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 P³² and H³-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.     

Effect of terrazol on the ultrastructure of Mucor mucedo]

Terrazol, a systemic fungicide showing high specifity to oomycetes, inhibits the apical growth of hyphae and promotes at lower concentrations the thickening of the cell wall in Mucor mucedo. As revealed by ultrastructural analysis, particularly the fine structure of some membrane systems is influenced. In the first place the inner membrane of the mitochondria is attacked leading to a complete lysis of mitochondria. However, the sensitivities within a given population are different. The plasmalemma enlarges, forms several invaginations, partly redraws from the cell wall, but remains intact. Only after an extensive treatment with relatively high concentrations of terrazol the nuclear envelope shows vesicles between the double membranes. The mechanism of action of terrazol is discussed.     

Action of tetracycline and sodium deoxycholate combinations on protein and nucleic acid synthesis in the cells of the NAG vibrio, Staphylococcus aureus and Escherichia coli]

The effect of tetracycline combination with sodium desoxycholate, a surface-active substance, on the synthesis of proteins and nucleic acids in the cells of NAG-vibrio, Staph. aureus and E. coli was studied by incorporation of 1-14C-glycine and 8-14C-adenine into proteins and nucleic acids. It was found that sodium desoxycholate suppressed the synthesis of proteins and nucleic acids in the cells of NAG-vibrio and Staph. aureus. Its combination with tetracycline resulted in summation or increase of the suppressive effects on proteins and nucleic acids as compared to the effect of the substances used alone. Sodium desoxycholate even in very high concentration, up to 12800 gamma/ml, had no effect on the synthesis of proteins and nucleic acids in the cells of E. coli and respectively it did not change the activity of tetracycline on combined use.     

Glycotargeting: Influence of the Sugar Moiety on Both the Uptake and the Intracellular Trafficking of Nucleic Acid Carried by Glycosylated Polymers

Nucleic acids (plasmids as well as oligonucleotides) used to specifically express or modulate the expression of a gene, must reach the cytosol and/or the nucleus. Several systems have been developed to increase their uptake and their efficiency. Glycosylated polylysines have been shown to specifically help nucleic acids to be taken up in cells expressing a given cell surface membrane lectin. However, it appeared that the efficiency of the imported nucleic acid was not directly related to the extent of the uptake. Indeed, some glycosylated polylysines bearing sugar moities which are poor ligands of the cell surface lectins of a given cell were found to be more efficient than those bearing better sugar ligands. The interpretation of this paradoxal result is discussed with regards to the nature of the compartment allowing the nucleic acid to cross the membrane and to be delivered in the cytosol on the one hand, and to the presence of intracellular lectins on the other hand.     

The influence of various substances on the gliding motility of Mycoplasma mobile 163K

Non-toxic concentrations of various substances were tested for their influence on the gliding motility of Mycoplasma mobile 163K. A significant inhibitory effect on motility was observed with agents acting on nucleic acid synthesis (mitomycin), protein synthesis (puromycin, chloramphenicol), energy metabolism (p-chloromercuribenzoate, iodoacetate) and with compounds reacting with the cytoplasmic membrane or contractile elements (albumin, cholesterol, EDTA, 2-propanol, procain, CaCl2, MgCl2, colchicin and KI). The surface-active compounds Triton X-100, Tego and SDS increased the gliding velocity significantly in some concentrations and incubation periods. The results suggest that the motility of M. mobile depends on a functional cytoplasmic membrane and that cytoskeletal elements are involved in the gliding mechanism.     

Visna virus synthesized in absence of host-cell division and DNA synthesis

Visna virus is similar to the avian and the murine oncornaviruses. Oncornavirus replication is dependent upon the provirus being integrated into the host cell's DNA but integration and subsequent oncornavirus synthesis is blocked when the host cells are prevented from synthesizing cellular DNA or dividing. The synthesis of visna virus is restricted in vivo and may be dependent upon the host cell's ability to synthesize cellular DNA or divide. Treatment of sheep choroid plexus (SCP) cells with ultraviolet light or with mitomycin C prior to infection irreversibly inhibited plexus (ScP) cells with ultraviolet light or with mitomycin C prior to infection irreversibly inhibited both cell division and cellular nucleic acid synthesis but did not inhibit visna virus synthesis. Similarly, the synthesis of visna virus in cultures of SCP cells which had been prevented from dividing by being deprived of serum and in cultures of SCP cells which were incapable of synthesizing host cell nucleic acids by being treated with miracil D or sodium hexachloroiridate was equivalent to the synthesis of visna virus in cultures of SCP cells which were allowed to both synthesize cellular nucleic acids and divide. The synthesis of visna virus in the presence of ethidium bromide further demonstrated that integration of the visna provirus into the host cell's DNA is not required for visna virus synthesis to occur.     

Mode of Action of Novobiocin in Escherichia coli

The mechanism of action of novobiocin was studied in various strains of Escherichia coli. In all strains tested except mutants of strain ML, the drug immediately and reversibly inhibited cell division, and later slowed cell growth. The previously described impairment of membrane integrity, degradation of ribonucleic acid (RNA), and associated bactericidal effect were found to be peculiar to ML strains. The earliest and greatest effect in all strains was an inhibition of deoxyribonucleic acid (DNA) synthesis; RNA synthesis was inhibited to a lesser extent, and cell wall and protein synthesis were affected later. The inhibition of nucleic acid synthesis was accompanied by an approximately threefold accumulation of all eight nucleoside triphosphates. Since novobiocin does not inhibit nucleoside triphosphate synthesis, degrade DNA, or immediately affect energy metabolism, it must inhibit the synthesis of DNA and RNA by direct action on template-polymerase complexes.     

玫瑰茄醇提物的抑菌活性及其作用机制

玫瑰茄具有多种药理活性,包括抗肿瘤、抗氧化和抑菌作用等,但目前关于玫瑰茄的抑菌作用研究较少,有关抑菌机制方面的研究尚未见报道. 本文通过测定玫瑰茄醇提物对大肠杆菌和金黄色葡萄球菌细胞膜、蛋白质和核酸的影响,及其与DNA的作用方式等,系统阐述玫瑰茄的抑菌作用机制. 电导率和大分子物质的测定结果显示,玫瑰茄醇提物只对菌体的细胞膜造成微小损伤,其抑菌作用的靶点不在细胞膜. SDS-PAGE和DAPI结果显示,玫瑰茄醇提物可抑制大肠杆菌和金黄色葡萄球菌蛋白质和核酸的合成. 琼脂糖凝胶电泳和紫外吸收光谱结果显示,玫瑰茄醇提物可与DNA结合,当DNA与药物的浓度比较低时,玫瑰茄醇提物与DNA以嵌入结合为主,当二者的浓度较高时,两者间发生的是氢键结合. 上述结果证明,玫瑰茄醇提物对大肠杆菌和金黄色葡萄球菌抑菌机制,主要是药物通过与DNA发生嵌入结合和氢键结合,使DNA不能进行正常的复制和转录,降低核酸的含量,进而影响蛋白质的合成,最终导致菌体生物学功能的丧失.     

Inhibition of synthesis of nucleic acids, proteins and respiration in isolated thymocytes by anthracyclines

The effect of anthracycline antibiotics such as carminomycin, daunomycin (rubomycin) and adriamycin on respiration and synthesis of nucleic acids and protein was studied comparatively. The anthracyclines inhibited the processes. By their efficacy in that respect they could be arranged in the following order: carminomycin greater than rubomycin greater than adriamycin. Thus, 50 per cent inhibition of nucleic acid synthesis in the thymocytes required 0.027, 0.044 and 0,173 mM of carminomycin, rubomycin and adriamycin respectively. Protein synthesis and respiration in the thymocytes were less sensitive to the effect of the anthracyclines than synthesis of nucleic acids. The study results were compared with the literature data on the effect of the compounds on respiration and synthesis of nucleic acids and protein in tumour and bacterial cells.     

Effects of Polychlorinated Biphenyls on Macromolecular Synthesis by a Heterotrophic Marine Bacterium

Growth rates and final cell yields of a polychlorinated biphenyl (PCB)-sensitive pseudomonad isolated from the open ocean were reduced in a dose-dependent manner by 10 to 100 μg of Aroclor 1254 per liter, a commercial mixture of PCB isomers added to its culture medium. Effects on growth rates were detected within 1 h (approximately one doubling time) of treatment. By 4 h posttreatment, the amounts of deoxyribonucleic acid and ribonucleic acid per cell in exponentially growing populations treated with sublethal doses of Aroclor were detectably lower than in appropriate controls. Corresponding cell protein values were slightly higher than in controls. Selective degradation of cell proteins or nucleic acids was not detected in cells whose growth was totally suppressed for 4 h by PCBs. Cells whose growth rate was inhibited 20 to 50% by Aroclor synthesized protein at normal rates for periods in excess of 5 h from the time the chlorinated hydrocarbons were added. In contrast, rates per cell of adenine uptake and adenine incorporation into deoxyribonucleic acid and total nucleic acids by the cells treated with PCBs were significantly lower than in control cells. Intracellular adenine pools of cells whose growth was inhibited to 20% of the control rate by PCBs were 30% smaller and appeared to require a longer interval to equilibrate than those of untreated cells. This may indicate impaired transport and/or efflux of this nucleic acid precursor through the membrane of affected cells. Inhibition of nucleic acid synthesis in this sensitive bacterium by PCBs could explain the observed inhibitory effects of the chlorinated hydrocarbons on its growth.     

Synthesis of nucleic acids in the Schwann cells as the early cellular response to nerve injury

Abstract— The early effect of mechanical injury of the sciatic nerve in cats and rats on the amount of nucleic acids and on the number of Schwann cells was studied. The content of RNA and DNA increased at both ends of a severed nerve as early as 2 hr after operation; at 8 hr and up to 24 hr, their level was about twice as high as at the beginning of experiment. The increase in nucleic acids seemed to be chiefly due to their biosynthesis in the Schwann cells although the possibility of a contribution of axonal RNA must also be taken into consideration. The division of Schwann cell nuclei was observed not earlier than 36 hr after operation. The injury stimulates the synthesis of nucleic acids not only in the Schwann cells directly damaged, but also in those from neighbouring internodes. The increase in DNA and RNA observed 24 hr after a fresh lesion had been made in a degenerating nerve was smaller than the increase observed 24 hr after cutting or crushing a normal nerve.     

Effects of protein inhibitors and auxin on nucleic Acid metabolism in peanut cotyledons

The accumulation of labeled phosphorus into newly synthesized nucleic acids or peanut cotyledon slices incubated with chloramphenicol, puromycin, or 2,4-dichlorophenoxyacetic acid (2,4-D) was reduced. Promotion of nucleic acid synthesis was not noted by any of these chemicals. Chloramphenicol completely inhibited the synthesis of the DNA-RNA fraction at 1.25 × 10⁻³ m while soluble and ribosomal RNA was inhibited by 70% and 80%, respectively. At the same concentration messenger RNA was inhibited by only 40%. These effects suggest that chloramphenicol inhibit nucleic acid synthesis in peanut cotyledons in a differential manner. Similar results were noted for DNA at low concentrations of 2,4-D. However, at high concentrations of 2,4-D, DNA as well as RNA fractions were inhibited in a similar manner at a given concentration. Puromycin did not differentially inhibit nucleic acid synthesis except at 2 × 10⁻³ m where DNA was least inhibited.     

Highly chlorinated Escherichia coli cannot be stained by propidium iodide

Several studies have shown that the staining by fluorochromes (DAPI, SYBR Green II, and TOTO-1) of bacteria is altered by chlorination. To evaluate the effect of chlorine (bleach solution) on propidium iodide (PI) staining, we studied Escherichia coli in suspension and biomolecules in solution (DNA, RNA, BSA, palmitic acid, and dextran) first subjected to chlorine and then neutralized by sodium thiosulphate. The suspensions and solutions were subsequently stained with PI. The fluorescence intensity of the PI-stained DNA and RNA in solution dramatically decreased with an increase in the chlorine concentration applied. These results explain the fact that for chlorine concentrations higher than 3 micromol/L Cl2, the E. coli cells were too damaged to be properly stained by PI. In the case of highly chlorinated bacteria, it was impossible to distinguish healthy cells (with a PI-impermeable membrane and undamaged nucleic acids), which were nonfluorescent after PI staining, from cells severely injured by chlorine (with a PI-permeable membrane and damaged nucleic acids) that were also nonfluorescent, as PI penetrated but did not stain chlorinated nucleic acids. Our results suggest that it would be prudent to be cautious in interpreting the results of PI staining, as PI false-negative cells (cells with compromised membranes but not stained by PI because of nucleic acid damage caused by chlorine) are obtained as a result of nucleic acid damage, leading to an underestimation of truly dead bacteria.     

Chlorella pyrenoidosa 7-11-05 in batch and in homogeneous continuous culture under autotrophic conditions

The cell content of substances (proteins, nucleic acids and chlorophylls) which play a significant role in growth processes in algae was used to characterize the physiological state of a continuous culture ofChlorella pyrenoidosa. The ratio of the various components of the cell content did not alter significantly with changes in the dilution rate. An increase in the mean cell volume was accompanied by a proportional increase in the amount of the various components. Their respective dry weight concentrations rose with the dilution rate and after reaching a maximum either fell or remained constant. The specific rates of synthesis of the given substances tended to rise, i.e. maximum activity of the culture was not attained. Deoxyribonucleic acid synthesis appears to be the endogenous factor limiting growth of the culture. The proportion of the individual nucleic acid fractions was compared with protein synthesis.     

A direct effect of growth hormone on the incorporation of precursors into proteins and nucleic acids of perfused rat liver

1. The livers of rats were perfused in situ. When the amino acid concentration in the perfusing medium was that present in rat plasma, the addition of growth hormone to the medium stimulated the incorporation of labelled amino acids into liver protein only marginally and not to a statistically significant extent. When, however, the amino acid concentration was raised to three times that present in rat plasma, growth hormone significantly and substantially stimulated amino acid incorporation into protein within 30min. of perfusion of normal rat liver. 2. A significant effect of growth hormone on labelling of normal rat-liver protein was seen with concentrations not much greater than those reported to be present in rat plasma. 3. The labelling of nucleic acids of normal and hypophysectomized rat liver by [(3)H]orotic acid was enhanced by addition of growth hormone to the perfusing medium when normal concentrations of amino acids were used. 4. At elevated concentrations of amino acids, growth hormone stimulated labelling of nucleic acids of hypophysectomized rat liver at 30 and 60min. of perfusion. Under these conditions, nucleic acids of normal rats were labelled to about the same extent in control and hormone-treated livers at 30min. and, because of a fall in the radioactivity of the control livers, there was more labelled nucleic acids in growth-hormone-treated livers at 60min. than in the control livers. 5. Growth hormone, unlike insulin, had no inhibitory effect on the release of glucose by the perfused liver. 6. It is concluded that growth hormone can stimulate the incorporation of precursor into proteins and nucleic acids of liver directly and without the mediation of other organs or of insulin.     

Light Effects on the Nucleic Acids of Excised Cotton Cotyledons

The effects of light and glucose in the nutrient medium on the nucleic acid metabolism of excised 8-day cotton (Gossypium hirsutum var. Acala 44) cotyledons were determined. The rates of synthesis as affected by light and glucose were determined by brief exposures to C(14)-labeled orotic acid. The nucleic acids were fractionated by homogenizing in Tris-HCl buffer and centrifuging to obtain soluble and microsomal RNA (20,000 x g supernatant) and a particulate nucleic acid fraction (20,000 x g precipitate) or by extracting in phenol followed by 10% NaCl extraction at 100 degrees . The phenol extract was analyzed by density gradient centrifugation.Light and glucose caused parallel changes in nucleic acid levels of the various fractions, in orotic acid-6-C(14) absorption and in rates of synthesis of nucleic acids. Light and glucose appear to enhance binding of the ribosome nucleic acid so that it becomes less extractable in Tris-HCl buffer or phenol. The bound nucleic acids were labeled at a slightly higher rate than the total nucleic acids extracted by Tris-HCl or phenol. However, light treatment for 48 hours promoted a very high labeling rate in the soluble, low molecular weight nucleic acid as shown by density gradient centrifugation of the phenol extractable fraction.It was concluded that a part of the nucleic acid changes were brought about by light acting through the photosynthetic production of carbohydrate. This conclusion was strengthened by the observation that herbicide inhibitors of photosynthesis and limited atmospheric CO(2) concentrations partially inhibited the nucleic acid changes. However, glucose did not cause changes in nucleic acid levels as large as those caused by light and changes were observed to occur in light even though the endogenous sugar levels were maintained at a low level by the inhibition of photosynthesis with herbicides. The data indicated that light may produce changes in nucleic acid levels by other mechanisms additional to those regulating the sugar level in the tissue.     

Comparison of the effects of bile acids on cell viability and DNA synthesis by rat hepatocytes in primary culture

Bile acid-induced inhibition of DNA synthesis by the regenerating rat liver in the absence of other manifestation of impairment in liver cell viability has been reported. Because in experiments carried out on in vivo models bile acids are rapidly taken up and secreted into bile, it is difficult to establish steady concentrations to which the hepatocytes are exposed. Thus, in this work, a dose-response study was carried out to investigate the in vitro cytotoxic effect of major unconjugated and tauro- (T) or glyco- (G) conjugated bile acids and to compare this as regards their ability to inhibit DNA synthesis. Viability of hepatocytes in primary culture was measured by Neutral red uptake and formazan formation after 6 h exposure of cells to bile acids. The rate of DNA synthesis was determined by radiolabeled thymidine incorporation into DNA. Incubation of hepatocytes with different bile acid species - cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), in the range of 10-1000 microM - revealed that toxicity was stronger for the unconjugated forms of CDCA and DCA than for CA and UDCA. Conjugation markedly reduced the effects of bile acids on cell viability. By contrast, the ability to inhibit radiolabeled thymidine incorporation into DNA was only slightly lower for taurodeoxycholic acid (TDCA) and glycodeoxycholic acid (GDCA) than for DCA. When the effect of these bile acids on DNA synthesis and cell viability was compared, a clear dissociation was observed. Radiolabeled thymidine incorporation into DNA was significantly decreased (-50%) at TDCA concentrations at which cell viability was not affected. Lack of a cause-effect relationship between both processes was further supported by the fact that well-known hepatoprotective compounds, such as tauroursodeoxycholic acid (TUDCA) and S-adenosylmethionine (SAMe) failed to prevent the effect of bile acids on DNA synthesis. In summary, our results indicate that bile acid-induced reduction of DNA synthesis does not require previous decreases in hepatocyte viability. This suggests the existence of a high sensitivity to bile acids of cellular mechanisms that may affect the rate of DNA repair and/or proliferation, which is of particular interest regarding the role of bile acids in the etiology of certain types of cancer.     

Altered nucleic acid partitioning during phenol extraction or silica adsorption by guanidinium and potassium salts

Nucleic acids were found to partition into the phenol phase during phenol extraction in the presence of guanidinium at certain concentrations under acidic conditions. The guanidinium-concentration-dependent nucleic acid partitioning patterns were analogous to those of the nucleic acid adsorption/partitioning onto silica mediated by guanidinium, which implied that phenol and silica interact with nucleic acids through similar mechanisms. A competition effect was observed in which the nucleic acids that had partitioned into the phenol phase or onto the silica solid phase could be recovered to the aqueous phases by potassium in a molecular weight–salt concentration-dependent manner (the higher molecular weight nucleic acids needed higher concentrations of potassium to be recovered, and vice versa). Methods were developed based on these findings to isolate total RNA from Escherichia coli. By controlling the concentrations of guanidinium and potassium salts used before phenol extraction or silica adsorption, we can selectively recover total RNA but not the high molecular weight genomic DNA in the aqueous phases. Genomic DNA-free total RNA obtained by our methods is suitable for RT-PCR or other purposes. The methods can also be adapted to isolate small RNAs or RNA in certain molecular weight ranges by changing the salt concentrations used.     

Study of the antiviral action of gentamicin]

Experimental data on the effect of various concentrations of gentamycin on reproduction of VEE and Sindbis viruses in tissue culture are presented. It was found that gentamycin had no cytotoxic effect on the primary tripsinized chick embryon fibroblasts (CEF) when used in doses of 10, 20 or 30 mg/ml and only when used in a dose of 50 mg/ml it induced 50 percent destruction of the cell layer. Multiplication of the VEE and Sindbis viruses in the culture of CEF was inhibited in the presence of gentamycin by 1.5--3.5 lg PFU/ml. Two stages in the virus inhibiting effect of gentamycin were determined on the model of VEE, i. e. the stage of inhibition in the absence of visible damages of the cells and the stage associated with their destruction. The doses of gentamycin higher than 3 mg/ml inhibited in parallel the virus specific synthesis and synthesis of the cell proteins and nucleic acids. At the same time, when gentamycin was used in a dose of 10 mg/ml, no impairement of the cell viability was observed and the cell capacity to produce high titers of the model virus was reduced after incubation without the antibiotic for 24 hours. The antiviral activity of gentamycin were therefore determined by revers inhibition of the cell metabolic activity.