Incorporation of Labeled Precursors into the RNA and Proteins of Lactic Dehydrogenase Virus

Lactic dehydrogenase virus was grown in primary mouse embryo cells and labeled with (3)H-uridine and (3)H-amino acids. Concentrated and purified virus was banded by isopycnic centrifugation in sucrose gradients, and infectivity and radioactivity were found to correspond at a density of 1.17 g/cm(3). The extracted viral RNA was resolved by electrophoresis in polyacrylamide-agarose mixed gels, and the mol wt was estimated to be 6.0 x 10(6).     

Specific Incorporation of Precursors into DNA by Feeding Labeled Bacteria to Paramecium aurelia

SYNOPSIS. Studies were carried out on the introduction of labeled precursors into the DNA of Paramecium aurelia (syngen 4, stock 51) by way of the bacteria that are used for food. A thymine-requiring strain of Escherichia coli (15 T⁻) was labeled by growth in either H³-methyl thymidine or 2-C¹⁴ bromouracil, washed free of the exogenous label, and fed to the paramecia. The tritium label from the bacteria was incorporated almost exclusively into the DNA of the paramecia, whereas it was much less specifically incorporated when introduced directly from the medium. The Cu label from bromouracil was also incorporated mainly into the DNA of the paramecia although a small amount appeared in RNA. The formation of labeled food vacuoles was followed. Food vacuoles were formed at a nearly constant rate, with the total number of vacuoles increasing throughout the cycle. The lifetime of the vacuoles was about 2.5 hours. Incorporation of the label into the DXA of the paramecia begins within a few minutes of the formation of the first labeled vacuole. DNA synthesis begins about 1.5 hr after the previous fission (total cell cycle about 5.8 hr) and progresses at a nearly constant rate throughout the remainder of the cycle.     

Incorporation of Precursors into Toxoplasma DNA

SYNOPSIS. DNA synthesis of Toxoplasma gondii differs from that of other obligate intracellular parasites in that the parasite can synthesize DNA independently of the host cell and can incorporate preformed pyrimidines as well as pyrimidine precursors. However, pyrimidine precursors such as orotic acid are preferentially utilized over preformed pyrimidines such as thymidine. There is little apparent utilization of purine precursors.     

Arsenic Uptake and Accumulation in Rice (Oryza sativa L.) at Different Growth Stages following Soil Incorporation of Roxarsone and Arsanilic Acid

Experiments were conducted with rice (Oryza sativa L.) by adding 0, 10, 20, 30, 40, 50 mg kg^-1 of arsenic (As) to soil (with roxarsone and arsanilic acid, presented as As concentrations) at a field with an isolation chamber. The aims were to evaluate the effects of As- (roxarsone or arsanilic acid) contaminated soil on rice agronomic parameters and uptake of As in different plant parts of the rice plant. The results showed that As (roxarsone or arsanilic acid) could significantly reduce plant height, effective tiller number, straw weight and grain yield (P < 0.01). As concentrations in different parts of the plant varied with the growth stages, and behaved similarly. At the maturing stage, the level in different parts peaked in all treatments, with tissue As concentrations showing the pattern: root > leaf > stem > husk > grain. In addition, at the mature stage, the As concentrations in different parts of the rice plant increased with increasing concentrations of roxarsone and arsanilic acid. The highest concentration of As found in grain was 0.82 mg kg^-1, which did not exceed the statutory permissible limit for rice grain (1.0 mg As kg^-1), and in the leaf and stem it was approximately 6.0 mg kg^-1, which was significantly higher than that in the controls. The results showed that rice could accumulate As from contaminated soil (roxarsone or arsanilic acid), which may be transferred to human beings via the food chain.     

Malaria parasites (Plasmodium lophurae) Developing Extracellularly in vitro: Incorporation of Labeled Precursors*

SYNOPSIS. P. lophurae were removed from their host duck erythrocytes and incubated in vitro in certain modifications of the red cell extract medium previously described. The extent of incorporation, into material precipitable with trichloracetic acid, of ¹⁴C-labeled precursors supplied after 15–16 hr of incubation, was determined and compared with effects on structure of the parasites. A decreased concentration of erythrocyte extract, which always resulted in increased numbers of degenerate parasites and decreased development to multinucleate forms, also decreased the incorporation of methionine-methyl-¹⁴C and orotic-acid-6-¹⁴C. It did not affect incorporation of proline-U-¹⁴C or of choline-1,2-¹⁴C. With a 1/3rd strength red cell extract, omission of coenzyme A, which increased the proportion of degenerate parasites and usually decreased the multinucleate forms, decreased the incorporation of all 4 substrates, in keeping with the inability of the parasites to synthesize CoA. On the other hand, omission of ATP and pyruvate, which had an even greater deleterious effect on structure of the parasites than omission of CoA, had no effect on incorporation of methionine or orotic acid and probably none on that of choline. Incorporation of adenine was reduced in presence of ATP or AMP, suggesting competition at an uptake site. Incorporation of proline, however, was higher with ATP and pyruvate present, in keeping with the better development of the extracellular parasites. The uptake of proline may depend on an ATPase in the outer of the 2 membranes surrounding the parasite.     

Incorporation of Precursors into Ribonucleic Acid, Protein, Glycoprotein, and Lipoprotein of Avian Myeloblastosis Virions

Freshly explanted leukemic myeloblasts produce avian myeloblastosis virus (AMV) at a constant rate without any obvious cytopathic effect; therefore, subviral components are continually synthesized at a steady rate. The incorporation of various radioactive precursors into virions was monitored by determination of radioactivity in purified virus after density equilibrium sedimentation in preformed sucrose gradients. The kinetics of incorporation of (3)H-uridine have shown that there is an average time interval of 3 to 4 hr (half-life) between the time viral ribo-nucleic acid (RNA) is synthesized and the time it is released as a mature virus particle; this represents the average time interval spent by AMV-RNA in an intracellular pool. Studies with (14)C-phenylalanine have revealed that some protein synthesis takes place at or near the cell surface immediately prior to maturation and release of virus. (14)C-glucosamine also appears to be incorporated into the outer viral envelope shortly before maturation. On the other hand, there is an average lag of about 16 to 20 hr before (14)C-ethanolamine incorporated into intracellular lipoprotein appears in free virions; this probably reflects the kinetics of replacement of cellular surface membrane. Actinomycin D inhibits AMV-RNA within 30 min but permits the maturation of AMV to continue for at least 2 hr. AMV released in the presence of actinomycin D contains AMV-RNA synthesized before the addition of the drug.     

Incorporation of Radioactive Precursors into DNA and RNA of Plasmodium knowlesi in vitro

SYNOPSIS. Cultures of the intra-erythrocytic stages of Plasmodium knowlesi incubated in vitro utilized all the pre-formed radioactive purines tested (adenine, adenosine, deoxvadenosine, guanine, guanosine and hypoxanthine) but none of the pyrimidines (thymine, thymidine, uracil, uridine, cytidine and deoxycytidine). They did, however, utilize the pyrimidine precursor orotic acid.
All precursors analysed, including deoxyadenosine, were incorporated into both DNA and RNA (in the ratio of ∼1:3) but 19% was incorporated into other unidentified compounds. ³Hadenosine was incorporated into adenine and guanine residues of both DNA and RNA.
No unambiguous evidence was obtained for any periodicity in the synthesis of DNA or RNA in our cultures, even tho cultures remained as synchronous in vitro as they are in vivo. An estimate is presented of the amount of DNA made during one cycle in vitro.     

Incorporation In Vivo and In Vitro of Radiolabeled Sphingolipid Precursors into Paramecium tetraurelia Lipids

ABSTRACT Paramecium tetraurelia contains high concentrations of ethanolamine sphingolipids, especially in its ciliary membrane. Three ethanolamine sphingophospholipids with different long chain bases (dihydrosphingosine, sphingosine and phytosphingosine), and their phosphonyl analogs, were previously identified and characterized. In the present study, radiolabeling experiments on lag- and log-phase cells were performed to investigate the extent of sphingolipid biosynthetic capacities of the ciliate. Long chain bases of sphingolipids are formed by an initial condensation reaction of serine with a fatty-coenzyme A. Thus, radiolabeled palmitic acid, stearic acid and serine were used as precursor compounds in these experiments. The results indicated that (1) sphingolipid precursors were incorporated into every major lipid fraction. (2) ethanolamine sphingophosphonolipids accumulated faster than the ethanolamine sphingophospholipids, (3) in contrast to these sphingolipids, the glycerolipid, phosphatidyethanolamine. accumulated faster than its phosphono analog, and (4) palmitic acid, but not stearic acid, was incorporated into the long chain bases of ethanolamine sphingophospho- and sphingophosphonolipids. consistent with an earlier report demonstrating that these lipids contain only C,g long chain bases. Since P. tetraurelia takes up serine and other water-soluble substrates very slowly, and catabolizes fatty acids rapidly, label is randomized in intact cells. Thus, cell-free protocols provide useful experimental systems for studies of sphingolipid biosynthesis than do intact organisms, when the uptake of precursor substrates are slow.     

Polyamines and Root Formation in Mung Bean Hypocotyl Cuttings : II. Incorporation of Precursors into Polyamines

The incorporation of [¹⁴C]arginine and [¹⁴C]ornithine into various polyamines was studied in mung bean (Vigna radiata [L.] Wilczek) hypocotyl cuttings with respect to the effect of indole-3-butyric acid on adventitious root formation.

Both [¹⁴C]arginine and [¹⁴C]ornithine are rapidly incorporated into putrescine, spermidine, and spermine, with similar kinetics, during 5- to 24-hour incubation periods. The incorporation of arginine into putrescine is generally higher than that of ornithine. The biosynthesis of putrescine and spermidine from the precursors, in the hypocotyls, is closely related to the pattern of root formation: a first peak at 0 to 24 hours corresponding to the period of root primordia development, and a second peak of putrescine biosynthesis at 48 to 72 hours corresponding to root growth and elongation. Indole-3-butyric acid considerably enhances putrescine biosynthesis in both phases, resulting in an increase of the putrescine/spermidine ratio.

It is concluded that the promotive effect of indole-3-butyric acid on putrescine biosynthesis, from both arginine and ornithine, supports the hypothesis that auxin-induced root formation may require the promotion of polyamine biosynthesis.

     

Kinetics of Incorporation of DNA Precursors from Ingested Bacteria into Macronuclear DNA of Paramecium aurelia12

SYNOPSIS. The kinetics of transfer of tritium-labeled material from the DNA of ingested bacteria into macronuclear DNA of Paramecium was examined by autoradiography. Bacteria labeled with tritiated thymidine were almost immediately incorporated into food vacuoles, thus becoming available for digestion and a potential source of labeled DNA precursors. Soluble label derived from food vacuoles appeared in low concentrations in the cytoplasm soon after cells were transferred to medium with labeled bacteria; incorporation of labeled precursors into macronuclear DNA began within 5 min. Labeled food vacuoles remained as potential sources of tritiated DNA precursors for a long and variable period after removal of labeled cells to non-labeled medium. The activity of the soluble cytoplasmic DNA precursors decreased parallel to the loss of labeled food vacuoles and no soluble DNA precursors were carried over from one macronuclear DNA synthetic period to the next. Labeling experiments were designed, using this information, which allowed determination of the pattern of macronuclear DNA synthesis and nuclear mass increase during the cell cycle. Macronuclear DNA synthesis began 25–30% of the way thru the cell cycle, continued at a constant rate during the middle half, and decreased in rate during the last quarter. Macronuclear mass increased in an approximately linear fashion, beginning with the onset of DNA synthesis and doubling by the time of karyokinesis.     

Phosphoglycerides of Trichophyton terrestre and One Phenotype Selected from the Apollo 16 Microbial Ecology Evaluation Device

Total lipid extracted from wild-type Trichophyton terrestre CDC-X285 was found to be 2.0 percent of the dry cell weight. The total lipid contained the following phospholipid components identified by silicic acid-impregnated thin-layer and paper chromatography: phosphatidyl inositol, phosphatidyl choline, phosphatidyl serine, and phosphatidic acid. The total lipid extracted from the phenotype T. terrestre 7048-1 isolated from the Apollo 16 Microbial Ecology Evaluation Device (MEED) was found to vary according to the time at which the phospholipids were extracted. The Trichophyton phenotype was selected from a cuvette housed in the MEED exposed to specific space parameters including ultraviolet light of known wavelengths and energy levels in deep space. The phospholipid components, identified in the phenotype were phosphatidyl ethanolamine and cardiolipin. The major lipid fraction was composed of digalactosyl diglyceride and monogalactosyl diglyceride. An unusual lipid was detected in the phenotype, which appeared to be sterol glycoside.     

Incorporation of radioactive glucosamine into macromolecules at nerve endings

Mice were injected intracerebrally with [¹⁴C]glucosamine, and incorporation into macromolecules in various subcellular fractions of brain was studied at a number of times after administration of the precursor. The [¹⁴C]glucosamine was rapidly incorporated into macromolecules of all the subcellular fractions of brain including both the soluble and particulate fractions of isolated nerve endings. Incorporation into macromolecules in the soluble fraction of nerve endings was quite extensive 3 hr after administration of the precursor and the specific acitvity of this fraction fell thereafter. In contrast there was only slight incorporation of [¹⁴C] leucine into the soluble protein from isolated nerve endings in the first few hours after administration, whereas the other subcellular fractions were maximally labelled at that time. The data suggests that, unlike protein which is largely transported to nerve endings in the axoplasm, there is extensive incorporation of carbohydrate into macromolecules in nerve endings. Whereas the protein component of a glycoprotein or mucopolysaccharide may be transported to the nerve ending from the perikaryon, the structure and function of this protein may be modified at the nerve ending by further incorporation of glucosamine, sialic acid and possibly other carbohydrates. The carbohydrate-containing macromolecules could influence nerve ending function immediately after these final synthetic reactions since these reactions occur at the nerve ending and not in the perikaryon.     

Thermotolerance of Pearl Millet and Maize at Early Growth Stages: Growth and Nutrient Relations

Thermotolerance of pearl millet (Pennisetum glaucum cv. ICMV-94133) and maize (Zea mays cv. Golden) was assessed at germination and vegetative stage. Final percentage of germinated seeds and rate of germination (number of days to 50 % germination) decreased due to high temperature (45 °C) similarly in the both species. In contrast, at the vegetative stage, high temperature (38/27 °C) caused a significant reduction in shoot dry mass of maize, whereas this attribute remained almost unchanged in pearl millet. Relative growth rate and net assimilation rate (NAR) increased significantly in pearl millet due to high temperature, but in contrast, in maize NAR was slightly reduced. Concentrations of N, P, and K in the shoots of both species increased at high temperature, but N accumulation was more pronounced in pearl millet than in maize. High temperature caused a marked increase in both shoot and root Ca²⁺ concentration in maize, but it did not affect that of pearl millet. S concentration in the shoots of maize decreased significantly due to high temperature, whereas that in pearl millet remained unaffected. Shoot Na⁺ concentration of both species was not significantly affected by high temperature. High temperature caused a significant increase in uptake of N, P, and K⁺ in pearl millet, but the uptake of Ca²⁺, Mg²⁺, Na⁺ and S remained unaffected in this species. In contrast, in maize, a significant increase in uptake of K⁺ and Ca²⁺, and a decrease in uptake of N, S, Mg²⁺, and Na⁺ were found at high temperature. Overall, maize showed lower tolerance to high temperature compared with pearl millet.     

Synthesis of Vaccinia Viral Proteins in Cytoplasmic Extracts: I. Incorporation of Radioactively Labeled Amino Acids into Polypeptides

Polypeptide synthesis has been studied in cell-free systems prepared from vaccinia virus-infected and uninfected HeLa cells. Cytoplasmic extracts containing endogenous messenger ribonucleic acid were used. Amino acid incorporation into hot trichloroacetic acid-precipitable material was linear for 15 to 20 min at 37 C. The initial rate of protein synthesis was approximately 15% of the rate in intact cells. Optimal conditions for polypeptide synthesis were similar in cell-free systems prepared from infected or uninfected cells. Requirements for an energy source and Mg(++) were demonstrated. The optimal Mg(++) concentration was 4 to 5 mm. Ribonuclease, puromycin, and cycloheximide were inhibitory. The molecular weights of the polypeptides labeled in the cell-free systems, as determined by gel filtration in 5 m guanidine hydrochloride, ranged from 16,000 to above 68,000. Polyacrylamide gel electrophoresis indicated that the polypeptides labeled in cell-free extracts of uninfected and infected cells were different. The latter closely corresponded in electrophoretic mobility with the viral polypeptides made in intact, infected cells.     

Inhibition of Growth and of Thymidine Incorporation into DNA in Tetrahymena by Chlorpromazine,Pimozide and Penfluridol1

The antipsychotic drugs chlorpromazine, pimozide, and penfluridol caused a 50% inhibition of growth of Tetrahymena at concentrations of 4.5, 5.5, and 1.5 μM, respectively. The degree of growth inhibition was dependent on the concentration of cells; higher drug concentrations were needed to produce inhibition of denser cell cultures. Binding studies with penfluridol showed that 50% growth inhibition resulted when approximately 50 μmoles of drug were bound per 10⁶ cells. A 20-min preincubation of cells with chlorpromazine (14.7 μM) inhibited DNA synthesis by 46%, and with penfluridol (4 μM) DNA synthesis was inhibited by 27%. The incorporation of labeled thymidine into the thymidine triphosphate pool was inhibited by chlorpromazine but not by penfluridol, indicating that the drugs produce their growth inhibitory effects by different mechanisms. TDP kinase activity was demonstrated in a particle-free fraction of the cells. Its enzymatic activity was not affected by added chlorpromazine, penfluridol, or calmodulin, suggesting that inhibition of DNA synthesis by these drugs may be a consequence of growth inhibition.     

Incorporation of Wild-Type and C-Terminally Truncated Human Epidermal Growth Factor Receptor into Human Immunodeficiency Virus-Like Particles: Insight into the Processes Governing Glycoprotein Incorporation into Retroviral Particles

Previous results have indicated that incorporation of surface glycoprotein into retroviral particles is not a specific process and that many heterologous viral and cellular glycoproteins can be incorporated as long as they do not have long cytoplasmic C-terminal regions which were presumed to be sterically inhibitory. In this study, this concept has been directly examined by analyzing the incorporation of the wild-type human epidermal growth factor receptor (Wt-EGFR) and of a C-terminally truncated mutant of Wt-EGFR (Tr-EGFR) into human immunodeficiency virus (HIV)-like particles. Incorporation was directly analyzed at the protein level and by immunogold labelling of enriched HIV-like particles. In agreement with the above concept, Tr-EGFR, with only 7 C-terminal amino acids (aa), was efficiently incorporated into HIV-like particles. Incorporation of the Wt-EGFR species, with 542 C-terminal cytoplasmic aa, was reduced by a factor of about 5 in comparison to that of the Tr-EGFR species. However, the Wt-EGFR species was still very significantly present in the HIV-like particles. A series of control experiments verified that this represents genuine incorporation of Wt-EGFR into the membrane of HIV-like particles. These observations allow further speculation as to the processes governing glycoprotein incorporation into retroviral particles and indicate that the internal virus structure of HIV (in particular the matrix layer [MA]) can accommodate much larger heterologous cytoplasmic domains in incorporated glycoproteins than previously assumed.     

Growth Medium-Dependent Glycine Incorporation into the Peptidoglycan of Caulobacter crescentus

The peptidoglycan (PG) is a macromolecular component of the bacterial cell wall that maintains the shape and integrity of the cell. The PG of Caulobacter crescentus, unlike that of many other Gram-negative bacteria, has repeatedly been shown to contain significant amounts of glycine. This compositional peculiarity has been deemed an intrinsic characteristic of this species. By performing a comprehensive qualitative and quantitative analysis of the C. crescentus PG by high-performance liquid chromatography (HPLC) and mass spectrometry (MS), we show here that glycine incorporation into the C. crescentus PG depends on the presence of exogenous glycine in the growth medium. High levels of glycine were detected at the fifth position of the peptide side chains of PG isolated from C. crescentus cells grown in the complex laboratory medium PYE or in defined medium (M2G) supplemented with casamino acids or glycine alone. In contrast, glycine incorporation was undetectable when cells were grown in M2G medium lacking glycine. Remarkably, glycine incorporation into C. crescentus peptidoglycan occurred even in the presence of low millimolar to sub-millimolar concentrations of free glycine. High glycine content in the PG had no obvious effects on growth rates, mode of PG incorporation or cell morphology. Hence, the C. crescentus PG is able to retain its physiological functions in cell growth and morphogenesis despite significant alterations in its composition, in what we deem to be unprecedented plasticity.     

Effects of Fungal Elicitors on Cell Growth and Artemisinin Accumulation in Hairy Root Cultures of Artemisia annua

The artemisinin accumulation in the hairy root cultures of Artemisia annua L. was enhanced via a treatment of three fungal elicitors separately ( Verticillium dahliae Kleb., Rhizopus stolonifer (Ehrenb. ex Fr. ) Vuill and Colletotrichum dematium (Pers.) Grove). Among these three elicitors, V. dahliae had the highest inducing efficiency, but none of them manifests any noticeable effects on the cell growth of the hairy root cultures. The artemisinin content of the hairy root cultures treated with V. dahliae elicitor was 1.12 mg/g DW, which was 45% higher than the control (0.77 mg/g DW). The results showed that elicitation was dependent on the elicitor concentration, the incubation period and the physiological stage at which the hairy root cultures were treated. In addition, the authors found that for V. dahliae, the optimum concentration was 0.4 mg carbohydrate per millilitre medium, the strongest response of A. annua hairy root cultures to the elicitation was at the late exponential growth stage, and the highest artemisinin content of the hairy root cultures was on the 4th day post treatment.     

Correlation of Cold Tolerance at Different Growth Stages in Rice

Cold injury which influence rice production in different rice growing countries occurs at various growth stages. The degree of cold injury depends on the air or water temperature, the cropping pattern, the growth stage of the rice and other factors. It is generally accepted that cold tolerance of rice at one stage is different from another stage. However, Okabe and Toriyama reported that varieties seem to respond similarly to cold temperature at different growth stages. Some varieties have been found to be tolerant at different growth stages. The purpose of this experiment is to find whether cold tolerance scores at different growth stages of different kinds of rice are correlated or not.