The effect of synaptic stimulation on RNA and protein metabolism in the R2 soma of Aplysia

Excitatory synaptic stimulation of the R2 neuron in the abdominal ganglion of Aplysia californica causes an increased incorporation of ³Huridine into RNA. However, this could be the result of a change in precursor specific activity rather than an increase in RNA synthesis. We find that at low external uridine concentrations (1.5 μM) there is no increase in ³H-uridine incorporation correlated with synaptic stimulation. In addition, no change in incorporation of ³H-leucine into total protein or in the pattern of newly-synthesized proteins, resolved by electrophoresis on SDS-polyacrylamide gels, was detected with stimulation. Since the R2 neuron can be stimulated without a detectable change in RNA or protein synthesis, we conclude that the increase in incorporation observed at high external uridine concentrations (100 μM) could be caused by increased specific activity in a precursor pool rather than by an RNA synthesis change.     

IN VITRO INCORPORATION OF 3H-THYMIDINE, 3H-URIDINE, 3H-LEUCINE AND 3H-MELPHALAN BY PLASMACYTOMA PLASMA CELLS

Bone marrow plasma cells from fifteen cases of multiple myeloma, immunologically typed, were incubated with different tritiated compounds. The labelling index with tritiated thymidine is generally low, while the mean grain count is fairly normal in the active cells. The labelling index of ³H-uridine and ³H-leucine was very high, while the mean grain count per cell lies within the normal range. The results obtained with ³H-phenylalanine-mustard (melphalan), which is a drug used in the treatment of the plasmacytoma, show also incorporation values roughly comparable to those of ³H-leucine. The present data seem to support the clinical use of melphalan as a compound that is actively incorporated into the plasma cells of plasmacytoma although inhibition of protein synthesis due to specific binding to protein was not demonstrated.     

Metabolism ofAedes aegypti cells grown in vitro. 1. Incorporation of3H-uridine and14C-leucine

Summary Metabolic activity ofA. aegypti cells grown in vitro has been studied by incorporation of³H-uridine and¹⁴C-leucine. “Chase” experiments with unlabeled precursors, and the use of actinomycin D and puromycin, showed that³H-uridine was incorporated into cellular RNA, and that¹⁴C-leucine was incorporated into protein of these cells. Incorporation of³H-uridine was inhibited when actinomycin D was used at a concentration of 10 μg/ml, and¹⁴C-leucine incorporation was inhibited to the same extent by puromycin at a concentration of 100 μg/ml medium. Contribution No. 148.     

Some biochemical effects of insulin and steroid hormones on the rat prostate in organ culture

The effect of various steroids and insulin on expiants of the ventral prostate of adult rats in organ culture was investigated. Testosterone activated the incorporation of labeled precursors into RNA and protein and the formation of ¹⁴CO₂ from ¹⁴C-glucose by expiants. All these effects were mimiced by insulin. The testosterone action was suppressed by cyproterone in vitro. In addition to androgens, glucocorticoids activated the incorporation of ³H-uridine into RNA. Simultaneous addition of testosterone with hydrocortisone or insulin produced an augmented effect on the incorporation of ³H-uridine into RNA which exceeded that resulting from a simple summation of the individual hormone responses. Insulin facilitated likewise the action of testosterone on the incorporation of ¹⁴C-amino acids into protein. When all three hormones were added simultaneously to the culture medium the stimulation of the three biochemical parameters was maximal. It was therefore concluded that all three hormones are necessary for an adequate maintenance of the ventral prostate of the rat.     

Characterization of the female sterile (1) 1304 mutant of Drosophila melanogaster: pattern of RNA metabolism in the ovary

The female sterile mutant of Drosophila melanogaster, fs(1)1304 (1-19 +/- 2), has been characterized. Our studies show that the mutation affects the organization of nucleolar material in the ovarian nurse cells and the pattern of RNA metabolism in the ovary. Autoradiographic analysis of incorporation of 3H-uridine in vivo and analysis of 3H-uridine incorporation into high molecular weight RNA in vitro suggest that RNA from the ovaries of homozygous fs flies is degraded at a higher rate than that from heterozygous fs and wild-type ovaries. It is likely that the RNA class affected is ribosomal RNA. These data are discussed in the context of the functional role for the wild-type gene allelic to fs(1)1304, and it is suggested that one of the effects of the mutation may be on the biogenesis of ribosomes that are to be stored in the oocyte.     

Macromolecular synthesis and stability in growth-arrested BALB/c-3T3 cells

Concentrations of methylglyoxal bis-(guanylhydrazone) (mGBG) that inhibited serum-stimulated BALB/c-3T3 cells in late G1 caused a marked inhibition of 3H-leucine incorporation during a 20-min incubation. No decrease was observed in the incorporation of 3H-uridine during a 20-min incubation; however, the amount of acid-insoluble 3H-uridine in mGBG-treated cultures was decreased when the incubation period was longer than 20 min. The amount of the decrease in the accumulation of incorporated 3H-uridine was directly proportional to the length of the incorporation time. Between 10 and 12 h after quiescent BALB/c-3T3 cells were serum-stimulated in mGBG no additional 3H-uridine was accumulated. The stability of the incorporated 3H-uridine, as determined by acid-insoluble radioactivity remaining after the addition of actinomycin D, was less in cells cultured in mGBG. Exogenous spermine or spermidine reversed the inhibition of 3H-uridine accumulation in acid-insoluble material produced by mGBG as well as the decrease in stability of the incorporated 3H-uridine in acid-insoluble material. The effects of mGBG on both the incorporation of 3H-uridine and the stability of the incorporated 3H-uridine can apparently be accounted for by an effect on ribosomal RNA.     

Studies of oogenesis in the rotifer,Asplanchna

Summary Oocyte development in Asplanchna brightwelli was studied by observation through the transparent body wall of living females and by electron microscopy. During oogenesis, which requires four to six hours, the oocyte increases in volume approximately 1000-fold. Most of its cytoplasm appears to be derived from the vitellarium by direct flow through the cytoplasmic bridge. This flow is rapid enough to be easily observed in the living female at low magnifications. Ribosomes, mitochondria, cortical granules, and lipid droplets were observed in the bridge area in electron micrographs.RNA synthesis during oogenesis was studied by means of autoradiography. Very little synthesis could be demonstrated in oocyte nuclei at any period of oogenesis, whereas the vitellarium nuclei show active incorporation of ³H-uridine throughout the reproductive life of the adult female. Most of this RNA is subsequently transferred to developing oocytes.This research was supported by USPHS Grant GM 121183 to Dr. C. W. Birky, Jr.     

Effect of thiopyrimidine ribonucleosides on DNA and RNA synthesis in synchronized mammalian cell cultures

The uptake of ³H-uridine into RNA and of ³H-thymidine into DNA was investigated in synchronized Chinese hamster cells which had been exposed to thiopyrimidine ribonucleosides. The cells were synchronized at metaphase by reversal of colcemid inhibition; these cells were then labeled with either ³H-thymidine or ³H-uridine at selected times, and analyzed in autoradiographs. Incorporation of ³H-thymidine into DNA was not inhibited by administration to the cells of 2-thiouridine or 4-thiouridine (4 × 10⁻³ M). Exposure of the cells to the anti-metabolites for over 15 h significantly reduced the incorporation of ³H-uridine into nuclear RNA and completely blocked the labeling of cytoplasmic RNA. This finding is interpreted as an indication that RNA synthesis was inhibited in cells which continued to synthesize DNA. The inhibition of RNA synthesis hindered cell division and decreased cell viability. This lethal effect is similar to the “unbalanced growth” induced by inhibitors of DNA synthesis. The thiopyrimidine ribonucleosides, however, killed mammalian cells without inhibiting DNA synthesis.     

Incorporation of label from radioactive uridine into the stylar nucleic acids of Lilium longiflorum thunb. as affected by heat, 6-methylpurine and actinomycin D

Summary 5-³H-uridine injected into the stylar canal of detached lily stigma-styles was taken up initially into the rapidly-labeled-RNA of the nucleic acid profile of a methylated albumin kieselguhr (MAK) column but with increasing time was found in all portions of the RNA profile, but not in the DNA. Heat treatment of the style before injection of 5-³H-uridine greatly reduced the rate of incorporation of label into and the ultimate amount of label found in the RNA species of the lily style. Translocation of 5-³H-uridine through the ovary into heattreated pistils and the injection of 5-³H-uridine into styles which had been incubated for 1 or 2 days after heat treatment resulted in stylar nucleic acids more highly labeled than nucleic acids in control styles, with an incorporation pattern different than control styles. Heat treatment of lily pistils resulted in detectable changes in the proportion of stylar RNA species as separated on MAK columns and measured as absorbance units. Actinomycin D and 6-methylpurine treated styles incorporated label from a stylar injection of radioactive uridine in patterns different than each other, different than heat-treated styles and different than non-treated styles. 6-methylpurine and heat treatment of styles only slightly reduced the rate at which 5-³H-uridine was removed from the stylar canal into the stylar tissue.Paper number 8917 of the Scientific Journal Series, Minn. Agr. Exp. Sta., St. Paul, MN 55108.     

Replication of Western Equine Encephalomyelitis Virus II. Cytoplasmic Structure Involved in the Synthesis and Development of the Virions

Analysis of the cytoplasmic fraction of chick embryo cells during the exponential phase of Western equine encephalomyelitis (WEE) virus growth showed that the viral ribonucleic acid (RNA) labeled by a short pulse with ³H-uridine was associated with a structure which sedimented in sucrose density gradients with a coefficient of 65S. The RNA extracted from this structure sedimented in sucrose density gradients at 26S. After a longer period of exposure to ³H-uridine, the radio-active viral RNA was associated with a structure which sedimented in sucrose density gradients as would materials with coefficients of about 140S. The 140S structure contained viral RNA and viral protein. It was shown that the 140S structures are not virus-induced polysomes. The 140S structure contained predominantly the 40S type of viral RNA and some 26S type. Electrophoretic analysis of the disrupted virion revealed that at least two proteins (types I and II) were present in the purified virion. Only type II protein was present in the 140S structure. Unlike the virion, the 140S structure did not contain any lipid which could be detected by the incorporation of ¹⁴C-choline. These data suggest that the 140S structure represents the internal nucleoprotein part of the virion. The rate of appearance of labeled virus lags behind that of the formation of the 140S structure in infected cells. Pulse-chase experiments with ³H-leucine suggest that the 140S structure may represent a precursor to the virus particle. The results are discussed in terms of the maturation of WEE virus in the infected cells.     

Functional activity of uremic erythroblast incubated in autologous and homologous plasma.

The uptake of 3H-thymidine, 3H-uridine and 3H-leucine in the erythroid precursors of patients with chronic renal failure (CRF) was examined by radioautography. The pattern of incorporation of the radioactive precursors was similar to that observed in erythroblasts of control subjects, i.e., the uptake decreased with cell maturation. CRF erythroblasts incubated with normal, homologous plasma, showed significant increase in the uptake of the radioactive precursors, compared to the activity of these cells incubated in autologous plasms, the only exception being the incorporation of 3H-leucine in the proerythroblasts, in which the increase was not statistically significant. These results suggest that the impaired function of CRF erythroblasts related to DNA, RNA and protein synthesis is due not to a defective mechanism in the cells themselves, but most probably to the effect of factors present in uremic plasma, the nature of which remains to be detected.     

In vitro culture of growing mouse oocytes

A method is described for in vitro culture of naked growing mouse oocytes for at least four days. Tests for metabolic function indicate that the oocytes increase in volume, accumulate glucose-6-phosphate dehydrogenase, and maintain a steady level of 3H-uridine and 3H-leucine incorporation.     

An autoradiographic study of RNA synthesis during maturation and germination of pollen grains ofHyoscyamus niger

Summary The pattern of RNA synthesis during maturation and germination of pollen grains ofHyoscyamus niger was studied using³H-uridine autoradiography. Incorporation of label during pollen maturation was periodic with peak RNA synthesis occurring in the uninucleate, nonvacuolate pollen grains and in the vegetative cell of the bicellular pollen grains. During the early stages of germination, isotope incorporation occurred predominantly in the nucleus of the vegetative cell with little or no incorporation in the generative cell. With the appearance of the pollen tube, incorporation of³H-uridine in the vegetative cell nucleus decreased and completely disappeared at later stages of germination. No incorporation of isotope was observed in the sperms formed in the pollen tube by the division of the generative cell. From a comparison of the results of this study with those of previous works on RNA synthesis during pollen embryogenesis in cultured anthers ofH. niger, it is concluded that in contrast to embryogenic development, there is no requirement for sustained RNA synthesis by the generative cell nucleus for normal gametophytic development.     

Adaptation of the 3H-Leucine Incorporation Technique to Measure Heterotrophic Activity Associated with Biofilm on the Blades of the Seaweed Sargassum spp.

The ecological interaction between microorganisms and seaweeds depends on the production of secondary compounds that can influence microbial diversity in the water column and the composition of reef environments. We adapted the ³H-leucine incorporation technique to measure bacterial activity in biofilms associated with the blades of the macroalgae Sargassum spp. We evaluated (1) if the epiphytic bacteria on the blades were more active in detritus or in the biofilm, (2) substrate saturation and linearity of ³H-leucine incorporation, (3) the influence of specific metabolic inhibitors during ³H-leucine incorporation under the presence or absence of natural and artificial light, and (4) the efficiency of radiolabeled protein extraction. Scanning electron microscopy showed heterogeneous distribution of bacteria, diatoms, and polymeric extracellular secretions. Active bacteria were present in both biofilm and detritus on the blades. The highest ³H-leucine incorporation was obtained when incubating blades not colonized by macroepibionts. Incubations done under field conditions reported higher ³H-leucine incorporation than in the laboratory. Light quality and sampling manipulation seemed to be the main factors behind this difference. The use of specific metabolic inhibitors confirmed that bacteria are the main group incorporating ³H-leucine but their association with primary production suggested a symbiotic relationship between bacteria, diatoms, and the seaweed.     

Eimeria tenella: Parasite-Specific Incorporation of 3H-Uracil as a Quantitative Measure of Intracellular Development1

ABSTRACT. An assay has been developed using parasite-specific incorporation of ³H-uracil to assess the intracellular growth of Eimeria tenella in vitro. As shown by both scintillation counts and autoradiography, ³H-uracil was incorporated specifically into intracellular parasites from the onset of infection and continued throughout development of the first generation schizonts. Mature schizonts and first generation merozoites did not continue to incorporate additional ³H-uracil, indicating that RNA synthesis had halted in these stages. Based on these findings, a semi-automated microscale uracil incorporation assay was developed to determine parasite viability. This method should be useful for biochemical studies with intracellular parasites and for screening compounds for anticoccidial activity. The ease, rapidity, and quantitative nature of this assay contrasts favorably with standard morphometric approaches of determining parasite development. In addition, parallel studies using host cell incorporation of ³H-uridine have been introduced as a method of determining whether antiparasitic activity is direct or indirect in relation to effects on the host cell.