Effect of CO2 on labelling of nerve and liver cells by nucleic acid precursors

In vivo experiments in mice demonstrated that 5% CO₂ content in the air inhaled did not change the labelling in autoradiograms from animals injected with [³H]uridine, [³H]orotic acid, [³H]hypoxanthine, [³H]lysine or [³H]cytidine. At 20% CO₂ content there was a significant decrease in labelling of brain cells with [³H]uridine and [³H]cytidine, but not following [³H]lysine; there was no labelling of nerve cells with [³H]orotic acid or [³H]hypoxanthine, but a control group was not included. The labelling of choroid plexus and hepatocytes was independent of the CO₂ concentration. A comparison of in vivo and in vitro experiments at 20% CO₂ content showed a similar significant decrease in labelling of brain cells with [³H]uridine and [³H]cytidine. It is concluded that a metabolic change is the most appropriate explanation of the CO₂ effect.     

Labelling of the chromatid body by [3H]uridine in rat pachytene spermatocytes

In this study it is shown that a cytoplasmic cell organelle, the chromatoid body, becomes labelled with [³H]uridine in the pachytene spermatocytes. The chromatoid body becomes labelled when the cells are first labelled for 2 h in the presence of [³H]uridine and thereafter chased for 9 h in the presence of unlabelled uridine. This labelling is inhibited by the specific RNA polymerase II inhibitor α-amanitin. Based on this it is suggested that part of the RNA synthesized in the pachytene spermatocytes is stored in the chromatoid body and transported to the postmeiotic spermatids where it is used in the differentiation of the spermatids.     

Labelling of the chromatoid body by [H]uridine in rat pachytene spermatocytes

In this study it is shown that a cytoplasmic cell organelle, the chromatoid body, becomes labelled with [³H]uridine in the pachytene spermatocytes. The chromatoid body becomes labelled when the cells are first labelled for 2 h in the presence of [³H]uridine and thereafter chased for 9 h in the presence of unlabelled uridine. This labelling is inhibited by the specific RNA polymerase II inhibitor α-amanitin. Based on this it is suggested that part of the RNA synthesized in the pachytene spermatocytes is stored in the chromatoid body and transported to the postmeiotic spermatids where it is used in the differentiation of the spermatids.     

Migratory patterns of B lymphocytes. IV. Effect of anti-Ig on follicular localization of radioactively labeled murine spleen and bone marrow cells.

A study was made of the localization of nylon-wool-adherent (AD) and nonadherent (NA) murine spleen cells in lymphoid tissue of irradiated syngeneic recipients. Cells were labeled in vitro with [³H]uridine or ⁵¹Cr and injected intravenously. Localization in recipient tissues was expressed as percent of injected radioactivity. NA and AD [³H]uridine labeled cells gave spleen to lymph node (S:LN) ratios of 1.0 and 2.7, respectively. After treatment of AD cells with rabbit anti-mouse Fab + C at 37 °C, localization in S decreased markedly.NA cells primarily localized in LN paracortex and splenic periarteriolar sheaths. Untreated and NRS-treated AD cells localized in lymphoid follicles, whereas anti-Fab-treated AD cells did not. When ⁵¹Cr-labeled AD cells were treated with anti-Fab at 4 °C without C, there was a transient decrease in splenic localization at 24 hr followed by a recovery to the normal pattern at 48 hr after transfer. [³H]uridine-labeled bone marrow (BM) cells showed less localization in lymphoid tissue than did S cells. Some BM cells were seen in LN follicles, particularly at 48 hr after transfer, but this localization was not affected by prior treatment with anti-Fab + C. The possible role of surface Ig in the determination of follicular localization of B lymphocytes is discussed.     

Mechanism of Apparent Transcription Inhibition by Methyl Mercury in Cerebellar Neurons

Abstract: We have investigated the mechanism of inhibition of RNA synthesis by methyl mercury (MeHg) in isolated neonatal rat cerebellar cells. Each of the three component steps involved in the incorporation of exogenous [³H]uridine into cellular RNA was examined separately in whole-cell and/or subcellular preparations. Nuclear RNA polymerase activity was measured in preparations containing both free nuclei and whole cells. Incorporation of [³H]UTP into nuclear RNA was found to be unimpaired at concentrations of MeHg that inhibited whole-cell incorporation of [³H]uridine by > 75%. Cellular uptake of [³H]uridine was assayed in cerebellar cells treated with KCN to deplete ATP levels and block subsequent phosphorylation reactions of transported uridine. Uptake activity under these conditions was unaffected by MeHg. Measurement of intracellular phosphorylation of [³H]uridine indicated that inhibition of this activity closely paralleled that of RNA synthesis. Quantitation of individual uridine nucleotides by polyethyleneimine-cellulose TLC revealed reduced levels of UTP and UDP whereas levels of UMP were elevated, suggesting that impairment of phosphorylation was not the result of cellular ATP depletion but, more likely, a direct effect on phosphouridine kinase enzymes. This mechanism of MeHg-induced inhibition of RNA synthesis was confirmed by assays of uridine phosphorylation using cell-free extracts in which exogenous ATP was supplied.     

Evidence that multiple species of aminoacylated transfer RNA are present in regenerating optic axons of goldfish

This study reports that 4S RNA present in regenerating optic axons of goldfish is likely to be transfer RNA. Evidence is also presented which indicates that this transfer RNA is similar to transfer RNA found in tectal cells and that its aminocylation is likely to occur both in retinal ganglion cells prior to axonal transport as well as in the axon itself. Fish with regenerating optic nerves received intraocular injections of [³H]uridine followed 4 days later by intracranial injections of [¹⁴C]uridine. Radioactive tectal 4S RNA was isolated 6 days after [³H]uridine injections and chromatographed by BD cellulose chromatography. Optical density as well as radioactivity profiles for both [¹⁴C]4S RNA (from tectal cells) and [³H]4S RNA (90% of which originated from regenerating optic axons) were found to be similar toE. coli transfer RNA optical density profiles, indicating that the intra-axonal 4S RNA is likely to be transfer RNA. Moreover, comparisons of³H/¹⁴C suggest that intra-axonal and cellular 4S RNAs are composed of similar species of transfer RNA. Results of other experiments indicated that aminoacylation of axonally transported tRNA occurs both in the retina and in optic axons subsequent to axonal transport.     

Inhibition of nucleic acid synthesis in Ehrlich tumor cells by periodate-oxidized adenosine and adenylic acid

Periodate-oxidized adenosine and AMP were inhibitory to both RNA and DNA synthesis in Ehrlich tumor cells in culture. With periodate-oxidized adenosine, the inhibition of RNA synthesis paralleled the inhibition of DNA synthesis. Periodate-oxidized AMP, however, was more inhibitory to DNA synthesis than to RNA synthesis. With both compounds, there was a decrease in the conversion of [¹⁴C]cytidine nucleotides to [¹⁴C]deoxycytidine nucleotides in the acid-soluble pool. The borohy-dride-reduced trialcohol derivative of the periodate-oxidized adenosine compound was not inhibitory to DNA or RNA synthesis in the tumor cells. The incorporation of [³H]uridine into 28S and 18S ribosomal RNA was inhibited by both periodate-oxidized adenosine and AMP, but the incorporation of [³H]uridine in 45S, 5S, and 4S RNA was essentially unaffected by these compounds. Periodate-oxidized adenosine inhibited Ehrlich tumor cell growth in vivo.     

Incorporation of uracil into the growing strand of adenovirus 12 DNA.

The amount of rapidly labeled short DNA chains in adenovirus 12(Ad12)-infected cells was markedly increased in the presence of either uridine or deoxycytidine which could be converted to dUTP. When the infected cells were labeled with [³H]uridine or [³H] deoxycytidine and the labeled nucleotides in the short DNA chains from the Hirt supernatant were analysed by thin-layer chromatography, approximately 90 or 20% of the label was detected in dUTP. These results suggest that at least a portion of short DNA chains formed during Ad12 DNA replication is derived from an excision-repair mechanism of uracil containing nascent strands.     

Compartmentation of uridine 5′-triphosphate occurs during synthesis of cytoplasmic and mitochondrial ribosomal RNAs of cultured tomato cells but not during synthesis of cytoplasmic ribosomal and transfer RNAs

Compartmentation of uridine 5-triphosphate (UTP) was studied during the nucleolar synthesis of cytoplasmic ribosomal RNA (cyt-rRNA) and the synthesis of cytoplasmic transfer RNA (cyt-tRNA) in the nuclear matrix as well as the synthesis of mitochondrial ribosomal RNA (mt-rRNA) in tomato (Lycopersicon esculentum Mill. cv. Lukullus) cell-suspension culture using the approach of Wiegers et al. (Eur. J. Biochem. 64, 535–540, 1976). Before measurements were made, it was ensured that: (i) there was steady-state labeling of all RNAs studied as well as UTP; (ii) there was stability of cyt-tRNA and cyt-rRNA; (iii) there was no label randomization through degradation of [³H]uridine; (iv) there were significant differences in the specific radioactivity of UTP, the final immediate precursor of RNA, after supplying the cells with two different exogenous [³H]uridine concentrations.By comparing the steady-state specific radioactivity of UTP with that of cyt-tRNA and cyt-18S rRNA during constant [³H]uridine supply, we found that the three molecules had equal specific radioactivities which, however, differed significantly from that of the mt-rRNA. With a 20-fold higher uridine concentration, i.e. a 20-fold lower specific radioactivity of exogenous [³H]uridine, the specific radioactivity of cyt-rRNA, cyt-tRNA and UTP decreased proportionally whereas that of mt-RNA increased. These results argue against different UTP pools during synthesis of cyt-rRNA and cyt-tRNA, but indicate compartmentation of UTP during rRNA synthesis in the nucleus and the mitochondria of tomato cells.Abbreviations CMP cytidine 5-monophosphate - cyt-rRNA cytoplasmic ribosomal RNA - cyt-tRNA cytoplasmic transfer RNA - mt-rRNA mitochondrial rRNA - NC nitrocellulose - PAGE polyacrylamide gel electrophoresis - TLC thin-layer chromatography - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol - UDP uridine 5-diphosphate - UMP uridine 5-monophosphate - UTP uridine 5-triphosphate     

Influence of mycoplasma infection on the incorporation of different precursors into RNA components of tissue culture cells

Seven different tissue culture cells have been cultured with and without mycoplasma (M. hyorhinis) in the presence of various precursors of RNA. Total cellular RNA was isolated and analysed by electrophoresis on polyacrylamide gels. The results obtained with mycoplasma-infected cells can be summarized as follows:

1. 1. When cells are labelled with [8-³H]guanosine or [5-³H]uridine there is some incorporation into host cell 28S and 18S rRNA, but it is less than into mycoplasma 23S and 16S rRNA. [8-³H]guanosine or [5-³H]uridine are also incorporated into host cell and mycoplasma tRNA and mycoplasma 4.7S RNA, but the incorporation into host cell 5S rRNA and low molecular weight RNA components (LMW RNA) is reduced.

2. 2. [5-³H]uracil is not incorporated into host cell RNA but into mycoplasma tRNA, 4.7S RNA, a mycoplasma low molecular weight RNA component M₁ and 23S and 16S rRNA.

3. 3. [³H]methyl groups are incorporated into mycoplasma tRNA, 23S and 16S rRNA, but not into host cell 28S, 18S, 5S rRNA nor into mycoplasma 4.7S RNA.

4. 4. With [³²P]orthophosphate or [³H]adenosine as precursors, the labelling is primarily in the host RNA.

Mycoplasma infection influences the labelling of RNA primarily by an effect on the utilization of the exogenously added radioactive RNA precursors, since the generation time of mycoplasma infected cells is about the same as that of uninfected cells. Mycoplasma infection may completely prevent the identification of LMW RNA components.     

Identification of viral ribonucleoproteins in the cytoplasm of murine cells chronically infected by a retrovirus.

Ribonucleoproteins were isolated from the cytoplasm of Friend-Eveline cells which produce the Friend virus complex, after a short labelling with [³H] uridine. These particles moved with a sedimentation coefficient of 53S in sucrose gradient and had a buoyant density of 1.46 g/cm³ in CsCl gradient. Analysis of their RNA content showed that they possessed a 35S major species having the size of the viral genome subunit. Moreover, a positive hybridization was observed when RNA of the 53S particles was annealed with viral complementary DNA. No such particles were found in cultures of uninfected murine cells suggesting that 53S RNPs have a viral origin.     

Adverse effects of hyperbaric oxygen on [3H]uridine incorporation and uridine kinase activity in B104 rat neuroblastoma cells

The effects of hyperbaric oxygen on uracil nucleotide metabolism in B104 rat neuroblastoma cells were investigated. Cells exposed to 10 atm O₂ for 4 h incorporated markedly less [³H]uridine into the acid-soluble fraction and RNA compared to cells kept in ambient air. The acid-soluble fraction of the oxygen-treated cells contained less total [³H]uridine phosphates ([³H]UMP + [³H]UDP + [³H]UTP) than air-treated cells. Uridine kinase activity, assayed in cytosolic extracts from cells exposed to 10 atm O₂ for 4 h, was decreased by 46% compared to the air controls. The reduced enzyme activity which appears to account for the depressed [³H]uridine incorporation, may contribute to the lethal effects of oxygen in these cells.Abbreviations DMEM Dulbecco's Modified Eagle's Medium - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid     

An autoradiographic method of detectingA. laidlawii andM. hyorhinis in cell cultures

Summary The autoradiographic investigation of L cells and Chinese hamster cells for the presence of mycoplasmas (A. laidlawii andM. hyorhinis) using uridine/uracil (UdR/U) testing is a rapid and reliable method suitable for the serial checking of even a small number of cells. It depends on a reduced incorporation of [³H]uridine and an increased uptake of [³H]uracil into the RNA of mycoplasma-infected cells, shown in autoradiograms by the density of the grains and their distribution. Results obtained by the autoradiographic technique correspond approximately to specific activity values of RNA-infected cells after the incorporation of [³H]uridine and [³H]uracil.     

Uptake of bacterial DNA by Chlamydomonas reinhardi

Escherichia coli [³H]DNA supplied to vegetative cultures of wild-type (mt+) and CW15 (mt+; mutant lacking the cell wall) Chlamydomonas reinhardi could bind to the cell wall of the wild-type and to the cell membrane of CW15 mutant cells. The extent of this binding decreased with time and was to a large degree (over 90%) DNA-ase-sensitive. Nevertheless, about 0.01% of the bacterial DNA remained irreversibly associated with the cells when they reached stationary phase. The irreversible binding of the donor bacterial DNA to Chlamydomonas cells could be increased by treatment of the cultures with polycations such as DEAE-dextran, poly-L-lysine and poly-L-ornithine. Although the CW15 cells rapidly degraded bacterial DNA in the culture medium wild-type cells showed only a small effect on the molecular weight of the donor DNA.The acid-insoluble radioactivity irreversibly bound to WT (+) cells consisted mainly of oligonucleotides with a small proportion present as less depolymerized donor DNA. No radioactivity, however, was found to be associated with the recipient high molecular weight Chlamydomonas DNA.No labeled donor DNA could be recognized in the cells given bacterial [³H]DNA in early stationary phase. Instead, radioactivity found in Chlamydomonas DNA corresponded to reutilization of [³H]thymine derivatives released as a result of [³H] DNA degradation. No evidence for the integration of detectable amounts of donor DNA sequences into the host cell DNA was obtained.     

Isolation of metabolic cooperation-defective variants from mouse embryonal carcinoma cells

We describe the isolation from the HGPRT^? embryonal carcinoma cell line PC13TG8 of a variant, R5/3, defective in metabolic cooperation. This was achieved in two stages via an intermediate, R2/1, using a selective system in which the HGPRT^? embryonal carcinoma cells were co-cultured with HGPRT⁺ cells in 6-thioguanine. R5/3 cells show increased survival compared with PC13TG8 when retested under selection conditions, and a reduction in grain count index when tested by autoradiography as recipients of [³H]hypoxanthine-labelled nucleotides from wild-type donors and as donors of [³H]thymidine- and [³H]adenine-labelled nucleotides to suitably marked recipients. However, a low residual fraction of heavily labelled recipients is found in all autoradiographic experiments with R5/3 cells. This is not due to heterogeneity of either donor or recipient populations. We also describe the development of a colony-formation assay for metabolic cooperation based on the “kiss of life” phenomenon, in which R5/3 shows very poor survival compared with PC13TG8. R2/1 shows behaviour intermediate between PC13TG8 and R5/3 in all the tests described above. We conclude that two steps can be identified in the change of phenotype by which R5/3 is derived from PC13TG8, and that both steps modify the ability of the cells to form permeable junctions.     

Studies on compartmentation of uridine 5'-triphosphate during synthesis of cytoplasmic and plastid ribosomal RNA's in Euglena gracilis

• 1.1. Compartmentation of uridine 5'-triphosphate (UTP) was studied during synthesis of cytoplasmic ribosomal RNA (cyt-rRNA) and plastid ribosomal RNA (pl-rRNA) in photoorganotrophically grown cells of Euglena gracilis Z.
• 2.2. Using the approach of Wiegers et al. (1976) the steady state specific radioactivity of UTP was compared with that ofcyt-20S rRNA, cyt-25S rRNA, pl-16S rRNA and pl-23S rRNA under low and at 100-fold higher specific radioactivity of exogenously fed pHl-uracil.
• 3.3. The equal steady state specific radioactivities of all rRNAs at both feeding conditions argue against compartmentation of UTP during their synthesis.
• 4.4. At high specific radioactivity of exogenous [³H]-uracil the salvage-derived labelled UMP was shown to be diluted 15,000-fold by unlabelled UMP formed de novo, whereas this dilution factor was 100-fold lower at low specific radioactivity of [³H]-uracil indicating inhibition of the de novo synthesis of UMP.
• 5.5. Transport is suggested of uridine nucleotides into chloroplasts by the 15-fold higher specific radioactivity of intracellular [³H]-uracil than that of UTP as well as UMP residues in pl-rRNA.

     

Enhancement of phospholipid hydrolysis in vasopressin-stimulated BHK-21 and H9c2 cells

The hydrolysis of phospholipids in vasopressin-stimulated baby hamster kidney (BHK)-21 and H9c2 myoblastic cells was investigated. Phosphatidylcholine and phosphatidylethanolamine in these cells were pulse labelled with [³H]glycerol, [³H]myristate, [³H]choline or [³H]ethanolamine, and chased with the non-labelled precursor until linear turnover rates were obtained. When cells labelled with [³H]glycerol or [³H]myristate were stimulated by vasopressin, no significant decrease in the labelling of phosphatidylcholine was detected, but the labelling of phosphatidic acid was elevated. However, the labellings of phosphatidylethanolamine and its hydrolytic product were not affected by vasopressin stimulation. When the cells were pulse labelled with [³H]-choline, vasopressin stimulation caused a decrease in the labelled phosphatidylcholine with a corresponding increase in the labelled choline. The apparent discrepancy between the two types of labelling might be explained by the recycling of labelled phosphatidic acid back into phosphatidylcholine, thus masking the reduction in the labelled phospholipid during vasopressin stimulation. Alternatively, the labelled choline produced by vasopressin stimulation was released into the medium, thus reducing the recycling of label precursor back into the phospholipid and making the decrease in the labelling of phosphatidylcholine readily detectable. Further studies revealed that vasopressin treatment caused an enhancement of phospholipase D activity in these cells. The presence of substrate-specific phospholipase D isoforms in mammalian tissues led us to postulate that the differential stimulation of phospholipid hydrolysis by vasopressin was caused by the enhancement of a phosphatidylcholine-specific phospholipase D in both BHK-21 and the H9c2 cells.Abbreviations BHK-21 cells baby hamster kidney-21 cells     

Autoradiographic localization of [3H]hydroxytamoxifen to uterine oestrogen- and antioestrogen-binding sites

Summary Immature rats were injected subcutaneously with 0.36 g of [³H]hydroxytamoxifen ([³H]TAM(OH)) or 0.24 g of [³H]oestradiol in oil, and 4 h later uteri were processed for thaw-mount autoradiography. The specificity of [³H]TAM(OH) localization was determined by injecting a 200-fold excess of unlabelled TAM(OH) or a 20-, 200- or 2000-fold excess of oestradiol 1 h before injection of [³H]TAM(OH). After injection of [³H]TAM(OH) or [³H]oestradiol, autoradiograms showed concentration of radioactivity in nuclei of stromal, epithelial and myometrial cells, but this labelling varied among the cell types depending upon which compound was injected. After [³H]TAM(OH) injection, the decreasing order of labelling intensity was stroma, myometrium, epithelium; after [³H]oestradiol injection the decreasing order was stroma, epithelium, myometrium. Injection of TAM(OH) before [³H]TAM(OH) eliminated nuclear labelling in all the uterine cell types. Injection of oestradiol before [³H]TAM(OH) decreased nuclear labelling and resulted in the concentration of label in the cytoplasm of luminal epithelium which was not present when [³H]TAM(OH) was injected alone. Cytoplasmic labelling increased initially as the oestradiol competition dose increased, but the increase in labelling did not continue with increasing concentrations of oestradiol. The results indicate that antioestrogen and oestrogen localize to nuclei of the same uterine cell types, but that cellular uptake differs among the tissue compartments. The results also suggest that a high concentration of antioestrogen-binding sites exist in the cytoplasm of the uterine luminal epithelium.