The selective inhibitory effect of NH4Cl on estrogen-stimulated rat uterine in vitro RNA synthesis

Estrogen-stimulated in vitro RNA synthesis in rat uterine nuclear-myofibrilar fractions, isolated uterine nuclei, and nucleoli was selectively inhibited by NH₄Cl (400 μmoles) when added prior to the start of a 37 °C incubation. This inhibitory effect was not observed if the salt was added after the start (as early as 1 min) of a 37 °C incubation. The removal of NH₄Cl from reaction mixtures not yet incubated at 37 °C further reduced in vitro RNA synthesis in both control and hormone-treated nuclei. The data suggest that NH₄Cl (400 μmoles) added prior to, but not after, the start of a 37 °C incubation inhibited estrogen-stimulated rat uterine nucleolar RNA polymerase activity perhaps by removing a protein component(s) which is necessary for hormonal stimulation.     

The Impact of Temperature Stress on DNA and RNA Synthesis in Potentially Toxic Dinoflagellates <Emphasis Type="Italic">Prorocentrum minimum</Emphasis>

Biomarkers of temperature stress were studied as major characteristics crucial for the understanding complex processes that underlie the response of marine planktonic microorganisms to environmental factors and their sublethal effects. Using the potentially toxic dinoflagellates Prorocentrum minimum as a model object, the impact of temperature stress on viability, cell cycle, RNA synthesis and DNA replication in these protists was evaluated. It was shown by flow cytometry that stress evoked by a temperature increase from 25°C (control) to 37 or 42°C during 15 to 60 min did not cause any considerable alterations in the cell cycle, while cell death rate increased from ≤ 1% (control) to 2–12% at 37°C and 4–22% at 42°C. Along with a relatively low cell death rate, following a temperature increase to 37 and/or 42°C, P. minimum displayed the ability to boost the synthesis of DNA (1.7–1.9 and 1.2–1.6 times, respectively) and especially RNA (2.5–3.1 and 1.7–2.8 times, respectively) during the first 15–30 min after stress. At certain stages of the life cycle, this effect can be critical for maintaining the viability and normal development of the P. minimum population. The obtained results demonstrate that a significantly elevated synthesis of nucleic acids can serve as an indicator (biomarker) of sublethal environmental stress.     

Anticancer effects of 6-<Emphasis Type="Italic">o</Emphasis>-palmitoyl-ascorbate combined with a capacitive-resistive electric transfer hyperthermic apparatus as compared with ascorbate in relation to ascorbyl radical generation

The aim of the present study is to determine the anti-proliferative activity of 6-o-palmitoyl-l-ascorbic acid (Asc6Palm) that is a lipophilic derivative of l-ascorbic acid (Asc), on human tongue squamous carcinoma HSC-4 cells by combined use of hyperthermia in comparison to Asc. Asc6Palm or Asc were administered to HSC-4 cells for 1 h, to which hyperthermia at 42 °C was applied for initial 15 min. After further 1–72 h incubation at 37 °C, cell proliferation was determined with Crystal Violet staining. Ascorbyl radical (AscR) in HSC-4 cell suspension was measured by electron spin resonance (ESR), and cell morphology was observed with scanning electron microscopy (SEM). At 37 °C, 4 mM Asc or 0.35 mM Asc6Palm were enough to suppress proliferation of HSC-4 cells. By combined use of hyperthermia at 42 °C, cell proliferation was decreased when compared to 37 °C. After Asc of 4 mM was incubated with HSC-4 cell suspensions at 37 °C or 42 °C for 0–180 min, the signal intensity of ascorbyl radical (AscR) by ESR was not different regardless of the presence or absence of cells at 37 °C, whereas AscR signal was enlarged in the presence of HSC-4 cells at 42 °C. It was suggested that oxidation of Asc occurred rapidly in HSC-4 cells by hyperthermia, and thereby enhanced the anti-proliferative activity. By SEM observation, the surface of HSC-4 cells treated with Asc6Palm revealed distinct morphological changes. Thus, the combined regimen of Asc6Palm and hyperthermia is expected to exert a marked antitumor activity.     

Metabolism and mechanisms of action of 9-(tetrahydro-2-furyl)-6-mercaptopurine in Chinese hamster ovary cells

The mechanisms of action of 9-(tetrahydro-2-furyl)-6-mercaptopurine (THFMP) have been studied in Chinese hamster ovary (CHO) cells in tissue culture. THFMP is relatively unstable in physiological buffers, being facilely converted to 6-mercaptopurine (6-MP) even in the absence of cells. Consequently, THFMP undergoes metabolic conversions characteristic of 6-MP, namely formation of 6-thioIMP and incorporation into DNA as 6-thioguanine (6-TG) nucleotide. A number of purines are capable of preventing the toxicity of THFMP in wild-type cells in a manner similar to that of 6-MP. However, exogenous purines and pyrimidines did not prevent the toxicity of THFMP to cells deficient in the enzyme, hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8; HGPRTase). Cells lacking HGPRTase were 20–40-fold resistant to 6-TG and 6-MP but were only 2–4-fold resistant to THFMP. Furthermore, the time-course for killing CHO cells deficient in HGPRTase was different from that in wild-type cells containing the enzyme. There was no apparent effect of THFMP on the utilization of precursors for DNA, RNA or protein synthesis in the enzyme-deficient mutant cell line. The results suggest that THFMP is converted non-enzymatically to 6-MP and shares its mechanisms of action in wild-type cells containing HGPRTase, i.e., inhibition of de novo purine biosynthesis and incorporation into DNA as 6-TG nucleotide. However, the mechanism of action of THFMP in cells lacking HGPRTase is probably unique and is presently unknown.     

Nontemplate Polymerization of Free Nucleotides Into Genetic Elements by Thermophilic DNA Polymerase in vitro

DNA synthesis is the cornerstone of all life forms and is required to replicate and restore the genetic information. Usually, DNA synthesis is carried out only by DNA polymerases semiconservatively to copy preexisting DNA templates. We report here that DNA strands were synthesized ab initio in the absence of any DNA or RNA template by thermophilic DNA polymerases at (a) a constant high temperature (74°C), (b) alternating temperatures (94°C/60°C/74°C), or (c) physiological temperatures (37°C). The majority of the ab initio synthesized DNA represented short sequence blocks, repeated sequences, intergenic spacers, and other unknown genetic elements. These results suggest that novel DNA elements could be synthesized in the absence of a nucleic acid template by thermophilic DNA polymerases in vitro. Biogenesis of genetic information by thermophilic DNA polymerase-mediated nontemplate DNA synthesis may explain the origin of genetic information and could serve as a new way of biosynthesis of genetic information that may have facilitated the evolution of life.

Supplemental materials are available for this article. Go to the publisher's online edition of Nucleosides, Nucleotides, and Nucleic Acids to view the free supplemental file.     

Activity of some bromomethylated aromatic hydrocarbons on the in vitro synthesis of DNA and RNA

The effect of a series of bromomethylated polycyclic hydrocarbons on in vitro DNA and RNA synthesis has been studied by measurement of the incorporation of [³H]-dTMP or [¹⁴C]-AMP into new chains. The inhibition of RNA synthesis was less than 12% for 9-bromomethylanthracene, 9-methyl-10-bromomethylanthracene and 12-bromomethylbenzo(a)acridine, and more than 37% for 7-bromomethylbenzo(a)anthracene, 7,12-dibromomethylbenzo(a)anthracene and 7-bromomethylbenzo(c)acridine. Analogous results were found for the inhibition of DNA synthesis, except for 7-bromomethylbenzo(c)acridine which had little effect. Apart from this exception a good correlation was found between the inhibitory action of the bromo derivatives and the carcinogenicity of the non-halogenated parent hydrocarbons.     

Formation of 6-dimethylcarbamyloxy-dGuo, 6-dimethylamino-dGuo and 4-dimethylamino-dThd following in vitro reaction of dimethylcarbamyl chloride with calf thymus DNA and 6-diethylcarbamyloxy-dGuo following in vitro reaction of diethylcarbamyl chloride with calf thymus DNA

The rodent carcinogens dimethylcarbamyl chloride (DMCC) and diethylcarbamyl chloride (DECC) react with dGuo (pH 7.0–7.5, 37°C, 4 h) to form the O⁶-acyl derivatives 6-dimethylcarbamyloxy-2′-deoxyguanosine (6-DMC-dGuo) and 6-diethylcarbamyloxy-2′-deoxyguanosine (6-DEC-dGuo), respectively. Reaction of DMCC with dThd under identical conditions yielded 4-dimethylamino-thymidine (4-DMA-dThd). Compounds 6-DMC-dGuo and 6-DEC-dGuo undergo a nucleophilic aromatic substitution reaction with dimethylamine (DMA) to form 6-dimethylamino-2′-deoxyguanosine (6-DMA-dGuo) via displacement of the C-6 dialkylcarbamyloxy moiety. The substitution reaction did not take place when diethylamine or NH₃ were substituted for DMA. The structures of the new compounds 6-DMC-dGuo, 6-DEC-dGuo, 4-DMA-dThd and 6-DMA-dGuo were deduced from chemical analyses and syntheses, UV and nuclear magnetic resonance (NMR) spectra and electron impact, isobutane chemical ionization and source insertion isobutane chemical ionization mass spectra. It was postulated that 4-DMA-dThd was formed following reaction of the transient intermediate 4-DMC-dThd with DMA formed by hydrolysis of DMCC. Calf thymus DNA was reacted in vitro with DMCC (pH 7.0–7.5, 37°C, 4 h) and the modified DNA hydrolyzed enzymatically to 2′-deoxynucleosides. Compounds 6-DMC-dGuo, 4-DMA-dThd and 6-DMA-dGuo were identified in the hydrolysate by high-pressure liquid chromatography (HPLC). In an indentical manner 6-DEC-dGuo was identified following in vitro reaction of DECC with calf thymus DNA. Compounds 6-DEC-dGuo and 6-DMC-dGuo possess novel structures with respect to the types of adducts known to be formed between carcinogens and bases in DNA. The implications of these findings with respect to chemical mutagenesis and carcinogenesis is discussed. The structural relationship between N⁴-dimethyl-5-methylcytosine (4-dimethylamino-Thy) formed in DNA following in vitro reaction with DMCC and 5-methylcytosine, the only modified base found in vertebrate DNA is noted.     

Synthesis and antimycobacterial activity of 7-O-substituted-4-methyl-2H-2-chromenone derivatives vs Mycobacterium tuberculosis

A series of 7-O-alkoxy-4-methylumbelliferone derivatives were prepared using a convenient one step synthesis. Additionally the bromo- and azido derivatives 7-O-(4-bromobutoxy)-, 7-O-(6-bromohexyloxy)- and 7-O-(6-azidohexyloxy)-4-methylumbelliferone derivatives were prepared. In vitro evaluation of antimycobacterial activity determined % inhibition and MIC vs M. tuberculosis H₃₇Rv with toxicity (IC₅₀) assessed in VERO cells. The coumarins with longer alkyl chains (nonyl and decyl) showed the optimum inhibitory activity in this series (MIC 3.13?μg/mL) and IC₅₀>10?μg/mL.     

Glutamine synthetase in cultured whole retinas from the embryonic chick

Glutamine synthetase (GS) activity is enhanced in cultured whole retinas when a 72 h incubation at 37°C is preceded by storage at 4°C for 2–24 h. This enhancement occurs even in the absence of glucocorticoids and is maximal in retinas from 11 to 14 d embryos. In comparison, cortisol-induced increases in retinal GS activity at 37°C are optimal in retinas from 8 to 12 d embryos. This study, using cycloheximide (an inhibitor of protein synthesis) and cordycepin (an inhibitor of RNA synthesis), indicates that both protein and RNA synthesis are required for the 4°C storage enhancement of GS activity. The necessary RNA synthesis occurs within the first 48 h following transfer to 37°C and does not require concomitant protein synthesis. Uridine uptake, but not incorporation into trichloroacetic acid-precipitable material, is increased by initial 4°C storage when compared with whole retina controls incubated at 37°C for the total time. In contrast, both uptake and incorporation of amino acids are increased in 4°C-stored retinas for as long as 72 h subsequent to transfer from 4 to 37°C. This suggests that enhancement of GS activity may arise from a combination of elevated general protein synthesis and specific messenger-RNA synthesis following 4°C storage.     

Effects of ammonium chloride and chloroquine on endocytic uptake of liposomes by Kupffer cells in vitro

In this study we investigated the interaction of liposomes with rat Kupffer cells in maintenance culture by using the lysosomotropic amines ammonium chloride and chloroquine as inhibitors of intralysosomal degradation. The liposomes (large unilamellar vesicles) contained either the metabolically inert ³H-labeled inulin or the degradable ¹²⁵I-labeled bovine serum albumin. In control incubations, the cells released nearly all accumulated protein label and about 30% of the lipid label when they were incubated in the absence of liposomes, after an initial uptake period of 1 h in the presence of liposomes. This release of label was, for the greater part, suppressed in the presence of ammonia or chloroquine. When the inhibitors were present during the initial uptake period, a several-fold increase in the amount of protein label accumulating in the cells and a smaller, but still marked, increase in lipid label accumulation were observed. The effect of ammonia when present during uptake was readily reversible in contrast to that of chloroquine. Experiments with encapsulated inulin revealed that both lysosomotropic agents also affected the uptake process per se to some extent, probably as a result of impaired membrane/receptor recycling. Labeled liposomes adsorbed to the cells at 4°C were effectively internalized and processed intracellulary after shifting the temperature to 37°C, even when a 500-fold excess of unlabeled liposomes was present in the medium during the 37°C incubation. The observed effects of ammonia and chloroquine indicate that, after uptake, the liposomes are degraded within lysosomes, thus confirming our previous conclusion that endocytosis is the major uptake mechanism at 37°C. From the temperature-change experiments we conclude that, at 4°C, the liposomes are bound with high affinity to the cells, remaining firmly attached to the cell-surface structures which initiate their internalization when the temperature is raised to 37°C.     

Phenotypic plasticity and biogeographic variation in physiology of habitat‐forming seaweed: response to temperature and nitrate

Southeastern Australian waters are warming at nearly four times the global average rate (~0.7°C · century^?1) driven by strengthening incursions of the warm oligotrophic East Australian Current. The growth rate hypothesis (GRH) predicts that nutrient depletion will impact more severely on seaweeds at high latitudes with compressed growth seasons. This study investigates the effects of temperature and nutrients on the ecophysiology of the habitat‐forming seaweed Phyllospora comosa in a laboratory experiment using temperature (12°C, 17°C, 22°C) and nutrient (0.5, 1.0, 3.0 μM NO₃^?) scenarios representative of observed variation among geographic regions. Changes in growth, photosynthetic characteristics (via chlorophyll fluorescence), pigment content, tissue chemistry (δ¹³C, % C, % N, C:N) and nucleic acid characteristics (absolute RNA and DNA, RNA:DNA ratios) were determined in seaweeds derived from cool, high‐latitude and warm, low‐latitude portions of the species’ range. Performance of P. comosa was unaffected by nitrate availability but was strongly temperature‐dependent, with photosynthetic efficiency, growth, and survival significantly impaired at 22°C. While some physiological processes (photosynthesis, nucleic acid, and accessory pigment synthesis) responded rapidly to temperature, others (C/N dynamics, carbon concentrating processes) were largely invariant and biogeographic variation in these characteristics may only occur through genetic adaptation. No link was detected between nutrient availability, RNA synthesis and growth, and the GRH was not supported in this species. While P. comosa at high latitudes may be less susceptible to oligotrophy than predicted by the GRH, warming water temperatures will have deleterious effects on this species across its range unless rapid adaptation is possible.     

Autoradiographic study of L929 cells on the incorporation of labeled precursors of DNA, RNA, and proteins during thermic shock and return to 37 degrees C

An in vitro cultured mouse fibroblast (strain L929) was submitted to a thermic shock, i.e. 42 °C. The drastic decrease of the cellular protein synthesizing activities which subsequently occurred, was quickly reversible upon bringing back the temperature to 37 °C, thus revealing that no essential components of the L cell protein synthesizing device had been impaired during this kind of treatment. The concomitantly observed inhibition of the preribosomal RNA synthesis and maturation processes as well as the DNA biosynthesis seems functionally related to the above-mentioned inhibition of protein synthesis.     

The effect of temperature change on gene expression in Paramecium primaurelia

When paramecia grown at 24°C are transferred rapidly to 32°C, DNA and protein synthesis continue uninterrupted but at higher rates. Electron microscopic observations indicate that more of the macronuclear chromatin is transcribed at the elevated temperature. This interpretation is supported by hybridization experiments which show that the percentage of the macronuclear genome transcribed into poly(A)⁺ RNA is 24°C and 35% at 32°C. Kinetic analysis of cDNA-poly(A)⁺ RNA hybridizations reveals three abundance classes of poly(A)⁺ RNA and indicates that the number of genes expressing low abundance sequences is about 9000 at 24°C and 13000 at 32°C. The intermediately abundant and highly abundant classes are represented by 100–200 and 1–3 different kinds of RNA sequence, respectively. Cross hybridization shows that changes occur throughout the distribution of abundance classes of poly(A)⁺ RNA with increase in temperature.     

Ultrasound Doppler as an Imaging Modality for Selection of Murine 4T1 Breast Tumors for Combination Radiofrequency Hyperthermia and Chemotherapy

Noninvasive radiofrequency-induced (RF) hyperthermia has been shown to increase the perfusion of chemotherapeutics and nanomaterials through cancer tissue in ectopic and orthotopic murine tumor models. Additionally, mild hyperthermia (37°C-45°C) has previously shown a synergistic anticancer effect when used with standard-of-care chemotherapeutics such as gemcitabine and Abraxane. However, RF hyperthermia treatment schedules remain unoptimized, and the mechanisms of action of hyperthermia and how they change when treating various tumor phenotypes are poorly understood. Therefore, pretreatment screening of tumor phenotypes to identify key tumors that are predicted to respond more favorably to hyperthermia will provide useful mechanistic data and may improve therapeutic outcomes. Herein, we identify key biophysical tumor characteristics in order to predict the outcome of combinational RF and chemotherapy treatment. We demonstrate that ultrasound imaging using Doppler mode can be utilized to predict the response of combinational RF and chemotherapeutic therapy in a murine 4T1 breast cancer model.     

Regulation of chitin synthase activity inSaccharomyces cerevisiae: Effect of the inhibition of cell division and of synthesis of RNA and protein

The effect of pronase and trypsin on the activation or deactivation (degradation?) of chitin synthase ofSaccharomyces cerevisiae occurs faster in membranous preparations than in toluene-treated cells. When the temperature is raised, the former preparation is deactivated earlier than the latter one. The activity found in growing cells is not modified after inhibition of protein synthesis by cycloheximide or amino acid starvation or by the inhibition of RNA synthesis. It was possible to activate the chitin synthase ofS. cerevisiae cdc 25 grown at 23°C by means of pronase, whereas trypsin had no effect. After the cells were grown at 37°C, chitin synthase could not be activated either with trypsin or with pronase. This effect occurred independently of protein synthesis but did not take place when the cells were toluenized prior to the transfer at 37°C. These results suggest that the low catalytic levels and stability of the chitin synthase found in actively growing cells ofS. cerevisiae may be due to the restrictions introduced in the system by its membrane location.     

Effect of pisiferic acid and its derivatives on cytotoxicity macromolecular synthesis and DNA polymerase alpha of HeLa cells

1. Seventy-seven derivatives of pisiferic acid (2), an antimicrobial diterpenoid, were tested for cytotoxicity against HeLa cells and 9 derivatives were found to show cytotoxicity at less than 2 micrograms/ml dose (IC50). 2. Hydrophobicity was revealed to be an important factor for cytotoxicity of the derivatives. 3. Compound 2 inhibited predominantly DNA synthesis in HeLa cells as compared with RNA and protein synthesis. 4. No direct interaction between 2 and nucleic acid bases was indicated by a u.v. spectral method. 5. Several of the pisiferic acid species showed inhibitory action on HeLa DNA polymerase alpha, and the inhibitory activity was about 1/20 of aphidicolin.     

Thymidine nucleotide synthesis and catabolism by CHO cells and their changes during the cell cycle

At 0°C, CHO cells efficiently incorporated [³H]thymidine into the nucleotide fraction, but not into DNA. Upon reincubation of asynchronous cultures at 37°C, 15–25% of the radioactivity contained in the cellular nucleotide fraction was released, in the form of thymidine, into the culture medium. At 0°C, however, radioactivity of the nucleotide fraction was retained within the cells. Similarly, dTMP phosphatase (EC 3.1.3.35) in cell extracts was active at 37°C, but not at 0°C, whereas thymidine kinase (EC 2.7.1.21) was active at both temperatures. If synchronous cultures in Gl phase were prelabeled at 0°C and reincubated at 37°C, almost all radioactivity in the nucleotide fraction was released into the medium, whereas in S-phase cultures nearly all radioactivity of the nucleotide fraction was incorporated into DNA. In synchronous S-phase cultures treated with hydroxyurea, radioactivity in the nucleotide fraction was released into the medium at a rate considerably lower than that observed for Gl-phase cells. Rates of endogenous synthesis of thymidine nucleotides were calculated from changes of cellular thymidine nucleotide content, incorporation of thymidine nucleotides into DNA and release of thymidine into the medium during reincubation of prelabeled cultures in thymidine-free medium. The results obtained (see Table III) reveal marked differences between Gl and S phases with respect to the determinants of thymidine nucleotide metabolism.     

Hyperthermia and polyamine biosynthesis: decreased ornithine decarboxylase induction in skin and kidney after heat shock

The effect of hyperthermia treatments on ornithine decarboxylase (ODC) induction in mouse tissue was determined both in vitro and in vivo. In vitro, the addition of 12-0-tetradecanoylphorbol-13-acetate (TPA) to adult mouse skin pieces incubated at 37 degrees C in serum-free MEM led to a dramatic increase in epidermal ODC activity 5 hours following treatment. In contrast, incubation temperatures of 40 degrees C for the entire 5 hour incubation period rendered the skin pieces unresponsive to TPA for ODC induction. This inhibition of ODC induction was not the result of thermal skin kill, inactivation of TPA, or a general effect on epidermal protein synthesis. The inhibition of ODC induction could be reversed by switching the incubation temperature back to 37 degrees C. In vivo, raising the core body temperature in male mice to 41 degrees C for 1 hour resulted in a 78% decrease in kidney ODC activity. The kidney DNA synthesis and protein synthesis remained unaltered following the whole body hyperthermia treatments.