scholarly journals Inhibition of ribonucleic acid efflux from isolated SV40-3T3 cell nuclei by 3′-deoxyadenosine (cordycepin)

1979 ◽  
Vol 180 (2) ◽  
pp. 371-378 ◽  
Author(s):  
P S Agutter ◽  
B McCaldin
Keyword(s):  
Nuclear Envelope ◽  
Ribonucleic Acid ◽  
Actinomycin D ◽  
Cell Nuclei ◽  
Maximal Inhibition ◽  
Degree Of Protection ◽  
Substantial Degree ◽  
Stimulation Of ◽  
Nucleoside Triphosphatase

The effect of 3′-deoxyadenosine (cordycepin) on mRNA efflux from isolated SV40-3T3 cell nuclei has been studied and compared with its effect on the nucleoside triphosphatase activity in the isolated nuclear envelope. Inhibition of mRNA efflux occurs rapidly, but is dependent on the presence of ATP. Half-maximal inhibition occurs with 40 microM-cordycepin. The effect is not simulated by 2′-deoxyadenosine or by actinomycin D, and adenosine provides a substantial degree of protection against it. Cordycepin does not directly inhibit the nucleoside triphosphatase. The stimulation of this enzyme by poly(A) is not affected unless the poly(A) and cordycepin are incubated together with nuclear lysate in the presence of ATP; in this case the stimulation is significantly reduced. Possible interpretations of these results and their relevance for understanding the system in vivo for nucleo-cytoplasmic messenger transport are discussed.

scholarly journals Ribonucleic acid stimulation of mammalian liver nuclear-envelope nucleoside triphosphatase. A possible enzymic marker for the nuclear envelope

1977 ◽  
Vol 162 (3) ◽  
pp. 671-679 ◽  
Author(s):  
P S Agutter ◽  
J R Harris ◽  
I Stevenson
Keyword(s):  
Nuclear Envelope ◽  
Specific Activity ◽  
Assay Medium ◽  
Exogenous Rna ◽  
Mammalian Liver ◽  
High Concentrations ◽  
Enzymic Marker ◽  
Stimulation Of ◽  
Nucleoside Triphosphatase

1. The specific activity of rat and pig liver nuclear-envelope nucleoside triphosphatase (EC 3.6.1.3) decreases when the system is depleted of RNA. The activity can be restored by adding high concentrations of yeast RNA to the assay medium. 2. Exogenous RNA also increases the activity of the enzyme in control envelopes (not RNA-depleted). The effect appears to be largely specific for poly(A) and poly(G); it is not stimulated by rRNA or tRNA preparations, ribonuclease-hydrolysed RNA, AMP, or double- or single-stranded DNA. 3. Inhibitors of the enzyme, in concentrations at which half-maximal inhibition of the enzyme is achieved, do not affect the percentage stimulation of the enzyme by yeast RNA. 4. The simulation is abolished by the inclusion of 150 mM-KCl or -NaCl in the assay medium, but not by increasing the assay pH to 8.5. 5. The results are discussed in the light of the possible role of the nucleoside triphosphatase in vivo in nucleo-cytoplasmic ribonucleoprotein translocation. 6. It is proposed that poly(G)-stimulated Mg2+-activated adenosine triphosphatase activity should be adopted as an enzymic marker for the nuclear envelope.

Insulin sensitivity of rates of glycolysis and glycogen synthesis in soleus, stripped soleus, epitrochlearis, and hemi-diaphragm muscles isolated from sedentary rats

1983 ◽  
Vol 3 (7) ◽  
pp. 675-679 ◽  
Author(s):  
R. A. J. Challiss ◽  
J. Espinal ◽  
E. A. Newsholme
Keyword(s):  
Fatty Acid ◽  
Glucose Utilization ◽  
Glycogen Synthesis ◽  
Maximal Inhibition ◽  
Maximal Stimulation ◽  
Glucose Fatty Acid Cycle ◽  
Isolated Adipocytes ◽  
Stimulation Of

The effect of insulin concentrations on the rates of glycolysis and glycogen synthesis in four different in vitro rat muscle preparations (intact soleus, stripped soleus, epitrochlearis, and hemi-diaphragm) were investigated: the concentrations of insulin that produced half-maximal stimulation of the rates of these two processes in the four muscle preparations were similar – about 100 μunits/ml. This is at least 10-fold greater than the concentration that produced half-maximal inhibition of lipolysis in isolated adipocytes. Since 100 μunits/ml insulin is outside the normal physiological range in the rat, it is suggested that, in vivo, insulin influences glucose utilization in muscle mainly indirectly, via changes in the plasma fatty acid levels and the ‘glucose/fatty acid cycle’. Consequently the view that insulin stimulates glucose utilization in muscle mainly by a direct effect on membrane transport must be treated with caution.

scholarly journals ENZYME-MEMBRANE RELATIONSHIP IN PHENOBARBITAL INDUCTION OF SYNTHESIS OF DRUG-METABOLIZING ENZYME SYSTEM AND PROLIFERATION OF ENDOPLASMIC MEMBRANES

1966 ◽  
Vol 28 (2) ◽  
pp. 181-198 ◽  
Author(s):  
Sten Orrenius ◽  
Jan L. E. Ericsson
Keyword(s):  
Actinomycin D ◽  
Enzyme System ◽  
Drug Metabolizing Enzyme ◽  
Induction Process ◽  
Autophagic Activity ◽  
Metabolizing Enzyme ◽  
Parenchymal Cells ◽  
Phenobarbital Induction ◽  
Stimulation Of

The enzyme-membrane relationship in phenobarbital induction of synthesis of drug-metabolizing enzyme system and proliferation of endoplasmic membranes has been further studied. Ultrastructural observations suggest that newly formed endoplasmic membranes in rat liver parenchymal cells arise through continuous outgrowth and budding off from pre-existing cisternae and tubules of rough-surfaced endoplasmic reticulum. The membranes induced by phenobarbital treatment persist in the cytoplasm of the hepatocyte for up to 15 days after the last of a series of 5 phenobarbital injections; the phase of regression of the induced enzymes lasts for only 5 days. Disappearance of the membranes is gradual and does not seem to be associated with increased autophagic activity in the cell. A second series of injections of phenobarbital to previously induced rats—exhibiting normal drug-hydroxylating activity but an excess of liver endoplasmic membranes—is associated with a stimulation of the rate of Pi32 incorporation into microsomal phospholipid in vivo, similar to that found during the original induction process. Administration of Actinomycin D following a single phenobarbital injection delays the regression of the enhanced drug-hydroxylating activity. Finally, the effects of Actinomycin D and puromycin on the stimulated membrane formation are discussed.

scholarly journals Regulation of the synthesis of lutropin-induced protein in rat testis Leydig cells

1978 ◽  
Vol 170 (1) ◽  
pp. 9-15 ◽  
Author(s):  
Felix H. A. Janszen ◽  
Brian A. Cooke ◽  
Maria J. A. Van Driel ◽  
Henk J. Van Der Molen
Keyword(s):  
Protein Synthesis ◽  
Cyclic Amp ◽  
Leydig Cells ◽  
Actinomycin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Phosphodiesterase Inhibitor ◽  
Dibutyryl Cyclic Amp ◽  
Rat Testis ◽  
Stimulation Of

The mechanism of action of lutropin on the stimulation of the synthesis of a specific lutropin-induced protein in rat testis Leydig cells was investigated. Lutropin-induced protein has a mol.wt. of approx. 21000 and is detected by labelling the Leydig-cell proteins with [35S]methionine, followed by separation by polyacrylamide-gel electrophoresis and radioautography of the dried gel. The incorporation of35S into lutropin-induced protein was used as an estimate for the synthesis of the protein. Incubation of Leydig cells with dibutyryl cyclic AMP or cholera toxin also resulted in the stimulation of synthesis of the protein. Synthesis of lutropin-induced protein, when maximally stimulated with 100ng of lutropin/ml, could not be stimulated further by addition of dibutyryl cyclic AMP. Addition of 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, further increased synthesis of the protein in the presence of a submaximal dose of lutropin (10ng/ml) but not in the absence of lutropin or with maximal amounts of lutropin (100 and 1000ng/ml). Actinomycin D prevented the effect of lutropin on the stimulation of lutropin-induced protein synthesis when added immediately or 1h after the start of the incubation, but not when added after 5–6h. This is interpreted as reflecting that, after induction of mRNA coding for lutropin-induced protein, lutropin had no influence on the synthesis of the protein in the presence of actinomycin D. Synthesis of the protein was also stimulated in vivo by injection of choriogonadotropin into rats 1 day after hypophysectomy, and the time course of this stimulation of lutropin-induced protein synthesis in vivo was similar to that obtained by incubating Leydig cells in vitro with lutropin. From these results it is concluded that stimulation of lutropin-induced protein synthesis by lutropin is most probably mediated by cyclic AMP and involves synthesis of mRNA.

scholarly journals Importance of mammalian nuclear-envelope nucleoside triphosphatase in nucleo-cytoplasmic transport of ribonucleoproteins

1979 ◽  
Vol 182 (3) ◽  
pp. 811-819 ◽  
Author(s):  
P S Agutter ◽  
B McCaldin ◽  
H J McArdle
Keyword(s):  
Ionic Strength ◽  
Substrate Specificity ◽  
Nuclear Envelope ◽  
Nucleoside Triphosphate ◽  
Rna Transport ◽  
Isolated Nuclei ◽  
Cytoplasmic Rna ◽  
Cytoplasmic Transport ◽  
Nucleoside Triphosphatase

The nucleoside triphosphate-stimulated efflux of RNA from isolated nuclei was studied under a range of conditions, and the effects of these conditions on the process were compared with the properties of the nucleoside triphosphatase located in the pore complex. A marked similarity between the rate of efflux and the rate of nucleoside triphosphate hydrolysis was apparent, in terms of substrate specificity, sensitivity to treatment with insolubilized trypsin, kinetics and the effects of increased ionic strength and of many inhibitors. These results are taken, in view of earlier evidence, to suggest that the activity of the nucleoside triphosphatase is a prerequisite for nucleo-cytoplasmic RNA transport in vivo. There are some indications that the nuclear-envelope lipid is also involved in regulating the efflux process.

Binding of thyroid hormone to the goat testicular Leydig cell induces the generation of a proteinaceous factor which stimulates androgen release

1994 ◽  
Vol 143 (3) ◽  
pp. 549-556 ◽  
Author(s):  
N R Jana ◽  
S Bhattacharya
Keyword(s):  
Protein Synthesis ◽  
Leydig Cell ◽  
Leydig Cells ◽  
Actinomycin D ◽  
Competitive Inhibition ◽  
Binding Capacity ◽  
Cell Protein ◽  
Scatchard Analysis ◽  
Cell Nuclei ◽  
Stimulation Of

Abstract Leydig cells isolated from goat testis were sonicated and pure nuclear preparations obtained for 125I-3,5,3′-triiodothyronine (T3)-binding assay. Under optimum assay conditions of pH 7·2 at 37 °C and 90 min of incubation, binding of 125I-T3 to Leydig cell nuclei reached saturation at 1·2 nmol/l concentration. A Scatchard analysis of T3 binding exhibited a Kd of 0·535 × 10−9 mol/l and a maximum binding capacity of 1·25 pmol/mg DNA. Competitive inhibition studies showed T3 binding to be analogue specific. The physiological relevance of T3 binding to goat Leydig cell was examined by adding increasing concentrations of T3 to the Leydig cell incubation (1×10 cells/incubation). T3 (10, 25 and 50 ng/ml or 4, 10 and 20 ng/incubation) resulted a dose dependent increase in androgen release and in all cases stimulation of androgen release was statistically significant (P<0·01) compared with control. Stimulation of Leydig cell androgen release by T3 was significantly inhibited by actinomycin-D (P<0·01) and cycloheximide (P<0·01). T3 had additive stimulatory effects on LH-augmented androgen release from Leydig cells. T3 (50 ng/ml or 20 ng/incubation) effected a more than twofold increase in Leydig cell protein synthesis compared with control and both actinomycin-D and cycloheximide (50 μg/ml) inhibited it completely. The data indicated that the stimulatory effect of T3 on androgen release is mediated via T3-induced protein(s). Sub-cellular fractions obtained from T3-treated Leydig cells showed an increase in protein synthesis in mitochondrial and soluble supernatant fractions (100 k sup) and it was only 100 k sup which stimulated androgen release from Leydig cells in separate incubations. Treatment of 100 k sup with trypsin or heat abolished its stimulatory effect. Incubation of Leydig cells with T3 for different times showed an increase in protein synthesis prior to the stimulation of androgen release. The results therefore indicated that T3 binding to Leydig cells induced the generation of a proteinaceous factor(s) which in turn stimulated androgen release. Journal of Endocrinology (1994) 143, 549–556

Neurotensin inhibition of canine intestinal motility in vivo via α-adrenoceptors

1984 ◽  
Vol 62 (4) ◽  
pp. 403-411 ◽  
Author(s):  
Y. Sakai ◽  
E. E. Daniel ◽  
J. Jury ◽  
J. E. T. Fox
Keyword(s):  
Acetylcholine Release ◽  
Contractile Activity ◽  
Inhibitory Effect ◽  
Inhibitory Effects ◽  
Maximal Inhibition ◽  
Contractile Responses ◽  
Adrenergic Antagonists ◽  
Distal Site ◽  
Stimulation Of

Neurotensin given intra-arterially in bolus doses to the canine small intestine inhibited field-stimulated, atropine-sensitive contractile responses in the duodenum (mean effective dose (ED50) = 3.2 × 10−11 mol) and in the ileum (mean ED50 = 2.1 × 10−11 mol). Norepinephrine (ED50 = 3 × 10−9 mol) also inhibited these contractile responses. Phenylephrine (ED50 = 1.3 × 10−8 mol) was one-fourth as potent as norepinephrine and clonidine (ED50 = 8 × 10−10 mol) was at least as potent as norepinephrine, while isoproterenol (up to 8 × 10−8 mol) failed to show any inhibitory effects. Phentolamine (2 mg/kg) increased significantly the ED50 of neurotensin and norepinephrine. Prazosin (2 mg/kg) increased significantly the ED50 of norepinephrine in the duodenum but had no effect on the ED50 of neurotensin. Yohimbine (2 mg/kg) increased the ED50 values of neurotensin and adrenergic agonists. Both neurotensin and norepinephrine in doses causing maximal inhibition of field-stimulated responses decreased (by 40 to 60%) contractile responses to 9 × 10−10 mol (approximately the intra-arterial ED50 dose) of acetylcholine. Reserpine pretreatment markedly diminished the inhibition of spontaneous or field-stimulated phasic contractions by distention or field stimulation of a distal site. Reserpine also diminished the ED50 for neurotensin from 1 × 10−11 to 2 × 10−11 mol (p < 0.02), but did not abolish neurotensin's inhibitory effect. Tetrodotoxin (10–15 μg, intra-arterially) increased the dose of neurotensin required to inhibit spontaneous activity in the ileum but after this toxin, as after adrenergic antagonists or reserpine, maximal inhibition could still be obtained. These results suggested that neurotensin inhibited contractile activity of canine intestine by acting on neural receptors to release norepinephrine. Norepinephrine activated primarily α2-adrenoceptors and ultimately inhibited acetylcholine release. Neurotensin also inhibited contractions by activating a second, less sensitive receptor on smooth muscle.

scholarly journals Specific changes in the messenger ribonucleic acid content of the rat ventral prostate gland after androgenic stimulation. Evidence from the synthesis of aldolase messenger ribonucleic acid

1974 ◽  
Vol 144 (2) ◽  
pp. 413-426 ◽  
Author(s):  
W I P Mainwaring ◽  
F R Mangan ◽  
R A Irving ◽  
D A Jones
Keyword(s):  
Ribonucleic Acid ◽  
De Novo ◽  
Prostate Gland ◽  
Ventral Prostate ◽  
Mrna Synthesis ◽  
Hormonal Stimulation ◽  
Messenger Ribonucleic Acid ◽  
Stimulation Of

1. Aldolase was selected as a suitable marker for following the androgenic regulation of mRNA synthesis in the prostate gland. 2. Antibodies raised in rabbits against crystalline prostate aldolase were used to monitor the synthesis of this androgen-induced enzyme after hormonal stimulation of castrated animals, by using procedures in vivo and in vitro for the translation of prostate poly(A)-rich mRNA. 3. After androgenic stimulation in vivo the poly(A)-rich mRNA was isolated from the prostate gland and other tissues of castrated rats, and added to a protein-synthesizing system in vitro derived from Krebs II ascites-tumour cells. By using this approach it was found that androgens regulate the synthesis of aldolase mRNA in a highly tissue-specific manner. Stimulation of aldolase mRNA synthesis reached a maximum after 8h of androgenic treatment and then declined. 4. The androgenic control of aldolase mRNA synthesis was also investigated in vivo. After treatment of castrated animals with various steroids in vivo [35S]methionine was injected directly into the prostate gland, and labelled aldolase was selectively precipitated from isolated polyribosomes with anti-aldolase serum. The regulation of aldolase mRNA synthesis in the prostate gland was stringently steroid-specific and could only be evoked by androgens. After a single injection of testosterone, aldolase synthesis reached a maximum after 16h of hormonal stimulation and then declined. 5. Although androgens exert significant control over transcriptional processes in the prostate gland, and appear to regulate the synthesis of aldolase mRNA de novo, the possibility exists for additional means of control at the translational level of aldolase synthesis. The results are discussed in the context of the overall mechanism of action of androgens.

scholarly journals Corticotropin regulation of the synthesis of a specific rat adrenal cytosolic protein. Effects of hypophysectomy and actinomycin D

1979 ◽  
Vol 182 (3) ◽  
pp. 717-725 ◽  
Author(s):  
Alice Dazord ◽  
Dominique Gallet ◽  
Helene Cohen ◽  
Jose M. Saez
Keyword(s):  
Protein Synthesis ◽  
Cyclic Amp ◽  
Total Protein ◽  
Actinomycin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cytosolic Protein ◽  
And Control ◽  
Corticotropin Stimulation ◽  
Stimulation Of

The mechanism of corticotropin stimulation of the synthesis of a specific rat adrenal cytosolic protein was investigated. This protein (protein E) has a mol.wt. of approx. 30000. It is detected by polyacrylamide-gel electrophoresis of cytosol prepared from adrenal slices from rats treated with corticotropin in vivo and control rats, the slices being incubated with [3H]- and [14C]-leucine respectively. In rats 1–15 days after hypophysectomy, corticotropin, like dibutyryl cyclic AMP, induces an increase in protein E similar to that induced in control rats, even though both compounds no longer stimulate total protein synthesis. Corticotropin stimulation of protein E synthesis is mediated by cyclic AMP but not by corticosterone, since aminoglutethimide, a steroidogenic inhibitor, does not affect corticotropin stimulation, and dexamethasone alone has no effect. Actinomycin D, when injected in vivo 1h before or after corticotropin injection, prevents the effect of corticotropin on protein E synthesis, which is interpreted as evidence that mRNA synthesis is necessary for the stimulation of protein E synthesis. When injected more than 2h after corticotropin, actinomycin D does not prevent corticotropin stimulation of protein E synthesis, but completely blocks corticotropin stimulation of total protein synthesis. This is interpreted as meaning that, after stimulation of mRNA coding for protein E, corticotropin has no effect on the synthesis of protein E. On the other hand, corticotropin stimulation of protein E synthesis persists after hypophysectomy even though it no longer stimulates total protein synthesis. These data suggest that the factor(s) involved in the synthesis of protein E are more stable than those involved in total protein synthesis.

scholarly journals Studies of the ethylation of rat liver transfer ribonucleic acid after administration of l-ethionine

1972 ◽  
Vol 128 (1) ◽  
pp. 59-68 ◽  
Author(s):  
A. E. Pegg
Keyword(s):  
Rat Liver ◽  
Ribonucleic Acid ◽  
Major Part ◽  
Actinomycin D ◽  
Major Product ◽  
The Other ◽  
Methyl Donor ◽  
Principal Product

1. The ethylated nucleosides present in tRNA isolated from the livers of rats treated with 0.5g of l-ethionine/kg body wt. were investigated. Evidence that this tRNA contained N2-ethylguanine, N2N2-diethylguanine, N2-ethyl-N2-methylguanine, 7-ethylguanine, two ethylated pyrimidines and ethylated ribose groups was obtained. 2. Ethylation of bacterial tRNA was catalysed by extracts containing tRNA methylases prepared from rat liver by using S-adenosyl-l-ethionine as an ethyl donor, but the rate of ethylation was 20 times less than the rate of methylation with S-adenosyl-l-methionine as a methyl donor. 3. The principal product of such ethylation in vitro was N2-ethylguanine and traces of the other ethylated guanines and pyrimidines found in tRNA isolated from rats treated with ethionine in vivo were also found. 1-Ethyladenine was not formed, although 1-methyl-adenine is a major product of methylation of bacterial tRNA by these extracts, and 1-ethyladenine was not present in the rat liver tRNA isolated from ethionine-treated animals. 4. After injection of actinomycin D (15mg/kg body wt.) or l-methionine (1.0g/kg body wt.) before the ethionine, ethylation of tRNA was diminished by about 80% but not completely abolished. Administration of 1-aminocyclopentanecarboxylic acid (2.5g/kg body wt.) to inhibit the formation of S-adenosyl-l-ethionine inhibited ethylation of tRNA by 44%. 5. These results suggest that not all of the ethylation of tRNA that occurs in the livers of rats treated with ethionine is mediated by the action of tRNA methylases acting with S-adenosyl-l-ethionine as a substrate, but that this pathway does occur and accounts for a major part of the observed ethylation. 6. The results are discussed with reference to ethionine-induced hepatocarcinogenesis.

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