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).

Synthesis of acid-soluble spore proteins by Bacillus subtilis

1982 ◽  
Vol 152 (3) ◽  
pp. 1117-1125
Author(s):  
J M Leventhal ◽  
G H Chambliss
Keyword(s):  
Bacillus Subtilis ◽  
Maximum Rate ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Sodium Dodecyl ◽  
Molecular Weights ◽  
Spore Proteins ◽  
Major Acid

The major acid-soluble spore proteins (ASSPs) of Bacillus subtilis were detected by immunoprecipitation of radioactively labeled in vitro- and in vivo-synthesized proteins. ASSP synthesis in vivo began 2 h after the initiation of sporulation (t2) and reached its maximum rate at t7. This corresponded to the time of synthesis of mRNA that stimulated the maximum rate of ASSP synthesis in vitro. Under the set of conditions used in these experiments, protease synthesis began near t0, alkaline phosphatase synthesis began at about t2, and refractile spores were first observed between t7 and t8. In vivo- and in vitro-synthesized ASSPs comigrated in sodium dodecyl sulfate-polyacrylamide gels. Their molecular weights were 4,600 (alpha and beta) and 11,000 (gamma). The average half-life of the ASSP messages was 11 min when either rifampin (10 micrograms/ml) or actinomycin D (1 microgram/ml) was used to inhibit RNA synthesis.

ACTION OF KI, THYROXINE AND CYCLIC AMP ON [3H]URIDINE INCORPORATION INTO THE RNA OF THYROID SLICES

1976 ◽  
Vol 83 (2) ◽  
pp. 313-320 ◽  
Author(s):  
Mario A. Pisarev ◽  
Leonardo O. Aiello ◽  
Diana L. Kleiman de Pisarev
Keyword(s):  
Cyclic Amp ◽  
Inhibitory Action ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Protein Biosynthesis ◽  
Rna Degradation ◽  
Precursor Pool ◽  
Rna Precursor

ABSTRACT Potassium iodide (KI) has been shown to impair thyroid protein biosynthesis both in vivo and in vitro. The present study was performed in order to clarify its mechanism of action. Ribonucleic acid (RNA) synthesis was studied in beef thyroid slices with either [32P] or [3H]-uridine as labelled precursors. Both KI and thyroxine (T4) at 10−5 m significantly decreased RNA labelling under our conditions. In other experiments RNA degradation was examined in pulse-labelled and actinomycin D-treated slices. KI did not modify the degradation of the [3H]-RNA thus indicating that it interferes with the biosynthesis rather than with the degradation of RNA. Taking the perchloric acid soluble radioactivity as a rough index of the precursor pool the present results would indicate an action at this level. Both KClO4 and methylmercapto-imidazole relieved the gland from the inhibitory action of KI, supporting the view that an intracellular and organified form of iodine is responsible for this action. Since T4 also reproduced the effects of KI on RNA synthesis we would like to propose iodothyronines as the intermediates of this action. Cyclic AMP has been shown to stimulate thyroid protein biosynthesis. The present results demonstrate an action at the RNA level. Cyclic AMP increased both the PCA-soluble and RNA-linked radioactivity, thus suggesting an effect at the RNA precursor pool. KI at 10−5 m blocked the action of 2 mm cyclic AMP.

The Effect of Actinomycin D in Vivo Upon Peripheral Nucleoli and Other Nuclear Organelles in Oocytes of Triturus Cristatus

1972 ◽  
Vol 10 (3) ◽  
pp. 833-855
Author(s):  
M. H. L. SNOW
Keyword(s):  
Phase Contrast ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Triturus Cristatus ◽  
Granular Component ◽  
Azure B ◽  
Final Maturation ◽  
Ribosomal Precursor ◽  
20 Nm

Exposure of the ovaries of Triturus cristatus to actinomycin D at a concentration of 100 µg/ml causes characteristic changes in the peripheral nucleoli and other nuclear organelles in oocytes of 0.6-1.1 mm diameter. Viewed with the light microscope untreated oocytes contain nucleoli that stain uniformly with a variety of dyes. They also appear homogeneous tmder phase-contrast optics. After 2 or 4 h of in vivo incubation with actinomycin D, oocyte sections stained with Haidenhain's haematoxylin or viewed under phase-contrast optics show nucleoli composed of 2 regions. The more heavily stained or contrasted zone is crescent-shaped and directed away from the nuclear membrane. Neither sections stained with azure B bromide nor gallocyanin chrome alum show this feature. Ribonuclease digestion does not eliminate or alter it. Autoradiography with [3H]uridine indicates that all recently synthesized RNA is lost from the nucleolus during actinornycin D treatment. The zonation is not therefore a reflexion of RNA distribution. During recovery from actinomycin D poisoning there is a reduction in the degree of zonation shown by nucleoli which re-establish a normal appearance some 48 h after treatment. Electron microscopy of peripheral nucleoli in oocytes sampled during this treatment indicates that the zonation is not associated with reorganization of ultrastructural components. During incubation with actinomycin D the coarse granules (20 nm diameter) are completely lost from the nucleolus. There is associated shrinkage of the nucleolus which after treatment is found to consist entirely of fibrils (5 nm thick) and small granules. The reappearance of the coarse granules during recovery is completed in about 48 h. It is thought that the loss of the granular component during treatment represents the movement of the 30-S precursor and the 18-s ribosomal unit from the nucleolus. Some 20-30 µm inside the nucleus of untreated oocytes is a region containing many spheroidal bodies, less than 1.0 µm diameter. They have been termed micronucleoli and consist of granules 2.5-5 nm in diameter and fibrils of similar thickness. Actinomycin D treatment causes these components to segregate and eventually (within 24 h of treatment) the granular component is extruded. This component reappears during the second day after treatment. It is postulated that these micronucleoli represent the sites at which the 30-S ribosomal precursor undergoes its final maturation. The segregation of components induced by actinomycin D is probably the morphological manifestation of an abnormal metamorphosis of this precursor. Treatment with actinomycin D also induces the immediate formation within the nucleus of crystalline bodies composed of lamellae 16 nm wide, 4 nm thick and with a centre-to-centre spacing of 8-10 nm. They are not present 24 h after treatment. They are thought to represent a protein fraction normally associated with periods of intense RNA synthesis.

GIBBERELLIN, AS A REGULATOR OF PROTEIN AND RIBONUCLEIC ACID SYNTHESIS DURING SENESCENCE IN LEAF CELLS OF TARAXACUM OFFICINALE

1966 ◽  
Vol 44 (6) ◽  
pp. 739-745 ◽  
Author(s):  
R. A. Fletcher ◽  
Daphne J. Osborne
Keyword(s):  
Leaf Senescence ◽  
Ribonucleic Acid ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Acid Synthesis ◽  
Taraxacum Officinale ◽  
Leaf Discs ◽  
Protein And Rna Synthesis ◽  
Chlorophyll Protein ◽  
Gibberellin Treatment

The addition of gibberellin A3 (GA) to leaf discs of Taraxacum officinale Weber retards their senescence and delays the decline in the levels of chlorophyll, protein, and RNA. Incorporation of 14C leucine and 14C adenine into protein and RNA respectively was increased by GA. This enhancement of protein and RNA synthesis did not occur if the discs were supplied with actinomycin D before treatment with gibberellin. If, however, actinomycin D was added after the gibberellin treatment then the stimulatory effect of the hormone was maintained. These results suggest that the retarding action of gibberellins on leaf senescence could be mediated through a regulation of RNA synthesis that is DNA dependent.

scholarly journals Purines as ‘hyper-repressors’ of glucose transport. A role for phosphoribosyl diphosphate

1983 ◽  
Vol 214 (1) ◽  
pp. 133-144 ◽  
Author(s):  
R J Gay ◽  
H Amos
Keyword(s):  
Methylene Blue ◽  
Carbon Source ◽  
Chick Embryo ◽  
Glucose Transport ◽  
Culture Medium ◽  
Metabolic Inhibitors ◽  
Purine Analogues ◽  
Purine Base ◽  
Embryo Fibroblasts ◽  
In Cells

Under selected conditions the rate of glucose transport and the intracellular phosphoribosyl diphosphate (PPRibP) concentrations of chick-embryo fibroblasts are inversely correlated. This relationship holds when cells are incubated with mannose, fructose, xylose or various concentrations of glucose. The metabolic inhibitors 2,4-dinitrophenol, rotenone and Methylene Blue increased glucose transport and decreased PPRibP. The addition of any pyrimidine or purine base or ribonucleoside dramatically depleted PPRibP pools, regardless of the carbon source. Addition of guanine (10 microM) or hypoxanthine (100 microM) decreased transport in glucose-grown chick cells to barely detectable values, but did not affect increases observed in cells depressed by substitution of xylose for glucose. Guanosine, inosine and the purine analogues 6-thioguanine, 6-thioguanosine, 8-azaguanine and 6-methylmercaptopurine riboside sharply decreased transport in glucose-grown cells and blocked the increase in transport resulting from the replacement of glucose by fructose or xylose in the culture medium.

Adenylate Concentrations in Chara: Variability, Effects of Inhibitors and Relationship to Protoplasmic Streaming

1983 ◽  
Vol 10 (5) ◽  
pp. 373 ◽  
Author(s):  
R.J Reid ◽  
N.A Walker
Keyword(s):  
Adenylate Kinase ◽  
Inorganic Phosphate ◽  
Culture Conditions ◽  
Metabolic Inhibitors ◽  
Protoplasmic Streaming ◽  
Atp Concentration ◽  
Carbonyl Cyanide ◽  
In Cells

The concentrations of ATP, ADP and AMP In Chara vary considerably depending on the culture conditions. The ranges of cytoplasmic concentrations were: ATP, 1 6-3 4 mM; ADP, 0 1-1 1 mM; AMP <O 6 mM. The ATP concentration was lower in the dark than in the light by 5-25%. The cytoplasmic concentration of inorganic phosphate in cells from one culture was 21 2 mM. From these values AGATp lies in the range 47-52 kJ mol-I. Lowering of the ATP concentration by metabolic inhibitors CCCP (carbonyl cyanide mchlorophenylhydrazone), DCCD (N,N'-dicyclohexylcarbodiimide) and DCMU (3-(3-chloropheny1)-1, I-dimethylurea) was associated with almost parallel changes in the rate of protoplasmic streaming. The use of streaming rate as an in vivo indicator of ATP status is discussed. The results are consistent with the participation of adenylate kinase in the regulation of adenylate concentrations.

scholarly journals SYNTHESIS OF MESSENGER-LIKE RIBONUCLEIC ACID AND PROTEIN DURING MEIOSIS IN ISOLATED CELLS OF TRILLIUM ERECTUM

1963 ◽  
Vol 19 (1) ◽  
pp. 45-58 ◽  
Author(s):  
Yasuo Hotta ◽  
Herbert Stern
Keyword(s):  
Messenger Rna ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Morphological Changes ◽  
Reaction Rates ◽  
Meiotic Stage ◽  
Isolated Cells ◽  
Rna Analysis ◽  
Protein And Rna Synthesis

The synthesis of RNA and protein by cultures of isolated microsporocytes has been demonstrated. The variation in capacities of such cultures to perform syntheses is a function of meiotic stage and parallels the pattern of changes observed for microsporocytes in situ. A principal feature of this pattern is the induction of syntheses during pachytene and diplotene, stages at which the chromosomes are partly contracted. By use of Actinomycin D, chloramphenicol, pulse-labeling with P32-phosphate, and nucleotide analyses of RNA digests, part of the RNA synthesized has been shown to correspond to messenger RNA. Analysis of reaction rates and the overlappings of protein and RNA synthesis indicates that the spread of cytological events in Trillium is not purely a function of the low temperature at which it occurs but, presumably, arises from a complement of regulatory devices which govern the periodic onset of reactions within the cells. The main conclusion drawn from the whole of these studies is that the sequence of morphological changes associated with chromosome contraction and movement during meiosis is accompanied by a set of gene transcriptions. Although comparatively few genes are presumed to be active during meiosis, the action of such genes may be essential to a translation of some of the information embodying the meiotic sequence which has been stored in the genome in the course of evolution.

FIBRIN STIMULATION OF ENDOTHELIAL CELL (EC) PRODUCTION OF PGI AND TISSUE PLASMINOGEN ACTIVATOR (t-PA)

1987 ◽  
Author(s):  
L K Kaplan ◽  
T Mather ◽  
L DeMarco ◽  
S Solomon
Keyword(s):  
Rna Synthesis ◽  
Actinomycin D ◽  
Umbilical Vein ◽  
Cytochalasin D ◽  
Time Dependent ◽  
Time Intervals ◽  
Tissue Plasminogen ◽  
Protein And Rna Synthesis ◽  
Maximal Production ◽  
Stimulation Of

Many substances are known to stimulate EC production of PGI2 and t-PA. Additionally, it has been reported that fibrin can Be formed on the EC surface. In this study, the possibility that fibrin generated on the surface of cells can stimulate production of PGI2 and t-PA was examined. Human umbilical vein ECs were incubated for various time intervals with citrated human plasma clotted on the cells by the addition of CaCl2 . Control dishes contained plasma without Ca++ or serum. Time-dependent generation of PGI2 and t-PA was seen over 22-24 hours. Maximal production of PGI2 occurred when fibrin on the cells was formed from 10 to 50% plasma, with serum comprising the remainder of the incubation volume, while maximal t-PA production occurred with clots formed from 100% plasma. Fibrin I formed by addition of batroboxin to citrated plasma stimulated less synthesis of t-PA than did fibrin formed by thrombin action, and it did not stimulate PGI2 production. Thrombin clots were significantly more adherent to the cells than were batroboxin clots. PG^ synthesis induced by fibrin was fully inhibited by indomethacin, approximately 50% inhibited by actinomycin D and cycloheximide, and 20% inhibited by trifluoperazine, but was unaffected by cytochalasin D and vinblastine. Stimulation of t-PA synthesis by fibrin was unaffected by indomethacin, completely inhibited by actinomycin D and cycloheximide, and 60%, 80% and 40% blocked by cytochalasin D, vinblastine, and trifluoperazine, respectively. Thus, thrombin-induced fibrin clots stimulated PGI2 synthesis, and both thrombin and batroboxin clots stimulated t-PA synthesis. Protein and RNA synthesis were essential to stimulation of t-PA synthesis but inhibition of these processes only partially inhibited stimulation of PGI2 synthesis. Integrity of the cytoskeleton was necessary for full stimulation of t-PA synthesis, but not for stimulation of PGI2 synthesis. Thus the mechanisms of stimulation of these two cellular products were different. Increased PGI2 production could serve to limit further fibrin formation by preventing platelets from contributing to the coagulation process and increased t-PA could stimulate lysis of existing fibrin.

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.

THE LACTOGENIC EFFECTS OF PROLACTIN AND GROWTH HORMONE ON MAMMARY GLAND EXPLANTS FROM VIRGIN AND PREGNANT SPRAGUE-DAWLEY RATS

1973 ◽  
Vol 57 (2) ◽  
pp. 253-264 ◽  
Author(s):  
R. C. HALLOWES ◽  
D. Y. WANG ◽  
D. J. LEWIS
Keyword(s):  
Growth Hormone ◽  
Dna Synthesis ◽  
Rna Synthesis ◽  
Culture Medium ◽  
Actinomycin D ◽  
Sprague Dawley ◽  
Pregnant Rats ◽  
Sprague Dawley Rats ◽  
Ovine Prolactin ◽  
Casein Synthesis

SUMMARY Explants of mammary glands from mature virgin and pregnant Sprague—Dawley rats were maintained in organ culture for up to 96 h. The effects of insulin, corticosterone, ovine prolactin and bovine growth hormone on the synthesis of DNA, RNA and casein in the explants were studied. DNA synthesis in explants from virgin rats was maintained by insulin but was not increased by the addition of the other hormones tested. DNA synthesis in explants from pregnant rats was increased by insulin, and the addition of corticosterone and either prolactin or growth hormone to the culture medium increased this synthesis. The rate of RNA synthesis in explants from virgin rats was similar in medium 199 with or without additional hormones. RNA synthesis in explants from pregnant rats was increased by the addition of insulin or insulin plus corticosterone to the medium. In explants from both virgin and pregnant rats the maximal rate of hormone-stimulated DNA or RNA synthesis occurred during the first 24 h of culture. Casein synthesis, as measured by the uptake of 32P-labelled orthophosphate by explants from virgin and pregnant rats, was increased by insulin plus corticosterone plus either prolactin or growth hormone. The rate of casein synthesis was maximal between 48 and 72 h and was reduced by actinomycin D. In the pregnant rats no significant differences were demonstrated between the effects of the hormones on DNA, RNA or casein synthesis in explants from the 5th, 10th, 16th or 19th day of pregnancy.

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