specific Binding ◽

Hepatoma Cells ◽

Ah Receptor ◽

Short Term ◽

Mouse Hepatoma ◽

Receptor Sites ◽

Short Term Exposure ◽

Cytosolic Extract ◽

Tetrachlorodibenzo P Dioxin

The aromatic hydrocarbon (Ah) receptor behaves as a ligand-dependent transcription factor in the induction of cytochrome P450IA1. In cells exposed to the Ah receptor ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the Ah receptor undergoes a transformation from a form with low affinity for nucleic acids (cytosolic receptor) into a form that preferentially associates with the cell nucleus (nuclear receptor). We followed the fate of the Ah receptor in mouse hepatoma cells during short-term exposure to [3H]TCDD by analyzing both cytosolic and nuclear fractions for specific binding. Nuclear Ah receptor levels increased over the first 2 h of treatment and then decreased to about 50% of maximal concentrations by 5 h after start of treatment. The decrease in nuclear receptor was not accompanied by a reappearance of detectable Ah receptor in the cytosolic fraction; further incubation with [3H]TCDD in cytosols from lysed cells did not label any additional receptor sites in cytosolic extract. By the 6th h of incubation, the total receptor population in the cell was only about 15–20% of that detected at the start of the incubation. The levels of specific binding detected were unaffected by up to 20 h of incubation with the vehicle DMSO, confirming that the presence of TCDD is required for the observed downregulation to occur. These results indicate that there is a substantial ligand-dependent loss in total Ah receptor during short-term exposure of cells to TCDD in culture.Key words: Ah receptor, 2,3,7,8-tetrachlorodibenzo-p-dioxin, dioxin toxicity, Hepa-1 mouse hepatoma cells.

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An mRNA Stability Complex Functions with Poly(A)-Binding Protein To Stabilize mRNA In Vitro

Molecular and Cellular Biology ◽

10.1128/mcb.19.7.4552 ◽

1999 ◽

Vol 19 (7) ◽

pp. 4552-4560 ◽

Cited By ~ 155

Author(s):

Zuoren Wang ◽

Nancy Day ◽

Panayiota Trifillis ◽

Megerditch Kiledjian

Keyword(s):

Mrna Stability ◽

Binding Protein ◽

Major Protein ◽

Globin Mrna ◽

Ideal System ◽

Complex Functions ◽

Cytosolic Extract ◽

Protein Components ◽

The Stability

ABSTRACT The stable globin mRNAs provide an ideal system for studying the mechanism governing mammalian mRNA turnover. α-Globin mRNA stability is dictated by sequences in the 3′ untranslated region (3′UTR) which form a specific ribonucleoprotein complex (α-complex) whose presence correlates with mRNA stability. One of the major protein components within this complex is a family of two polycytidylate-binding proteins, αCP1 and αCP2. Using an in vitro-transcribed and polyadenylated α-globin 3′UTR, we have devised an in vitro mRNA decay assay which reproduces the α-complex-dependent mRNA stability observed in cells. Incubation of the RNA with erythroleukemia K562 cytosolic extract results in deadenylation with distinct intermediates containing a periodicity of approximately 30 nucleotides, which is consistent with the binding of poly(A)-binding protein (PABP) monomers. Disruption of the α-complex by sequestration of αCP1 and αCP2 enhances deadenylation and decay of the mRNA, while reconstitution of the α-complex stabilizes the mRNA. Similarly, PABP is also essential for the stability of mRNA in vitro, since rapid deadenylation resulted upon its depletion. An RNA-dependent interaction between αCP1 and αCP2 with PABP suggests that the α-complex can directly interact with PABP. Therefore, the α-complex is an mRNA stability complex in vitro which could function at least in part by interacting with PABP.

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Nutrient-sensing mTOR-mediated pathway regulates leptin production in isolated rat adipocytes

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00230.2002 ◽

2003 ◽

Vol 284 (2) ◽

pp. E322-E330 ◽

Cited By ~ 67

Author(s):

Cecilia Roh ◽

Jianrong Han ◽

Alexandros Tzatsos ◽

Konstantin V. Kandror

Keyword(s):

Endoplasmic Reticulum ◽

Food Intake ◽

Mammalian Target Of Rapamycin ◽

Heavy Fraction ◽

Nutrient Sensing ◽

Rat Adipocytes ◽

Cytosolic Extract ◽

Adipose Cells ◽

Isolated Rat

Leptin biosynthesis in adipose cells in vivo is increased by food intake and decreased by food deprivation. However, the mechanism that couples leptin production to food intake remains unknown. We found that addition of leucine to isolated rat adipocytes significantly increased leptin production by these cells, suggesting that postprandial leptin levels may be directly regulated by dietary leucine. The effect of leucine was inhibited by rapamycin and not by actinomycin D. Besides, leucine administration did not increase the amount of leptin mRNA in adipocytes. Therefore, we concluded that leucine activates leptin expression in adipose cells at the level of translation via a mammalian target of rapamycin (mTOR)-mediated pathway. Because leptin is a secreted protein, its biosynthesis is compartmentalized on the endoplasmic reticulum. To analyze mTOR signaling in this subcellular fraction, we separated adipose cells by centrifugation into a heavy membrane fraction that includes virtually all endoplasmic reticulum and the cytosolic extract. Phosphorylation of the major mTOR targets, the ribosomal protein S6 and the translational inhibitor 4E-binding protein (BP)/phosphorylated heat- and acid-stable protein (PHAS)-1, was stimulated by leucine in the cytosolic extract, whereas, in the heavy fraction, S6 was constitutively phosphorylated and leucine only induced phosphorylation of 4E-BP/PHAS-1. We also found that 60–70% of leptin mRNA was stably associated with the heavy fraction, and leucine administration did not change the ratio between compartmentalized and free cytoplasmic leptin mRNA. We suggest that, in adipose cells, a predominant part of leptin mRNA is compartmentalized on the endoplasmic reticulum, and leucine activates translation of these messages via the mTOR/4E-BP/PHAS-1-mediated signaling pathway.

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Isotopic Fractionation During Reduction of Nitrate and Nitrite by Extracts of Spinach Leaves

Functional Plant Biology ◽

10.1071/pp9850631 ◽

1985 ◽

Vol 12 (6) ◽

pp. 631 ◽

Cited By ~ 38

Author(s):

SF Ledgard ◽

KC Woo ◽

FJ Bergersen

Keyword(s):

Nitrate Reductase ◽

Nitrite Reductase ◽

Spinacia Oleracea ◽

Reductase Activity ◽

Isotopic Fractionation ◽

Cytosolic Extract ◽

Nitrite Reductase Activity ◽

Reconstituted System ◽

No2 Reduction ◽

Nitrate And Nitrite

The isotopic fractionations of nitrogen during the reduction of NO3- and NO2- in a cytosolic fraction and in a chloroplast preparation from spinach (Spinacia oleracea L.) leaves were determined. The reduction of NO3- to NH3 was studied using a reconstituted system containing cytosolic extract and intact chloroplasts, while a chloroplast system was used for NO2- reduction. In the reconstituted systems the ratio of nitrate reductase activity to nitrite reductase activity had a large effect on the relative amounts of NO2- and NH3 formed. Ammonia predominated when the nitrate reductase to nitrite reductase activity ratio was 1 : 5 and this ratio was used in the isotopic fractionation studies. Significant isotopic fractionation of N was observed in the reconstituted system but not in the chloroplast system. This indicates that the observed isotopic fractionation was associated with the reduction of NO3- to NO2- by nitrate reductase. The isotopic fractionation (i.e. δ15Nproduct - δ15Nsubstrate) for this reaction was - 15‰.

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Purification and characterization of protein kinase C from the nematode Caenorhabditis elegans

Biochemical Journal ◽

10.1042/bj2820219 ◽

1992 ◽

Vol 282 (1) ◽

pp. 219-223 ◽

Cited By ~ 8

Author(s):

T Sassa ◽

J Miwa

Keyword(s):

Protein Kinase C ◽

Caenorhabditis Elegans ◽

Protein Kinase ◽

Column Chromatography ◽

Purification And Characterization ◽

C Elegans ◽

Kinase C ◽

Sds Page ◽

Cytosolic Extract

Protein kinase C (PKC) of Caenorhabditis elegans was identified by enzymatic activity and [3H]phorbol 12,13-dibutyrate binding after DEAE-Sephacel column chromatography of a crude cytosolic extract. Ca(2+)-dependent activation of nematode PKC was observed in the presence of phosphatidylserine. The enzyme was maximally activated by 1,2-dioleoylglycerol or phorbol 12-myristate 13-acetate in the presence of phosphatidylserine and Ca2+. Hydroxyapatite column chromatography showed only one peak of PKC activity with histone H1 and myelin basic protein as substrates. The enzyme was purified to near homogeneity by sequential chromatography on polylysine-agarose and phosphatidylserine affinity columns. The purified protein showed a molecular mass of 79 kDa on SDS/PAGE. The substrate specificity of the C. elegans enzyme was shown to be different from that of mammalian PKCs. Here we describe some of the properties of the nematode enzyme.

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