Kinetics of velvet tobacco mottle virus satellite RNA synthesis and encapsidation

Synthesis of circular (RNA 2) and linear (RNA 3) molecules of velvet tobacco mottle virus (VTMoV) satellite RNA (sat RNA) has been studied by incubating strips of tissues excised from systemically infected Nicotiana clevelandii in solutions of [14C]uridine. After a short lag, RNA and virus synthesis proceeded at a constant rate for at least 24 hr, during which time most of the synthesis was directed to the production of RNAs 2 and 3. The kinetics of [14C]uridine incorporation into the sat RNA molecules after increasing times of incubation and during pulses of [14C]uridine followed by chase incubation with excess [12C]uridine suggest that RNA 3 is a percursor of RNA 2. However, not all the RNA 3 synthesized was shown to end up as RNA 2, even after 72 hr of incubation. Several lines of evidence are presented supporting the conclusion that VTMoV-infected cells contain large pools of unencapsidated sat RNA. It is suggested that the sat RNA may have a greater affinity for the VTMoV replicase than the helper viral RNA which results in copious production of the sat RNA.     

Consequences of adenosine deaminase deficiency on thymocyte metabolism

Using 2′-deoxycoformycin inhibition of adenosine deaminase as a model of adenosine deaminase deficiency, the effects of 10 μM 2′-deoxyadenosine (dAdo) on the metabolism of concanavalin A (Con A)-stimulated rat thymocytes were studied. When dAdo and Con A were added simultaneously, a strong inhibition of the incorporation of [³H]thymidine (84%); [³H]uridine (98%) and L-[³H] leucine (46%) in the acid-insoluble fraction, and of [¹⁴C]formate (78%) and H¹⁴CO₃^? (43%) uptake is observed after 48 h of incubation. When dAdo is added after 12 h of Con A stimulation, no such inhibition is observed, but when added after 24 h of stimulation, there is an enhancement of blastogenesis as measured by nucleic acid, protein, and purine and pyrimidine base synthesis. More detailed studies of thymocytes stimulated by Con A for 0–72 h, followed by short-term incubation periods with dAdo (1–5 h), revealed that thymocyte metabolism becomes progressively less sensitive to dAdo-mediated inhibition during the course of blastogenesis.     

Alternate radiolabeled markers for detecting metabolic activity of Mycobacterium leprae residing in murine macrophages.

This study demonstrated the utility of using 4% NaOH as a murine macrophage cell-solubilizing agent to discriminate between host macrophage metabolism and that of intracellular Mycobacterium leprae. A 4% concentration of NaOH had no deleterious effect on labeled mycobacteria. Thereby, alternate radiolabeled indicators of the metabolic activity of intracellular M. leprae could be experimented with. Significant incorporation of 14C-amino acid mixture, [14C]leucine, [14C]uridine, and carrier-free 32P was observed in cultures containing freshly extracted ("live") strains of M. leprae as compared with control cultures containing autoclaved bacilli.     

TNF-alpha secretion and macrophage mortality induced by cobalt and chromium ions in vitro-qualitative analysis of apoptosis

Metal ion toxicity is a major cause for concern in metal-metal hip replacements. A previous study in our laboratory demonstrated that Co(2+) and Cr(3+) induce macrophage apoptosis in vitro at 24h, with the implication of a caspase-3 pathway. The aim of the present study was to look at the effect of a prolonged incubation time on macrophage response with regards to TNF-alpha secretion and macrophage mortality, more specifically apoptosis. J774 macrophages were exposed for up to 48 h to 0-10 ppm Co(2+) and 0-500 ppm Cr(3+). ELISA results demonstrated that Co(2+ )and Cr(3+) induced a concentration- and time-dependent increase of TNF-alpha secretion, but a decrease at the highest concentrations of Cr(3+) (350-500 ppm). This decrease was most likely due to a high toxicity of Cr(3+) at such concentrations. Higher levels of TNF-alpha were observed with Co(2+) than Cr(3+), demonstrating a higher stimulatory effect of this ion. Trypan blue and flow cytometry results demonstrated that both Co(2+) and Cr(3+) ions induce macrophage mortality in a dose- and time-dependent manner. The number of cells decreased when ion concentrations increased, especially at 48 h. In parallel with the TNF-alpha results, Co(2+) was more toxic than Cr(3+) since the maximal effects were reached with lower concentrations (8-10 ppm vs. 350-500 ppm, respectively). DNA analysis demonstrated that both Co(2+) and Cr(3+) ions induce macrophage apoptosis, with a stronger signal at 24h than at 48 h, suggesting the presence of more necrosis after 48 h. PARP cleavage, another marker of apoptosis, was observed at both 24 and 48 h, with a maximum intensity at 48 h and with the highest concentrations of ions. In conclusion, this study demonstrates that both Co(2+) and Cr(3+) ions can induce the release of TNF-alpha and macrophage mortality in a dose- and time-dependent manner. More specifically, Co(2+) and Cr(3+) ions induced apoptosis after both 24 and 48 h incubation, although DNA analysis suggested the presence of necrosis at 48 h. The relative importance of apoptosis and necrosis in the induction of macrophage mortality by these metal ions remains to be investigated.     

Effect of corticosteroids on committed lymphocytes.

Human peripheral blood lymphocytes were cultured for 48 hr with concanavalin A. The amount of [3H]thymidine incorporated during the last 4 hr of culture, as well as the percentage of rosettes of activated lymphocytes generated, were assayed at the 48th hr. Adding 0.1 M alpha-methyl-D-mannoside (MAM) at progressively later times after the initiation of culture caused progressively less suppression because of the commitment phenomenon. This suppression was not exceeded by the addition of 10(-4) M preparations of corticosteroids and was statistically the same as that induced by a combination of both corticosteroids and MAM. The addition of PGE2 alone and in combination with methylprednisolone also failed to affect [3H]thymidine incorporation by committed lymphocytes.     

Quantitative estimation of cytotoxic activity of immune lymphocytes using 51Cr-labelled peritoneal macrophages as target cells.

Peritoneal macrophages labelled with 51Cr in suspension and cultivated for 48 hr on glass pretreated with poly-L-lysine were used as target cells for determination of the cytotoxic effect of immune lymphocytes. 51Cr release from such target cell from different strains of mice is 15.5 +/- 0.8% of the total target cell radioactivity after 20 hr incubation with normal allogeneic lymphocytes. The use of 2% sodium dodecylsulphate ensures 100 percent solubilisation of labelled target cells growing on the glass surface and permits the cytotoxic effect to be determined by both 51Cr release and measuring the label retained by the intact cells. The two methods proved to be accurate, reproducible and in accord with each other as well as with the method of direct cell counting. Determination of released 51Cr enables the cytotoxic effect of lymphocytes to be measured after 4 hr incubation with the macrophage monolayer. SaI and Mc11 ascitic sarcomas display different sensitivities to the cytotoxic effect of immune lymphocytes and require different optimum conditions for its development.     

Cucumber mosaic virus-associated RNA 5. IX. The overtaking of viral RNA synthesis by CARNA 5 and dsCARNA 5 in tobacco

Cucumber mosaic virus (CMV) and CMV-associated RNA 5 (CARNA 5)-related RNA synthesis was monitored under conditions of semisynchronous infection using a differential temperature inoculation technique (Dawson and Schlegel, 1973). Leaf strips sampled at specific intervals between 0 and 100 hr after temperature shift were vacuum infiltrated with [32P]phosphoric acid and actinomycin D and incubated during 4-hr periods. Total nucleic acid extracts were analyzed on polyacrylamide gels to compare the relative rates of 32P incorporation into CMV-RNA 3, CARNA 5, and dsCARNA 5. During the first 24 hr there was a rapid increase in the rate of 32P incorporation into all three RNAs. During the next 10-20 hr the rate of 32P incorporation into RNA 3 declined to minimal levels while that of CARNA 5 stayed at a plateau or declined slowly. In contrast, the rate of 32P incorporation into dsCARNA 5 increased steadily well beyond the first 48 hr after temperature shift. The distribution of radioactivity among its (+) and (-) strands was determined by isolating the dsCARNA 5 in each nucleic acid extract obtained from the 4hr-labeled tissues and determining its radioactivity after hybridization in the presence and in the absence of a large excess of unlabeled CARNA 5. It appeared that throughout the 100-hr experiment about 60% of the radioactivity of dsCARNA 5 was in its (+) strands. The rates of 32P incorporation into virus and CARNA 5-related RNAs were also compared in leaf strips from plants inoculated with the genomic CMV-RNAs alone and with a mixture of genomic RNAs and CARNA 5. Although in the infection with the genomic RNAs alone the synthesis of CARNA 5 and dsCARNA 5 was not prevented, the increase in their relative rates of synthesis seemed significantly slower and the relative rate of RNA 3 synthesis much greater than when the inoculum contained detectable CARNA 5.     

Effects of oxygen metabolites on rat alveolar type II cell viability and surfactant metabolism

Neutrophil-derived reactive oxygen metabolites have been implicated as one mechanism for the cellular injury in the adult respiratory distress syndrome. Previous studies have demonstrated that alveolar lung fluid of patients with adult respiratory distress syndrome has abnormal composition and surface active properties. To examine the effects of oxygen metabolites on the viability and metabolism of type II alveolar pneumocytes, the cellular source of surfactant, isolated rat type II pneumocytes were exposed to reactive oxygen metabolites generated by the enzymatic action of xanthine oxidase upon hypoxanthine. Utilizing a 51Cr release assay to detect cellular death, we found that oxygen metabolites were lethal to type II cells in a dose-dependent manner. To demonstrate that oxygen metabolites were responsible for the toxicity, we assessed the protective effects of catalase and superoxide dismutase, scavengers of hydrogen peroxide and the superoxide anion, respectively. At a xanthine oxidase concentration of 50 mU/ml, catalase reduced the percentage of 51Cr release from 58.9 +/- 3.1% (SEM) to 7.2 +/- 2.3% (p less than 0.0001), whereas superoxide dismutase was without protection (58.9 +/- 3.1% versus 54.2 +/- 1.8% (p greater than 0.05). To determine whether oxygen metabolites also impair surfactant metabolism, we measured the incorporation of [3H]palmitate into the surfactant component disaturated phosphatidylcholine by type II pneumocytes. We found that sublethal amounts of generated oxygen metabolites caused a progressive decrease in the amount of [3H]palmitate incorporated into disaturated phosphatidylcholine. For example, using a xanthine oxidase concentration of 5 mU/ml (which causes no increased 51Cr release), we found that [3H]palmitate incorporation into disaturated phosphatidylcholine fell from a control level of 3.53 +/- 0.22 X 10(5) to 0.66 +/- 0.10 X 10(5) dpm/10(6) cells/4 hours (p less than 0.0001). Both catalase and superoxide dismutase protected the [3H]palmitate incorporation of oxygen metabolite-exposed type II cells. We conclude that reactive oxygen metabolites are injurious to type II pneumocytes and may result in impaired surfactant synthesis even at sublethal doses. Thus, oxygen metabolites generated by stimulated phagocytic cells may be responsible in part for the decreased surfactant that has been observed in adult respiratory distress syndrome.     

RNA synthesis in activated macrophages I. Poly(I) · poly(C)-induced triggering of cytolytic activity is associated with decrease in RNA synthesis

The effects of polyinosinic, polycytidylic acid [poly(I) · poly(C)] on the activation and RNA metabolism in murine peritoneal macrophages (MΦ) elicited by proteose-peptone (pMΦ) was investigated. Poly(I) · poly(C) triggered the cytolytic activity of pMΦ and augmented their glucose oxidation. In contrast, a profound depression of [³H]uridine incorporation into RNA was observed in poly(I) · poly(C)-activated pMΦ. The degree of depression of RNA labeling paralleled the dose of poly(I) · poly(C) used to activate the pMΦ and the expression of tumoricidal activity. This decrease in [³H]uridine incorporation into MΦ RNA could not be accounted for by decreased permeability of the activated MΦ to [³H]uridine, or by instability of the labeled RNA. Moreover, analysis of the specific activity of the intracellular uridine triphosphate (UTP) pool and studies on the labeling of MΦ RNA with [³²P] orthophosphate indicated that the decreased RNA labeling was not due to changes in the specific activity of UTP. We concluded that poly(I) · poly(C)-activated pMΦ exhibit a depressed rate of RNA synthesis. We suggest that the rate of RNA synthesis may be investigated as a potential new indicator for MΦ activation.     

Replication of tobacco mosaic virus : III. Viral RNA metabolism in separated leaf cells

The metabolism of RNA has been studied in cell suspensions prepared from tobacco mosaic virus (TMV)-infected tobacco leaves. The cell suspensions incorporated [³H]-uridine into encapsidated viral RNA at a constant rate for as long as 44 hr. Gel electrophoretic analyses of RNA extracted from infected and uninfected cells which had been exposed to [³H]uridine, showed that both incorporated label into ribosomal RNA species. However, RNA preparations from infected cells contained five additional species of RNA not observed in preparations from healthy cells; in addition to TMV RNA, label was detected in two species of double-stranded RNA identified as replicative form (RF) and replicative intermediate (RI), a single-stranded RNA component of low molecular weight (LMC) and one with a molecular weight greater than that of TMV RNA. Synthesis of all five species of virus-specific RNA was insensitive to actinomycin D indicating their independence of cellular DNA.     

Production of macrophage-activating and migration-inhibition factors in vitro by serologically selected and cloned Listeria monocytogenes-specific T cells of the Lyt 1+2- phenotype

Lyt-selected Listeria-immune T lymphocytes from peritoneal exudates and cloned T cells were cocultured with heat-killed listeriae and peritoneal macrophages from nonimmune donors. Supernatants were assayed for: activation of macrophages for tumoristatic and tumoricidal activity via macrophage-activating factors and migration-inhibition factor activity. Peptone-induced peritoneal macrophages were activated by incubation with the supernatants for 24 h. For examination of cytocidal activity, 51Cr-labeled EL4 tumor cells were subsequently added, and 51Cr release was determined. Cytostatic activity was measured by adding unlabeled EL4 tumor cells to the pretreated macrophages and determining [3H]thymidine incorporation 24 h later. Migration-inhibition factor production was examined in an agar microdroplet assay. Only Listeria-specific T cells of the phenotype Lyt 1+2- proved active in these assays, whereas T cells of the phenotype Lyt 1-2+ were not active. When T-cell clones were used, a single clone was capable of inducing macrophage-activating and migration-inhibition factor production at cell concentrations of ca. 10(3)/ml.     

Human cytomegalovirus induces DNA-dependent RNA polymerases in human diploid cells.

Endogenous RNA polymerase activity was enhanced in human embryonic cells infected with human cytomegalovirus (HCMV) as determined by the incorporation of [³H]UTP into nuclear monolayers. The increase in activity was first detected about 18 hr postinfection, and reached a level 3-fold higher by 48 hr. This kinetics of enhancement coincided with the kinetics of [³H]uridine incorporation into RNA in the infected, intact cells. The treatment of HCMV-infected cells with either cycloheximide or actinomycin D during the first 6 hr of infection practically abolished the subsequent enhancement of endogenous RNA polymerase activity, suggesting a key role of a HCMV-induced early protein(s) in the stimulation of endogenous RNA polymerase activity. Multiple forms of cellular RNA polymerases from HCMV- or mock-infected cells were separated by DEAE-Sephadex chromatography and assayed using calf thymus DNA as a template. In HCMV-infected cells, there was a marked increase in the activity of three major classes of RNA polymerases; the activity corresponding to RNA polymerase II increased 16-fold and those corresponding to polymerases I and III 6- and 3-fold, respectively.     

Standardization of tissue culture conditions for spontaneous thymidine-2-14C incorporation by unstimulated normal human peripheral lymphocytes: circadian rhythm of DNA synthesis.

The rate of DNA synthesis by human lymphocytes was studied in vitro by measuring unstimulated thymidine-2-14C incorporation (spontaneous lymphocyte blastogenesis; SLB). Freezing lymphocytes and extracting DNA after thawing did not alter the radioactive label count rate and was as efficient as extracting DNA immediately after culture. Omission of fetal calf serum also did not alter the rate of DNA synthesis. Standards established as optimal for studies of SLB were: cell concentration, 1.0 times 10(6)/ml/tube; 14C-TdR concentration, 0.4 mjCi/tube; duration of incubation, 8 hr. In sets of identical samples obtained by specimen division, the variation in counts was 6%. To achieve reproducibility of results; it was essential to count the lymphocytes, and then to ensure that each tube contained almost precisely known numbers of cells. Diurnal variations in the rate of DNA synthesis by circulating lymphocytes of healthy men were measured in vitro by SLB at 2-hr intervals for 24 hr. Leukocyte counts, hematocrit, hemoglobin, plasma cortisol, and body temperature were monitored concurrently. The DNA synthesis rate varied in a 24-hr cycle with peaks at 10 A.M. and 11:00 P.M.., depressions at 4 A.M. and 4 P.M. The rate was correlated with body temperature and hematocrit level, and inversely related to the absolute eosinophil count.     

Nucleic acid metabolism in the rat following short-term and prolonged ingestion of chrysotile asbestos or cigarette-smoke condensate.

Rats which have ingested a natural diet containing chrysotile asbestos (50 mg/day) both in the short (one week) and long term (5-15 months) show a statistically significant increase in the incorporation of [3H]-thymidine into DNA in the small intestine mucosa, colon and rectum, stomach and spleen and a significant decrease in the incorporation of this radiolabel into liver DNA. However, short-term ingestion of similar quantities of chrysotile produced no significant change in the incorporation of [3H]-uridine into RNA in any tissues, although prolonged ingestion of the mineral induced alterations in RNA metabolism in the lung and liver. By contrast, animals ingesting a natural diet containing cigarette-smoke condensate show a significantly high incorporation of [3H]-uridine into RNA in the mucosa, submucosa, spleen and heart following short-term exposure. Long-term ingestion of this diet produces similar changes in the submucosa, spleen and lung. The apparent specificity and the mechanism whereby ingested chrysotile interferes with DNA metabolism in some body tissues is discussed.     

Specific activity of several enzymes of nucleotide metabolism in mouse spleen after immunization

The specific activity of uridine kinase and rate of UMP formation de novo in the particle-free extract of mouse spleen increased within 8 hr after the intraperitoneal injection of SRBC. This heightened activity was found up to 72 hr and by 144 hr returned to or below control levels. The thymidine kinase activity increased up to 48 hr and then decreased to normal levels by 144 hr.

Activities of both uridine kinase and thymidine kinase were additive after the simultaneous injection of SRBC and BGG.

     

RNA-nascent DNA complexes in Ehrlich ascites tumor cells in vivo

When mice bearing Ehrlich ascites tumors were given a single intraperitoneal injection of [5-³H]uridine, the ascites cells incroporated radioactivity into DNA for 4 hr, while incorporation into total cellular RNA ceased after 20–30 min. Nascent DNA, isolated by Cs₂SO₄ isopycnic centrifugation 30 min, 4 hr or 50 hr after [5-³H]uridine injection contained RNA fragments. Treatments with hydroxyurea, an inhibiton hibitor of DNA synthesis, reduced the incorporation of [5-³H]uridine into the DNA-RNA complex to 43% of controls. Treatment with actinomycin D, an inhibitor of DNA-dependent RNA synthesis, abolished the incorporation of [5-³H]uridine into the DNA-RNA complex. Finally, when DNA was pulse-labeled with [¹⁴C]deoxythymidine and then denatured by heat-treatment, it banded in a region with a higher density than that of reference DNA. However, if treated with alkali, it banded at the density of reference DNA, again suggesting the presence of RNA fragments in nascent DNA. The length of the RNA chains in this DNA-RNA complex was estimated to be in the order of 70 nucleotides.     

A possible role of PMN in a casein-induced enhancement of PFC response to sheep erythrocytes in mice.

The effect of inflammation induced by sodium caseinate or aluminum hydroxide on the splenic plaque-forming cells (PFC) response to sheep red blood cells (SRBC) was studied in mice. Direct and indirect splenic PFC responses were enhanced when suboptimal SRBC doses (3 x 10(6)) were injected intraperitoneally (i.p.) within 9 hr of i.p. inflammatory stimulation; antigen administration 48 hr or more after such stimulation resulted in a slight suppression of the direct response. The inflammation had no effect on the secondary immune response, nor did intravenous antigen administration enhance the PFC response. Enhancement occurred when early (3 hr), casein-induced peritoneal exudate cells (PEC, consisting mostly of neutrophils) were adoptively transferred at the same time as antigen. Treatment of the 3-hr PEC with anti-Thy-1 and complement did not decrease their PFC-enhancing capability. Late (96-hr) PEC, consisting mostly of macrophages, manifested only a slight enhancing effect. We suggest that enhancement of the splenic PFC response in the presence of an ongoing inflammation, may be partially attributable to neutrophil function.     

The effect of 5-fluorouracil on [3H]nucleoside incorporation into the DNA of mouse embryos and maternal tissues

Pregnant albino mice (ICR) were administered, ip, 40 mg/kg 5-fluorouracil (5-FU) on Day 10 of gestation. This dosage produced 96.3% embryolethality and 100% of the surviving fetuses were malformed. This dosage also produced a pronounced inhibition of 6-[³H]dUrd incorporation into the DNA of the embryo, chorioallantoic placenta, maternal intestine, and maternal spleen. There was no recovery in the embryo or placenta over the 48 hr time period analyzed. In the maternal tissues, however, incorporation levels returned to control values by 48 hr after 5-FU administration. 5-FU had no effect on 6-[³H]dThd incorporation into the DNA of any of the mitotically active tissues with the exception of the embryo at the 48 hr time point. Concomitant histological studies of the embryo neural tube showed that 40 mg/kg 5-FU produced mitotic inhibition 2 hr after treatment. Cell death began at 4 hr with heteropycnosis in the DNA synthetic zone of the primitive ependymal layer. As with the incorporation experiment, there was no histological recovery in the neural epithelium and the pycnosis became progressively more severe with time after 5-FU treatment.     

Cell cycle-specific effects of glucocorticoids on phytohaemagglutinin-stimulated lymphocytes.

Effects of steroid hormones and colchicine on the response of pig lymphocytes to phytohaemagglutinin (PHA) were assessed by measurement of [6-3H]thymidine incorporation. At steroid concentrations of 1 microM and below, only glucocorticoids and progesterone inhibited PHA-stimulated [6-3H]thymidine incorporation but at 100 microM inhibition was also produced by oestrogens, androgens and physiologically inactive steroids. Measurement of [6-3H]thymidine incorporation 18-24 hr, 6-12 hr or 0-6 hr after the delayed addition of the synthetic glucocorticoid, dexamethasone, to PHA-stimulated lymphocytes revealed a succession of alternating phases of sensitivity and insensitivity to the effects of the steroid which suggested that it was acting, perhaps indirectly, in a cell cycle stage-specific manner to arrest the progression of activated lymphocytes from G1 to S. Similar effects were observed with colchicine, but 100 microM 11-epicortisol inhibited [6-3H]thymidine incorporation in a non-cycle-specific manner. Glucocorticoid receptor levels in pig lymphocytes were increased 2-5-fold within 24 hr of PHA stimulation.     

Processing of SV40 RNA is associated with the nuclear matrix and is not followed by the accumulation of low-molecular-weight RNA products

Nuclear matrices from SV40-infected cells were prepared by treating purified nuclei with DNase and salt to extract DNA and histones. After a 10-min pulse with [5,6(-3)H]uridine over 85% of the viral RNA was found in association with the nuclear matrix. Following a 3-hr chase with glucosamine and unlabeled uridine, 2-4 S nuclear viral components accumulate, but they are not associated with the nuclear matrix. The 2-4 S components had been characterized previously as viral RNA processing products (N. H. Chiu, M. F. Radonovich, M. M. Thoren, and N. P. Salzman, J. Virol. 28, 590-599, 1978). The results of the present study identify the 2-4 S components as DNA rather than RNA, thus indicating that the synthesis and the majority of the processing products of the viral RNA are associated with the nuclear matrix.