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Biomedical subjects

I Tamm

Publications and source records attributed to I Tamm.

At least 109 records · Page 6Linked to original sources
PubMed1977

Interferon messenger RNA content of human fibroblasts during induction, shutoff, and superinduction of interferon production.

Translation of injected mRNA in oocytes of Xenopus laevis has been used as a highly sensitive and quantitative assay for interferon mRNA. Injection into oocytes of polyadenylylated RNA extracted from poly(I).poly(C)-induced human diploid fibroblasts (FS-4) leads to the synthesis of biologically active human fibroblast interferon over a period of 24-32 hr. There is a linear relationship between the amount of mRNA injected and the interferon yield obtained over a range of 1-20 ng of injected RNA. Injection of 40-80 ng of mRNA into each of 15 oocytes, homogenized in 0.3 ml of incubation medium, gave a titer of 128-256 interferon reference units/ml of homogenate.FS-4 cells at the peak of interferon production-i.e., approximately 2.5 hr after the beginning of induction with poly(I).poly(C)-gave mRNA that yielded 24-48 interferon reference units/ml in the oocyte assay (30 ng of RNA injected per oocyte). An equivalent amount of mRNA from FS-4 cells in the shutoff phase, approximately 6 hr after induction, gave </=4 interferon reference units/ml. In contrast, mRNA extracted from FS-4 cells that had been induced and maintained in the presence of 40 muM 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole for 6 hr produced 64-128 interferon reference units/ml. Polyadenylylated RNA obtained from uninduced FS-4 cells did not lead to detectable interferon synthesis (<4 interferon reference units/ml). These data provide a direct verification of the hypothesis that the shutoff of interferon production in FS-4 cells involves a regulatory event leading to the posttranscriptional inactivation or degradation of interferon mRNA. Because the inactivating mechanism is sensitive to inhibition by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, a selective inhibitor of nuclear heterogeneous RNA and mRNA synthesis, it is likely that synthesis of an RNA molecule is necessary for the shutoff of interferon production.

Animals↗
PubMed1976

5,6-Dichloro-1-Beta-D-ribofuranosylbenzimidazole inhibits initiation of nuclear heterogeneous RNA chains in HeLa cells.

The nucleoside analog 5, 6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB) at 75 to 150 micromolar concentrations inhibits the synthesis of nuclear heterogeneous RNA (hnRNA) in HeLa cells by 60 to 70 percent. The sedimentation profile of hnRNA labeled with (3H)uridine for 45 seconds after brief treatment (45, 90, or 180 seconds) with DRB showed a progressive decrease in the labeling of shorter hnRNA molecules relative to longer molecules. Prior exposure of the cells to actinomycin D, an inhibitor of RNA chain elongation, did not alter the sedimentation profile of hnRNA. These results suggest that DRB preferentially inhibits the initiation of hnRNA chains so that after exposure to DRB for a brief period the longer nascent chains still remain to be finished and thus incorporate a greater share of the pulse label. By progressively increasing the time of exposure to DRB, and measuring the rate of increase in the average size of the labeled, nascent RNA, it was estimated that the chains were growing at rates between 50 and 100 nucleotides per second.

Benzimidazoles↗
PubMed1976

The inhibition by DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) of hnRNA and mRNA production in HeLa cells.

5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB; 75 muM) inhibited only 60-75% of nuclear heterogeneous RNA (hnRNA) synthesis in HeLa cells, while the appearance of labeled cytoplasmic poly(A)-containing messenger RNA (mRNA) was reduced by approximately 95%. DRB-sensitive and resistant transcripts spanned the entire size range of hnRNA molecules. The relative poly(A) content of DRB-resistant hnRNA molecules was the same as that of normal hnRNA. In contrast to the effects of 3'deoxyadenosine, DRB appeared to inhibit the initiation of hnRNA chains, but did not directly interfere with labeling of poly(A). We present an interpretation of these results and propose how the results might be used to gain more information about nuclear RNA metabolism.

Adenosine↗
PubMed1976

An evaluation of messenger RNA competition in the shutoff of human interferon production.

The process that shuts off poly(I)-poly(C)-induced interferon production in a strain of diploid human fibroblasts (FS-4)-involves the synthesis of new RNA, presumably nuclear heterogeneous RNA. When cultures in the shutoff phase of interferon production are treated with actinomycin D (5 mug/ml) or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB 40 muM), the rate of interferon production continues to decline for a further 3-4 hr, but then tends to level off. The treated cultures maintain interferon production at a reduced level for at least 10 hr. The residual rate of interferon production is higher in cultures which received actinomycin D or DRB early in the shutoff phase as compared to the rate in cultures treated late.

Benzimidazoles↗
PubMed1976

Action of dichlorobenzimidazole riboside on RNA synthesis in L-929 and HeLa cells.

5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) inhibits RNA synthesis in L-929 cells (mouse fibroblast line) and HeLa cells (human epitheloid carcinoma line) within 2 min of addition of the compound to the medium. By removing DRB from the medium, the inhibition is promptly and completely reversed after treatment of cells for as long as 1 h or even longer. The inhibitory effect of DRB on the overall rate of RNA synthesis is similar in L and HeLa cells and is markedly concentration-dependent in the low dose range (5-20 muM or 1.6-6.4 mug/ml), but not as higher concentrations of DRB. At a concentration of 12 muM, DRB has a highly selective inhibitory effect on the synthesis of nuclear heterogenous RNA in L cells. At higher concentrations, there is also inhibition of 45 S ribosomal precursor RNA synthesis, but at all concentrations the effect on heterogeneous RNA synthesis in L cells in considerably greater than that on preribosomal RNA synthesis. In HeLa cells, too, DRB has a selective effect on heterogeneous RNA synthesis, but quantitatively the selectivity of action is somewhat less pronounced. In both L and HeLa cells, the inhibition of synthesis of nuclear heterogeneous RNA is incomplete even at very high concentrations of DRB (150 muM). Thus, while DRB is a selective inhibitor of nuclear heterogeneous RNA synthesis, not all such RNA synthesis is sensitive to inhibition. It is proposed that messenger precursor RNA synthesis may largely be sensitive to inhibition by DRB. In short-term experiments, DRB has no effect on protein synthesis in L or HeLa cells. DRB has a slight to moderate inhibitory effect on uridine uptake into L cells and a moderate to marked effect on uptake of uridine into HeLa cells.

Benzimidazoles↗
PubMed1975

Enhancement of human interferon production by neutral red and chloroquine: analysis of inhibition of protein degradation and macromolecular synthesis.

Two lysosomotrophic drugs, neutral red and chloroquine, enhance polyinosinic:polycytidylic acid-induced interferon production by a strain of diploid human fibroblasts (FS-4). Treatment of cells with neutral red or chloroquine between 2.5 and 3.5 h after induction increases interferon yields 16- to 64- and 4- to 16-fold, respectively, in the subsequent 20.5 h. The two drugs inhibit the rates of protein degradation and of RNA and protein synthesis. In addition, neutral red is a very potent inhibitor of uridine transport into cells. Normalized dose-effect curves show that interferon superinduction is correlated with the inhibition of macromolecular synthesis, but not with that of protein degradation. Treatment of cells with chloroquine at low concentration (25 mug/ml) for a prolonged period of time (24 h) caused approximately 40% reduction in the rate of protein degradation. The usual rapid shutoff of interferon production and the effectiveness of effectiveness of actinomycin D superinduction are not altered by this treatment. This strongly suggests that inhibition of intralysosomal protein degradation does not significantly contribute to interferon superinduction. Degradation of the rapidly and the slowly turning over proteins was unaffected by actinomycin D under conditions of treatment known to enhance interferon production. Treatment with cycloheximide (5 or 50 mug/ml for 5 h) inhibited the rate of degradation of the rapidly turning over component by 10% and the slow component by 30-40%, which suggests that the two components turn over by distinct cellular mechanisms.

Cells, Cultured↗
PubMed1975

Human interferon production: superinduction by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole.

Polyinosinic.polycytidylic acid [poly(I.C)] induced production of interferon by a strain of diploid human fibroblasts (FS-4), measured between 5 and 24 hours from induction, is enhanced up to 128-fold by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a reversible inhibitor of nuclear heterogeneous RNA synthesis. A normalized dose-effect plot shows a close correlation between the superinducing effect of DRB and inhibition of RNA synthesis. Cultures that contained DRB continue to produce interferon for up to 4 days. Removal of the drug at any time during this period leads to a prompt shutoff of interferon production.

Benzimidazoles↗
PubMed1975

Cell injury with viruses.

The inhibitions of cellular protein and RNA synthesis in picornavirus-infected cells are early events which require only limited synthesis of virus-specific proteins. The inhibition of cellular protein synthesis appears to be due to blocking of the association of ribosomes with host messenger RNA. Inhibition of cellular RNA synthesis involves inactivation of the template-enzyme complex and affects ribosomal RNA synthesis before messenger RNA synthesis. Inhibition of cellular DNA synthesis also occurs early and may be secondary to inhibition of cellular protein synthesis. Early chromatid breaks may be related to inhibitions of cellular protein and nucleic acid synthesis. Stimulation of phospholipid synthesis in picornavirus-infected cells also requires only limited synthesis of virus-specific proteins. In contrast, release of lysosomal enzymes, proliferation of smooth cytoplasmic membranes, cellular vacuolization, retraction, and rounding, and diffuse chromosomal changes related to karyorrhexis and pyknosis are all dependent on considerable synthesis of virus-specific proteins during the middle part of the picornavirus growth cycle. The early virus-induced alterations in cellular biosynthetic processes are not the direct or sole cause of the subsequent marked pathologic changes in the membranes and chromosomes of the cell.

Cell Transformation, Neoplastic↗
PubMed1973

Benzimidazole derivatives: new enhancers of influenza virus multiplication.

The enhancing activity of 5-methyl-2-D-ribobenzimidazole on influenza B (Lee) virus yield in chorioallantoic membranes from 10-day old embryonated eggs was compared with that of eight other polyhydroxyalkyl-benzimidazoles. No marked differences in activity were noted with the following six derivatives: 5,6-dimethyl-2-D-ribo; 2-D-gluco; 5-methyl-2-D-gluco; 5,6-dimethyl-2-D-gluco; 5-methyl-2-D-galacto; and 5-methyl-2-L-rhamno. None caused morphological damage to the membranes at a concentration of 3.5 mM. The solubility of the 5-methyl-2-D-arabo and 5-methyl-2-D-manno derivatives was too low to permit quantitative comparisons, but both were active and nontoxic at a concentration of 1.75 mM. 5-Hydroxy-1-methylbenzimidazole and 5-methoxy-1-methylbenzimidazole are more active than 5-methyl-2-D-ribobenzimidazole both with respect to specific activity and maximal enhancement at the highest tolerated dose. The hydroxyl substituent is superior to the methoxyl grouping. Substitution at position 5 is superior to substitution at position 6 with respect to the tolerated dose level and therefore the maximal effect obtainable, but the 6-hydroxy-1-methyl derivative showed the highest specific activity. 5-Methoxy-1-methylbenzimidazole increases the yield to a comparable extent as measured by infectivity and hemagglutination titrations. The responses of membranes from individual chicken embryos to the enhancing action of 5-methoxy-1-methylbenzimidazole and 5-methyl-2-D-ribobenzimidazole are similar. 5-Methoxy-1-methylbenzimidazole restores the capacity of membranes from older chicken embryos to produce a large amount of virus after a small inoculum. This derivative increases the yield of virus in membranes treated before infection only. Maximal enhancement is obtained with prolonged treatment, starting before, and continuing after infection. 5-Methoxy-1-methyl-benzimidazole increases the yield of virus from COFAL-negative embryos in which the control yield is very low. Combined treatment with moderate doses of 5-methoxy-1-methylbenzimidazole and 5-methyl-2-D-ribobenzimidazole gives an additive effect.

Animals↗
PubMed1973

DNA replication: direction and rate of chain growth in mammalian cells.

Using pulse labeling techniques with [(3)H]thymidine or [(3)H]cytidine, combined with DNA fiber autoradiography, we have investigated the direction and rate of DNA chain growth in mammalian cells. In general, chain elongation proceeds bidirectionally from the common origin of pairs of adjacent replication sections. This type of replication is noted whether the DNA is labeled first with [(3)H]thymidine of high specific activity, followed by [(3)H]thymidine of low specific activity or the sequence is reversed. Approximately one-fifth of the growing points have unique origins and in these replication units, chain growth proceeds in one direction only. Fluorodeoxyuridine and hydroxyurea both inhibit DNA chain propagation. Fluorodeoxyuridine exerts its effect on chain growth within 15-23 min, while the effect of hydroxyurea is evident within 15 min under conditions where the endogenous thymidine pool has been depleted by prior treatment with fluorodeoxyuridine. Puromycin has no effect on chain growth until 60 min after addition of the compound, even though thymidine incorporation is more than 50% reduced within 15 min. After 2 h of treatment with puromycin, the rate of chain growth is reduced by 50%, whereas thymidine incorporation is reduced by 75%. Cycloheximide reduces the rates of DNA chain growth and thymidine incorporation 50% within 15 min, and, on prolonged treatment, the decrease in rate of chain growth generally parallels the reduction in thymidine incorporation.

Animals↗
PubMed1973

Reovirus: effect of noninfective viral components on cellular deoxyribonucleic acid synthesis.

We determined the effects of noninfective reovirus components on cellular deoxyribonucleic acid (DNA) synthesis. Reovirus inactivated by ultraviolet light inhibited cellular DNA synthesis, whereas reovirus cores and empty capsids did not. Both cores and empty capsids were adsorbed to cells. Adenine-rich ribonucleic acid (RNA) from reovirus, adsorbed to cells in the presence of diethyl-aminoethyl-dextran, produced a partial inhibition of DNA synthesis. RNA was synthesized in the presence of actinomycin D after infection with ultraviolet light-irradiated reovirus, and this RNA synthesis was not due to multiplicity reactivation of virus infectivity. These data suggest that viral structural proteins do not inhibit DNA synthesis and that the inhibition produced by ultraviolet-irradiated virus may be mediated in part or in toto by a newly synthesized viral product.

Animals↗