Rate Of Precursor Incorporation Research Articles

Genotoxicity of chemical and physical agents in cultured human tissues and cells

In vitro model systems have recently been developed to investigate the toxicity of chemical, microbial and physical agents in normal human tissues and cells from many of the major tissue sites of high cancer incidence. Pathobiological endpoints used in these studies include alterations in incorporation rates of precursors into DNA, RNA and protein, in the clonal growth rate of cultured cells and in DNA structure (e.g. single-strand breaks, DNA-protein crosslinks and chemical-DNA adducts) and the induction of differentiation, chromosomal and karyotypic abnormalities, mutations and neoplastic transformation. These systems have been used to study a variety of complex mixtures and individual substances, including cigarette smoke components such as benzo[ a]pyrene and N-nitrosamines, formaldehyde, fecapentaenes, asbestos, and nickel and chromium ions. In addition, increasing awareness of the role of oncogenes in human carcinogenesis has led to studies involving transfection experiments with oncogenes and hepatitis B viral genes in normal human cells.

Lung surfactant.

Aspects of pulmonary surfactant are reviewed from a biochemical perspective. The major emphasis is on the lipid components of surfactant. Topics reviewed include surfactant composition, cellular and subcellular sites as well as pathways of biosynthesis of phosphatidylcholine, disaturated phosphatidylcholine and phosphatidylglycerol. The surfactant system in the developing fetus and neonate is considered in terms of phospholipid content and composition, rates of precursor incorporation, activities of individual enzymes of phospholipid synthesis and glycogen content and metabolism. The influence of the following hormones and other factors on lung maturation and surfactant production is discussed: glucocorticoids, thyroid hormone, estrogen, prolactin, cyclic AMP, beta-adrenergic and cholinergic agonists, prostaglandins and growth factors. The influence of maternal diabetes, fetal sex, stress and labor are also considered. Nonphysiologic and toxic agents which influence surfactant in the fetus, newborn and adult are reviewed.

Ribonucleic acid metabolism during planarian regeneration.

A method for the extraction of total RNA of the planarian Polycelis tenuis is described. This technique has been applied to the study of RNA synthesis in the course of regeneration. Synthesis of RNA begins 7 hrs after sectioning and proceeds in two phases. The first phase, from 7 to 21 hr is characterized by an increased rate of (3H) orotic acid incorporation into RNA up to the 18th after sectioning followed by a decrease between 18 and 21 hrs. The rate of precursor incorporation then had risen again in the second phase to the 30th hr and finally decreased slowly until completion of regeneration. Studies of the pool of nucleotide precursors, show that the observed variations of orotic acid incorporation into RNA reflect variations in RNA synthesis. Electrophoretic analysis of RNA labelled for 2 hrs at various times during regeneration shows that the product formed during the first phase of regeneration is mainly rRNA and low molecular weight RNA. The second phase of the regeneration process, from the 24th hr, is characterized by the appearance of both ribosomal and polydisperse RNAs. These heterogeneous RNA species are detected up to the 46th hr of regeneration, after which time rRNA again becomes predominent. The significance of the two phases of RNA metabolism of which this is the first observation during planarian regeneration is discussed in connection with other biochemical events which have been described during regeneration processes in planarians and other animals.

A scanning electron microscope study of the surface features of HeLa S3 suspension-culture cells treated with methylmercury(II)

HeLa S3 suspension-culture cells were exposed for 12 hr to varied concentrations of CH3Hg(II) and the changes in their surface features that accompany the exposure observed with the aid of a scanning electron microscope. Also investigated in the presence of CH3Hg(II) was the cells' ability to incorporate suitable radioactive-labeled precursors into DNA, RNA, and protein and to prevent trypan blue stain from entering the cell interior. Investigated further was the cells' ability to recover from the effects of the methylmercury treatment once the chemical had been removed from the medium. The results show that suitable concentrations of CH3Hg(II), within a narrow range, inhibit intracellular DNA, RNA, and protein syntheses abruptly, followed as abruptly by a decline in cell viability. At the ultrastructural level, the metabolic inhibition is characterized by extensive cellular “bubbling,” yielding a multitude of cellular shapes and surface structures, by the thinning out and ultimate disappearance of microvilli, and, in cell death, by a seeming collapse of the exoplasmic bubbles due, presumably, to osmotic shock. At CH3Hg(II) concentrations below a certain threshold, the cells recover completely as shown by a rate of precursor incorporation and by surface features that are both indistinguishable from those of the control. In general, the surface features of the cells are considerably more sensitive towards the effects of the methylmercury treatment than are, for instance, intracellular activities such as DNA, RNA, or protein synthesis.

Differentiation-dependent decline of DNA synthetic activities in Naegleria gruberi.

DNA of Naegleria gruberi strain NEG, grown in axenic culture, forms a band at a density of 1.6912 in CsCl gradient and has a GC content of 31.8%. Incorporation of [3H]thymidine into DNA is much reduced in differentiating Naegleria immediately after the stimulation to transform, primarily because of the reduction in thymidine uptake by differentiating cells. In addition, there is a marked decrease in the rate of incorporation of [3H]thymidine and [3H]uracil into DNA at from 45 to 60 min after the stimulation for differentiation. This decrease in the rate of precursor incorporation into DNA appears to be due to the differentiation-dependent cessation of nuclear DNA synthesis. The differentiated phenotype (the flagellate) emerges at approximately 70 min after the stimulation, and over 90% of the population differentiates within the next 30 min. Synthesis of mitochondrial DNA is detectable until 190 min after the stimulation. Since the S phase of Naegleria lasts approximately 180 min, some cells in the population must cease synthesizing nuclear DNA in the middle of the S phase.

The effect of the cytostatic agent 9-α- d-arabinofuranosyladenine on the nucleic acid metabolism in L5178 Y cells

The influence of 9-α- d-arabinofuranosyladenine (α-araAdo) on cell metabolism of mouse lymphoma cells (L5178y) has been studied on cellular as well as on subcecllular level. α-AraAdo strongly inhibits the cell proliferation of L5178y cells. Starting with 3 × 10 3 cells/ml and an incubation period of 72 hr, the drug reduces the cell proliferation to 50 per cent at a concentration of 12 μM. The cells die because of “unbalanced growth”. The cytostatic effect of α-araAdo can be abolished by coincubation with deoxyadenosine but not by adenosine. At cytostatic concentrations α-araAdo inhibits selectively the incorporation rate of thymidine into DNA but not the incorporation rates of precursors into RNA or protein. α-AraAdo is intracellularly phosphorylated to α-araATP. The compound has no effect on the extent of intracellular phosphorylation of exogenous administered adenosine or deoxyadenosine. α-AraAdo is incorporated into DNA but not into RNA; one molecule of α-araAdo is incorporated per 14,000 molecules of deoxyadenosine.

Insulin stimulation of fatty acid synthesis in human breast cancer in long term tissue culture.

The effect of various steroid and peptide hormones on the rate of incorporation of (14C)acetate into fatty acids was studied in MCF-7 cells, a human breast cancer cell line in continuous tissue culture. Physiologic concentrations of insulin elicited an increase in the rate of precursor incorporation. Addition of human placental lactogen and dexamethasone with insulin caused no further increment. Other hormones, such as 17beta-estradiol and 15a-dihydrotestosterone, had no effect on the rate of net labeled-acetate incorporation, although they stimulated the rate of general macromolecular synthesis. The stimulation by insulin appeared to be independent of glucose concentration, since the effect was seen in the absence of glucose. The insulin-induced increase was observed as early as 1 h following hormone addition and appeared to be maximal by 4 h. Inhibitors of RNA and protein synthesis had no effect on the insulin-mediated stimulation of precursor incorporation into fatty acids. Thus, these human cell lines in long term tissue culture may be of value as a model system in studying the action of physiologic concentrations of insulin.

Regulation of the secretory cycles of mucous and serous cells in the human bronchial gland.

Studies of the human bronchial gland in organ culture have allowed us to elucidate some of the factors controlling the synthesis and secretion of bronchial mucus. The secretory cycles of mucous and serous cells appear to differ, the former alternately accumulating and discharging secretory material, while in the latter synthesis and discharge occur simultaneously. Parasympathomimetic agents increase the secretory rate of mucous and serous cells by stimulating discharge, but have no effect on the rate of precursor incorporation into intracellular glycoproteins. Glycoprotein synthesis inhibitors reduce the incorporation of precursors into intracellular macromolecules but do not reduce the rate of discharge of preformed glycoproteins. The rate of glycoprotein synthesis appears to be greater in mucus-secreting cells of hypertrophied glands than of normals. Ouabain reduces the incorporation of glucose and threonine, but not glucosamine, into mucous and serous cells but has no effect on the rate of discharge. In hypertrophied glands, ouabain--sensitive threonine, but not glucose, transport appears to be increased. Exposure of rats to tobacco smoke causes an increase in the size of tracheal and laryngeal glands and in the secretory rate of mucous cells. The anti-inflammatory agent, phenylmethyloxadiazole, prevents both tobacco smoke-induced effects, presumably by reducing the secretory activity of mucous cells.

Normal production of cartilage glycosaminoglycan in mice homozygous for the chondrodysplasia gene

AbstractBiochemical analysis of a lethal recessive mutation affecting chondrogenesis in mice further delineated the mechanism by which this pleiotropic gene acts. Cartilage from late fetuses homozygous for chondrodysplasia (cho/cho) were terminally labeled in vitro with Na235SO4, [14C] glucosamine, and [14C] glucose. Rates of precursor incorporation; tissue contents of uronic acid, amino sugar, and protein; and molecular weight of glycosaminoglycan were determined and found normal compared with control littermate cartilage. Samples from mutants, agitated overnight in distilled water and assayed for loss of incorporated 35S, showed between 47 and 77% of control activity. The rinse water from mutant cartilage contained correspondingly more 35S than did control. Chromatographic analysis of both tissue‐bound and water‐diffusible macro‐molecules confirmed that glycosaminoglycan and protein backbone were intact. Because the molecular content and rates of precursor incorporation were normal the results suggested that the cho gene does not alter net synthesis of glycosaminoglycan. The observation that water facilitates extraction of a glycosaminoglycan‐protein complex of normal molecular weight suggests that the defect in chondrogenesis results from defective interaction of proteoglycan and some other matrix constituent. In view of a previous report that collagen fibrils of mutant cartilage are atypically large and natively banded the site of chondrogenic defect might be the interaction between proteoglycan and collagen.

Effect of insect hormones on RNA polymerases of mass-isolated imaginal discs of Drosophila melanogaster cultured in vitro.

Four chromatographically separable DNA-dependent RNA polymerases (nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) were partially purified from imaginal discs of Drosophila melanogaster. Their properties are similar to those described for RNA polymerases I and II isolated from other eukaryotes. In vitro incubation of discs with beta-ecdysone, juvenile hormone, or cycloheximide resulted in increased activity of RNA polymerase I. The increase was irreversible with beta-ecdysone incubation and removal but reversible with juvenile hormone incubation and removal. With beta-ecdysone, the rate of the increase in polymerase I activity paralleled the kinetics of ecdysone binding to discs and increases in the rate of precursor incorporation into RNA. A model to explain the increased acticity of RNA polymerase I is presented.

Substrate incorporation into hepatic lipids and proteins in vitro in patients with pre-beta hyperlipoproteinemia.

Fifty-three patients operated on for uncomplicated gallstone disease have been studied concerning the hepatic synthesis rate in vitro of glycerides and proteins. Thirteen of the patients had pre-beta hyperlipoproteinemia. Five of them and four normolipoproteinemic patients were fed a sucrose-enriched diet for two weeks prior to the operation. In the non-sucrose-fed hyperlipoproteinemic patients the liver concentration of triglycerides (TG) and the incorporation rate of precursors into TG were increased. A significant correlation was found between the synthesis rate of TG in liver tissue and the plasma TG concentration in these hyperlipoproteinemic patients. After sucrose feeding of patients with hyperlipoproteinemia the concentration of phosphoglycerides (PG) the incorporation rate of labelled precursors in PG were significantly lower than in normolipoproteinemic patients and in hyperlipoproteinemic patients on an ordinary diet. The incorporation rate of leucine into hepatic proteins and the hepatic protein concentration were the same in non-sucrose-fed controls, sucrose-fed, and non-sucrose-fed hyperlipoproteinemic patients. The results indicate an increased vulnerability of the hepatic PG and protein metabolism for dietary sucrose in patients with pre-beta hyperlipoproteinemia.

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.

The effects of local x irradiation on the relative rates of incorporation of thymidine-methyl-5-3H and cytidine-5-3H into mitochondrial and nuclear DNA of Morris hepatoma 3924A.

RECENT STUDIES have shown that not all the DNA of animal cells is confined to the nuclei; approximately 1 per cent is found in the mitochondria (1). Since the successful development of complex organelles such as mitochondria appears to depend upon integration of the genetic information coded by cytoplasmic (probably mitochondrial) and nuclear genes, one might expect that uneven distribution of the cytoplasmic particles and mitochondria at cell division would have a profound but controlled effect on the orderly reading of genetic messages and hence upon differentiation and, possibly, upon neoplastic transformation. Although the genetic function of mitochondrial DNA is not clear, the genetic continuity of the mitochondria has been demonstrated in certain respiratory mutants of yeast and Neuro-spora (2–4). The phenotypic changes observed in cancer cells may be related to modification of the chemical structure of DNA, the transcription system, or the translation process. Thus, carcinogenesis may involve either mutational or regulatory mechanisms controlling the expression of genetic information. Since mitochondria are one of the principal cytoplasmic controllers of cell metabolism, knowledge of the fundamental nature of nucleic-acid synthesis and DNA replication in the mitochondrion and nucleus is a prerequisite to understanding the interrelationship between the DNA synthesis in these organelles and the growth of tumor cells. Radiation has been employed to learn more about these processes under nonequilibrium conditions. Preliminary studies have disclosed differing rates of incorporation of the two labeled DNA precursors, thymidine and cytidine, into mitochondrial and nuclear DNA (5). Furthermore, preliminary studies have shown less effect of radiation on the rates of precursor incorporation into mitochondrial DNA than into nuclear DNA (6). This study is designed to show the relative rates of incorporation of labeled thymidine and cytidine into a rapidly growing tumor, in view of its possible ramifications in the areas of neoplastic growth and disease. Materials And Methods Hepatoma 3924A, a solid, hypotetra-ploid tumor with a chromosome number of 72–73 and a tumor transfer time of approximately 0.6 month, was used in this experiment. This is a rapidly growing, undifferentiated tumor which demonstrates changes in the enzymatic activity involved in carbohydrate and lipid metabolism (7). The tumor was transplanted bilaterally in rats, subcutaneously in the back. The animals were kept under uniform controlled conditions of temperature, humidity, and lighting. The length and width of the tumors were periodically measured with a caliper and recorded until they reached 1–2 cm.

Lipid composition and turnover of rough and smooth microsomal membranes in rat liver

Subfractions of rat liver microsomes (rough, smooth I, and smooth II), isolated in a cation-containing sucrose gradient system, were analyzed. After removal of adsorbed and luminal protein, these subfractions had the same phospholipid/protein ratio, about 0.40. Both the classes and the relative amounts of phospholipids were similar in the three subfractions, but the relative amounts of neutral lipids (predominantly free cholesterol and triglycerides) were higher in smooth I and especially in smooth II than in rough microsomes. Various pieces of evidence indicate that the neutral lipids are tightly bound to the membranes. Glycerol-(3)H was incorporated into the phospholipids of the rough and smooth I microsomes significantly faster than into those of the smooth II membranes; (32)P incorporation followed a similar but less pronounced pattern. Acetate-(3)H was incorporated into the free cholesterol of smooth I microsomes only half as fast as into the other two subfractions. Injection of phenobarbital increased the cellular phospholipid and neutral lipid content in the rough and smooth I, but not in the smooth II microsomes. Consequently, the neutral lipid/phospholipid ratio of all three subfractions remained unchanged after phenobarbital treatment. It is concluded that the membranes of the rough and the two smooth microsomal subfractions from rat liver have a similar phospholipid composition, but are dissimilar in their neutral lipid content and in the incorporation rate of precursors into membrane lipids.

Reactivation of ribonucleic acid and protein synthesis during germination of Blastocladiella zoospores and the role of the ribosomal nuclear cap.

Freshly harvested zoospores of Blastocladiella emersonii begin to germinate about 15 min after inoculation into a defined growth medium at a density of 10(6) zoospores per ml. Flagellum retraction accompanies encystment, and dispersal of the ribosomal nuclear cap takes place shortly thereafter. The primary rhizoid begins to emerge at 25 to 30 min and starts to branch at ca. 60 min. The first nuclear division occurs between 120 and 190 min. The dry weight per cell increases linearly after 60 min, whereas the deoxyribonucleic acid per cell doubles between 120 and 240 min. A linear increase in total ribonucleic acid (RNA) is detectable beginning at 40 to 45 min, and in total protein beginning at 80 min; neither process is interrupted during nuclear division. Encystment and nuclear cap disorganization are associated with a sharp rise in the rates of precursor incorporation into RNA and protein. Cycloheximide at 20 mug/ml prevents leucine incorporation at all stages and inhibits development beyond the earliest encystment stage. Actinomycin D at 25 mug to 50 mug/ml prevents uracil incorporation, but it has no effect on leucine incorporation or development until 40 to 45 min. At the latter stage, actinomycin D causes a sharp developmental arrest and begins to inhibit leucine incorporation. It is concluded that early protein synthesis must occur on the ribosomes formed during the prior growth phase and conserved through the zoospore stage in the nuclear cap. The results further indicate that this synthesis is dependent upon messenger RNA already present in the zoospore before germination.

The effects of kinetin and contaminating bacteria on the incorporation of 32P-orthophosphate into various fractions of nucleic acid extracted from radish leaves

By comparing (32)P-orthophosphate incorporation into nucleic acid extracts of sterile and non-sterile radish leaf discs, it was shown that contaminating bacteria cause a 2-4 fold increase in the rate of precursor incorporation and alter the pattern of label distribution after fractionation of the extracts by sedimentation through sucrose gradients or chromatography on MAK(1) columns. Using sterile senescing radish leaf discs, a stimulation of (32)P-orthophosphate incorporation into various fractions of nucleic acid was observed as a result of kinetin treatment.