Isotopic Precursors Research Articles

Myocardial nucleotide synthesis from purine bases and nucleosides. Comparison of the rates of formation of purine nucleotides from various precursors and identification of the enzymatic routes for nucleotide formation in the isolated rat heart.

14 C-Labeled adenosine, inosine, hypoxanthine, and adenine were extracted by the isolated rat heart in amounts proportional to their concentration in the perfusion medium between 0.05 and 5 µM. With each of the precursor materials, nearly all of the radioactivity retained by the heart was identified as acid-soluble nucleotide. Nucleotide formation from the four isotopic precursors occurred at similar rates when the concentration of the precursors was below 1 µM. Above this concentration, the heart appeared to utilize adenosine for nucleotide synthesis at rates three to five times those for the other purines. Several experimental approaches were employed to determine the predominant enzymatic routes in the rat heart for the conversion of the nucleosides adenosine and inosine to nucleotides. The results indicated that adenosine was directly phosphorylated to 5'-adenosine monophosphate by a nucleoside kinase. Inosine appeared to proceed to the nucleotide, at least partially, through an initial conversion to hypoxanthine.

Gas chromatography—Mass spectrometry studies of tetramethyl uric acids

Of the 12 possible isomers of tetramethyl uric acid, 6 are obtained by methylation with diazomethane and 4 by on column methylation with trimethylphenyl ammonium hydroxide. Elution patterns on three different columns, quantitative data, and mass spectra are presented. In addition, elution data and the mass spectrum of 8-methoxy-1,3,8-trimethylxanthine are given. The results may be useful for studies of incorporation of [ 13C]glycine and other stable isotope precursors into uric acid.

Early estrogen effects on lipid metabolism in rat uterus.

The effects of short periods (1-4hr) of in vivo 17P-estradiol treatment on the subsequent in vitro incorporation of several isotopic precursors into rat uterine lipids were studied. Throughout this period, estrogen progressively accelerated 2-14C-acetate incorporation into all major lipid fractions examined. When phospholipids were doubly labeled with 2-14C-acetate and 32Porthophosphate, estrogenic stimulation of 32P incorporation occurred at a different and greater rate than 14C incorporation. Subsequent separation and identification of major uterine phospholipid classes allowed the finding that novel or selective differential synthesis of specific lipid classes did not occur; rather there was a similar and coordinate increase in the labeling of all classes. To elucidate the mechanisms involved in these responses, the synthesis of the largest class of uterine phospholipids was examined in detail. 90- min periods of estrogen treatment increased Me- 3H-choline incorporation into choline phospholipids by 70...

Hyaluronate turnover during chondrogenesis in the developing chick limb and axial skeleton

Hyaluronidase activity is first detected in leg and wing buds and the axial trunk region of chick embryos at the onset of cartilage differentiation and continues during subsequent chondrogenesis. Incorporation of isotopic precursors into hyaluronate is demonstrated in early stages of development of both limb bud and axial skeleton. The ratios of incorporation of acetate- 3H into hyaluronate and chondroitin sulfate in both regions is higher prior to the appearance of cartilage than subsequently and is greater at all the stages of axial chondrogenesis studied than at corresponding stages in the limb bud. A hypothesis is further discussed in which hyaluronate production accompanies mesenchymal cell movements and its removal is necessary for differentiation of these cells.

The appearance and quantitation of cytoplasmic ribonucleic acid in the early chick embryo

ABSTRACT This paper seeks to extend our knowledge about RNA synthesis in early embryogenesis to the domestic fowl, Gallus domesticus. Using this species for research, apart from increasing our knowledge of higher vertebrate embryology, has certain advantages such as rapid uptake of isotopic precursors and ease of microdissection in culture. The following results are presented: The cell number in the whole chick embryos is shown to be increasing logarithmically between the time of laying and the early neurula stage; with a doubling time of 7·4 h. The onset of ribosomal RNA synthesis has been shown to be during mid-cleavage of the chick embryo, while development is taking place in the oviduct and uterus of the mother. In a cumulative labelling experiment, embryos were labelled at the unincubated-egg stage, allowed to develop to various morphological stages up to neurulation, and their cytoplasmic RNA prepared and analysed by gel electrophoresis. The specific activity of the precursor pool for RNA synthesis was measured at several stages, using the same labelling conditions, and the results were used to quantitate the RNA synthesis from the incorporated radioactivity. Using these techniques, it was found that newly synthesized cytoplasmic RNA accumulates steadily in the whole chick embryo, reaching a level of 104 μg by the early neurula stage. On a per cell basis, however, the amount of newly synthesized cytoplasmic RNA seems to decrease slightly. These findings are discussed in the light of present knowledge about embryos of other vertebrates and certain invertebrates.

Protein synthesis during insect development

The present study examines changes in specific proteins during insect development. The investigation explores the careers of individual proteins, their specific activities, rates of synthesis, and when and where they are made and transported. Quantitative disk electropherograms of protein patterns in blood, fat body, wings, antennae, epidermis, gut, ovaries and egg, Malpighian tubules, and silk glands of the Hyalophora cecropia silkworm are examined for the late fifth-instar larva, two stages when the animal spins the cocoon, diapausing pupa, pharate adult, and the emerged adult. The separation of proteins with acrylamide gel electrophoresis, densitometric evaluation of stained bands, and the incorporation of radioactive precursors into the specific bands furnish excellent tools for examining and evaluating the macromolecules, and immunodiffusion detects the complexities of electrophoretic protein bands. The total soluble protein per unit weight is highest in blood, followed in decreasing amounts in fat body, wing, and epidermis. However, the capacity to synthesize proteins is found to be highest in the epidermis, followed by wing, antennae, midgut, ovaries and eggs, and fat body, respectively. All the larval and adult tissues show lower concentrations of proteins than the pharate adult. The peak protein concentration in each tissue is attained at a different physiological and chronological stage of development, and is often followed by marked morphological changes. The number of electrophoretically separable bands varies extensively in each tissue and stage of development, e.g., blood contains 8–16, fat body 2–16, epidermis 4→20, wings 5–20, antennae 3–12, gut has as many as 40, ovaries and eggs 8–12, Malpighian tubules 3–7, and silk glands 2–10. Qualitatively, some proteins from one tissue differ from others. The blood serves as a fluid transport medium for proteins released into it and removed from it, e.g., larval blood proteins, sequestered by pupal fat body, and a wing protein are released into it, whereas one is taken up by ovaries in the pharate adult. According to the dye-binding capacity, the protein bands are arbitrarily assigned to two groups, major and minor protein bands. Four major bands, R F' s 20, 25, 32, and 40, are separated using a 5 per cent gel concentration. The pattern of quantitative changes in each of the major bands is presented for the entire period of development in different tissues. Of numerous minor bands, few are typical of a tissue and specific stage of development. Their role and relationship to physiological events are discussed. On the basis of whether a protein band incorporates isotope precursor or not, synthetically active and inactive proteins are separated. Employing computations of specific activity, the rates of synthesis, degradation, transport, etc., are determined for a specific protein. The limitations of different techniques are discussed with possible solutions.

Selective and nonselective isolation of temperature-sensitive mutants of mouse L-cells and their characterization.

AbstractMutants of mouse L‐cells which are temperature‐sensitive for growth have been obtained by using both selective and nonselective isolation procedures on populations treated with the mutagen nitrosoguanidine. Selective isolation was carried out by utilizing a five‐day treatment with 3H‐TdR and ara‐C as selective agents at the nonpermissive temperature. Nonselective isolation was performed by isolating 1400 clones in the absence of selective agents and then testing them for temperature‐sensitivity. From this experiment we obtained a minimum estimate of 6 × 10−3 for the frequency of mutants in the mutagentreated population. The mutants were characterized by their plating efficiencies, growth in suspension culture, and uptake of isotopic precursors of DNA, RNA, and protein. A range in phenotypes was observed, and there appeared to be some differences between the mutants obtained by the two types of isolation procedures. In uptake experiments the most marked reductions in the rates of precursor incorporation were seen with 3H‐TdR, rather than 3H‐UR or 3H‐Leu. Different mutant lines showed considerable variation in the rate of cessation of DNA synthesis as well as the time required for termination of cell division. These experiments suggest that both types of isolation procedures are feasible for obtaining temperature‐sensitive mutants having a range of phenotypes.

T-even bacteriophage-tolerant mutants of Escherichia coli B. II. Nucleic acid metabolism.

T-even bacteriophage-tolerant mutants are strains of Escherichia coli which can adsorb T-even phages but cannot support the growth of infective virus. Under some conditions, the infected cells are not killed. Mutant cells infected by phage T6 are able to carry out several metabolic functions associated with normal virus development, including arrest of bacterial nucleic acid and protein synthesis, incorporation of isotopic precursors into viral nucleic acids and proteins, synthesis of early enzymes of deoxyribonucleic acid (DNA) metabolism, formation of rapidly sedimenting DNA intermediates, and formation of normal levels of early and late messenger ribonucleic acid species. Phage are unable to mutate to forms capable of growth on these mutants. The nature of the biochemical alteration leading to tolerance is still unknown.

Evaluation of the rates of biological processes from tracer kinetic data: III. The net synthesis lemma and exchangeable pools

Equations are developed for total isotope incorporation into the sum of all species of RNA in growing bacterial cultures. The model is approximate in that it considers only one intermediate pool, only one messenger fraction and only one intermediate on the path of the biosynthesis of the ribosome. However, there is no mathematical approximation involved in the treatment as there was in the previous paper. With the aid of a computer, the conditions necessary to observe linear uptake with no lag were determined. It is concluded that exchange of exogenous isotopic precursor across the cell membrane, together with the existence of a turning over class of RNA, accounts for the observed smaller lag in isotope incorporation than that predicted from the size of the intermediate pool. The effect of amino acid starvation and turnover of precursors of ribosomal and transfer RNA is discussed. With the computer program, data from the literature are used to estimate the turnover rate of messenger RNA.

Hyaluronate production and removal during corneal development in the chick

Glycosaminoglycan (acid mucopolysaccharide) synthesis was studied during development of the embryonic chick cornea by the introduction of isotopically labeled precursors both in ovo and to excised corneas in culture. Results obtained for both types of preparations were similar. Hyaluronate is the major glycosaminoglycan synthesized by the embryonic cornea between stages 24 and 35, a period during which the primary corneal stroma swells and is invaded by mesenchymal cells. Subsequent to the completion of invasion a rapid decline in incorporation of isotopic precursors into hyaluronate occurs concomitant with an increasing hyaluronidase activity. The hyaluronidase is present primarily between stages 35 and 38, when the cornea begins to dehydrate prior to becoming transparent.

The extracellular matrix of the regenerating newt limb: Synthesis and removal of hyaluronate prior to differentiation

Active synthesis of hyaluronate has been demonstrated in the early “dedifferentiated” blastema of the regenerating newt limb. Incorporation of isotopic precursors in vivo into hyaluronate is maximal at approximately 10 days of regeneration, whereas incorporation into chondroitin sulfate is maximal at 25–35 days. At 10 days, incorporation into hyaluronate is greatest in the most distal portion of the stump, grading off in the distal to proximal direction. Concomitant with a sharp decrease in incorporation into hyaluronate after 10–15 days of regeneration, hyaluronidase activity becomes measurable in the blastema and persists at a significant level until 40 days of regeneration, i.e., during the period of maximal chondroitin sulfate synthesis and cartilage differentiation. Turnover experiments indicated that hyaluronate was degraded rapidly at 25 days but not at 10 days of regeneration. Incorporation into hyaluronate was considerably greater than into chondroitin sulfate at all time points tested between 3 and 48 hours after administration of isotope to animals with 10-day regenerates. The reverse was true in animals with 25-day regenerates. It is concluded that hyaluronate is associated with blastema formation, and its possible role in the regulation of mesenchymal cell behavior is discussed.

Biosynthèse de la transcortine rôle du 3′, 5′-adénosine-monophosphate cyclique dans les mécanismes cellulaires de biosynthèse

The injection of 3′,5′-adenosine monophosphate increases the corticosteroidbinding activity of a glycoprotein, transcortin, at the subcellular level of rat liver cell. At the same time, incorporation of the isotopic precursor (D-[6-3H]glucosamine) into hepatic glycoproteins is increased. These results suggest that cyclic AMP is an inducer of transcortin biosynthesis.

CIS-dichlorodiammineplatinum (II) : Persistent and selective inhibition of deoxyribonucleic acid synthesis in vivo

Cis-dichlorodiammineplatinum(II) ( cis-[Pt(NH 3) 2Cl 2] 0) at 10 −4 M inhibits the incorporation in vitro of thymidine-methyl- 3H, uridine-5- 3H, and l-leucine 14C into the acid-insoluble fraction of Ehrlich ascites tumor cells from untreated mice only after a period ofpreincubation. Similarly, when mice bearing well developed tumors are given this compound intraperitoneally, subsequent assessment in vitro of incorporation of these isotopic precursors into tumor cells from injected mice shows marked impairment of incorporation of all three; the greatest inhibition occurs 6–12 hr after injection. After this depression, the rates of incorporation of uridine and l-leucine return to control values or somewhat greater, while a striking suppression of the rate of incorporation of thymidine persists for at least 96 hr. These data were interpreted as indicative of a possible metabolic transformation by the cells of the inorganic moiety from an inactive to an active form, although alternate tentative explanations were also proposed. It was suggested that if the persistent inhibitory action on DNA synthesis is directly related to the chemotherapeutic efficacy of this agent or a metabolic product thereof, a less frequent treatment regimen may be as effective as daily injections while evoking fewer toxic reactions.

Membranes of animal cells. V. Biosynthesis of the surface membrane during the cell cycle.

AbstractKB cells grown in suspension culture were synchronized by using a double thymidine block. At various times throughout the life cycle aliquots of cells were pulsed with 14C‐L‐leucine, 14C‐D‐glucosamine and 14C‐choline for one hour periods. Surface membranes, cell particulates and soluble proteins were isolated and their 14C specific activities were determined. It was found that there was a marked increase in the rate of incorporation into surface membrane just after division. The pattern of incorporation was the same for all three isotopic precursors. The rate of incorporation of isotopic precursors into soluble proteins was constant throughout the cycle. Some increase in rate of incorporation of isotope into the particulate fraction was observed during division.

In Vivo Studies on Glycosphingolipid Metabolism in Porcine Blood

Abstract Porcine plasma and erythrocytes were found to contain the same glycosphingolipids as humans, namely glucosyl ceramide, lactosyl ceramide, galactosylgalactosylglucosyl ceramide, and globoside, and the actual concentrations were very similar. The in vivo studies were carried out by using uniformly labeled 14C-d-glucose as the isotopic precursor and isolating the glycolipids from 30-ml samples of blood removed at frequent intervals over the 3-month study period. The results suggested that erythrocyte lactosyl ceramide, galactosylgalactosylglucosyl ceramide and globoside were synthesized in the bone marrow, did not exchange with their plasma counterparts, and were released into the plasma at the time of general erythrocyte catabolism. Erythrocyte glucosyl ceramide did not appear to be synthesized in the marrow and was in dynamic equilibrium with plasma glucosyl ceramide. The label was rapidly and randomly incorporated into all the plasma glycosphingolipids but after 9 days no labeling could be detected until the erythrocytes began to be catabolized (50 to 70 days). The relative amounts of label in the glycolipids during the 1st week and the latter period from 40 to 60 days confirmed that the rapid source of plasma glycosphingolipid biosynthesis is a minor pathway and showed that the release or catabolism of erythrocyte glycosphingolipids (especially globoside, the major component) is the major source of all four plasma glycosphingolipids.

Biosynthése de la transcortine Rôle de la thyroxine dans les mécanismes cellulaires de biosynthése

Biosynthesis of Transcortin. Role of Thyroxine in the Cellular Mechanism of Biosynthesis The injection of thyroxine increases the corticosteroid-binding activity of a glycoprotein, transcortin. The results of isotopic precursors' incorporation into glucidic fragments of glycoprotein, examined at the subcellular level (membranes) of rat liver cell, contradicts the stimulation of glycoprotein biosynthesis, but supports the activation of previously masked molecular sites.

Immunochemical analysis of Mycoplasma pneumoniae. 3. Separation and chemical identification of serologically active lipids.

SummaryLipids of Mycoplasma pneumoniae were fractionated by chromatography on columns of diethylaminoethylcellulose and silicic acid, and on thin layers of silica gel G.Hapten activity, measured in implement‐fixation tests with rabbit and with human antisera to M. pneumoniae, was found to be associated with four or more neutral glycolipids. These comprised digalactosyl‐ and trigalactosyldi‐ glycerides, a second dihexosyldiglyceride containing both glucose and golactose, and a trihexosyldiglyceride fraction containing both hexoses, which was probably a mixture of two or more substances. These glycolipids were labelled when the organisms were grown in the presence of radioactive glucose, glycerol or palmitate, but not when they were grown with labelled galactose. The label from glucose appeared in both the glucose and galactose moieties of these glycolipfds. A fraction, consisting mainly of alkali‐stable glycolipids. into which there was little or no incorporation of isotope during growth with labelled precursors, was also found to have hapten activity. Rabbit antisera to M. pneumoniae were found to cross‐react with glycolipids from other sources, including the ceramide lactoside of mammalian tissues (cytolipin H).Chloroform‐methanol extracts of M. pneumoniae contained large amounts of lecithin, which was not labelled during growth with the isotopic precursors used. The major phosphatide synthesized by the organisms was phosphatidylglycerol. This was accompanied by smaller amounts of another phosphatide, tentatively identified as a phosphatidyl monoglyceride, which fixed complement with Wassermann‐positive human sera. These two acidic phosphatides were found to be radioactive after the cells had been grown with labelled glycerol or palmitate.

Biochemical changes in tissues of goldfish acclimated to high and low temperatures—I. protein synthesis

1. 1. The initial rate of protein synthesis, as measured by the level of incorporation in 1 hr of 14C-leucine into protein, exhibited a compensation in gill and liver of goldfish acclimated at 5°C only when tested at 25°C. However, muscle of 5°C fish showed a greater incorporation than that of 25°C fish at 15°C and 25°C but not at 5°C. The Q 10 of the incorporation rate was higher for the 25°C-acclimated fish than for the 5°C-acclimated ones in the lower range of temperature, but this relationship was reversed in the higher thermal range. 2. 2. All the tissues reached a steady-state level of incorporation within 6–12 hr after the injection of the isotopic precursor in both the cold- and warm-adapted fish, when measured at 15°C. 3. 3. The maximal net synthesis of protein at 12 hr of incorporation was 100–500 per cent higher in the cold-acclimated tissues than in the warm-acclimated ones at 5°C and less so at 15°C. There were no significant differences between the cold- and warm-adapted gill and liver in the net protein synthesis at 25°C, although muscle showed a 50 per cent reduction in this process during warm acclimation. 4. 4. The 14C-count in the PCA-supernatants of the warm-acclimated tissues was higher than in the cold-acclimated, ones, when measured 1 hr after the injection of 14C-leucine at 15°C; this might signify a greater amino acid permeability of the cell membrane during warm acclimation. However, no difference was found in 14C-counts of the PCA-soluble pools of the corresponding tissues from cold- and warm-adapted fish, when measured either at 5° or 25°C, 12 hr after the administration of the isotope. Thus the availability of the precursor did not seem to be rate limiting for the protein synthesis, although a rapid metabolism of the amino acids in the warm-adapted fish tissues cannot be excluded. 5. 5. It is concluded that the translational increase in enzymatic activities and oxidative metabolism of goldfish during cold acclimation may be achieved by a complicated rotational (gill and liver) or a simple translational (muscle) augmentation of the maximal net synthesis of proteins.

Relationship between RNA synthesis, cell division, and morphology of mammalian cells. I. Puromycin aminonucleoside as an inhibitor of RNA synthesis and division in HeLa cells.

Logarithmically growing HeLa cell monolayers were treated with a range of concentrations of puromycin aminonucleoside (AMS). The effects of AMS were studied by the following means: microscope examination of treated cells; enumeration of the cell number using an electronic particle counter; analyses for DNA, RNA, and protein content; incorporation of P(32) and H(3)-thymidine into nucleic acids; and fractionation of nucleic acids by column chromatography. Taking the rate of incorporation of the isotopic precursor as a measure of nucleic acid synthesis, it was found that concentrations of the inhibitor which had a rapid effect on the rate of cell division inhibited the synthesis of all types of nucleic acids and of protein, but depressed ribosomal RNA synthesis most markedly. Lower concentrations of AMS selectively inhibited ribosomal RNA and, to a lesser extent, transfer RNA synthesis. Partial inhibition of ribosomal RNA synthesis with low doses had no effect on the rate of cell division within the period studied (3 generation times). The cell content of RNA returned to normal when the inhibitor was removed.

Ribonucleic acid and protein synthesis in Rhizophlyctis rosea zoospores.

LéJohn, Herbert B. (Purdue University, Lafayette, Ind.), and James S. Lovett. Ribonucleic acid and protein synthesis in Rhizophlyctis rosea zoospores. J. Bacteriol. 91:709-717. 1966.-The uniflagellate zoospores of Rhizophlyctis rosea display active motility and a high endogenous respiratory metabolism, but neither growth nor net ribonucleic acid (RNA) or protein synthesis can be measured by ordinary procedures. Nevertheless, synthesis can be detected with isotopic precursors. Uracil-C(14) is incorporated slowly into both the soluble and ribosomal RNA. Analysis of zoospore extracts (on diethylaminoethyl cellulose columns or sucrose gradients) after various periods of labeling suggested that most of the uracil incorporation represents slow synthesis of ribosomal precursor RNA and, ultimately, ribosomes. Actinomycin D caused an 80% inhibition of uracil incorporation. The most rapidly labeled RNA was susceptible to extensive degradation in cells treated with actinomycin, but the percentage of stable RNA increased with the time of incorporation before addition of the antibiotic. Neither the effects of actinomycin nor the results of chase experiments have established unequivocally the existence of turnover or the presence of a short-lived "messenger" fraction in motile spores. Both leucine and methionine were slowly incorporated into a spectrum of cellular proteins. The methyl group of C(14)-methylmethionine also served as a methyl donor for the methylation of soluble RNA but not of ribosomal RNA. The observations that some of the newly synthesized RNA and protein occur in the intact 82S ribosomes and that actinomycin inhibits the low level of protein synthesis provide some indirect evidence for a very low rate of "messenger" synthesis and turnover in zoospores.