Rate Of Nucleic Acid Synthesis Research Articles

Radiomodulatory effect of liposome encapsulated AK-2123 on tumor in mice exposed to hepatocarcinogen

An attempt was made to evaluate the whole body gamma-radiation effect on tumor in the presence of free and liposome encapsulated AK-2123, a hypoxic cell radiosensitizer that has widely been used in combination with a number of cancer therapies such as thermotherapy, chemotherapy and radiotherapy. Entrapment efficiency of AK-2123 into liposome was determined by LASER Raman spectroscopy. Cancer induction in mice was carried out by repeated exposure of N-nitrosodiethylamine (DEN) in combination with partial hepatectomy. Parameters such as marker enzymes activities (GGT and AChE), rates of nucleic acid synthesis, viability modification factor and the histology of liver tissues monitored, supported the induction of cancer in liver. In addition, the effect of free as well as liposome encapsulated AK-2123 on haemopoietic parameters were also studied. It was observed that AK-2123 after incorporation into liposome afforded more efficient radiomodulatory effects than that of free AK-2123 as determined by the above-mentioned parameters. Neither free AK-2123 nor liposome encapsulated AK-2123 showed any detectable toxic effects on the mice. Thus, it is seen that treatment of cancer with a combination of radiation, a radiomodifier and a drug delivery system, opens a wide scope for exploitation for the improvement of existing cancer therapies.

Pyrimidine nucleotide and nucleic acid synthesis in embryos and megagametophytes of white spruce (Picea glauca) during germination

Pyrimidine nucleotide synthesis was investigated in isolated germinating zygotic embryos and separated megagametophytes of white spruce by following the metabolic fate of 14C-labelled orotic acid, uridine, and uracil, as well as by measuring the activities of the major enzymes participating in nucleotide synthesis. The rate of nucleic acid synthesis in these tissues was also examined by tracer experiments and autoradiographic studies conducted with labelled thymidine, and by conventional light microscopy. From our results, it emerges that changes in the contribution of the de novo and salvage pathways of pyrimidines play an important role during the initial stages of zygotic embryo germination. Preferential utilization of uridine for nucleic acid synthesis, via the salvage pathway, was observed at the onset of germination, before the restoration of a fully functional de novo pathway. Similar metabolic changes, not observed in the gametophytic tissue, were also documented in somatic embryos previously. These alterations of the overall pyrimidine metabolism may represent a strategy for ensuring the germinating embryos with a large nucleotide pool. Utilization of 14C-thymidine for nucleic acid synthesis increased in both dissected embryos and megagametophytes during germination. Autoradiographic and light microscopic studies indicated that soon after imbibition, DNA synthesis was preferentially initiated along the embryonic axis, especially in the cortical cells. Apical meristem reactivation was a later event, and the root meristem became activated before the shoot meristem. Taken together, these results indicate that precise changes in nucleotide and nucleic acid metabolism occur during the early phases of embryo germination.

DNA, RNA, and protein synthesis in the cyanobacteriumSynechocystis sp. PCC 6803 adapted to different salt concentrations

A salt shock of 684mm NaCl reduced RNA and DNA synthesis to about 30% of the control level inSynechocystis. DNA synthesis recovered to the initial level within 4 h, while for recovery of RNA synthesis about 8 h were necessary. In cells completely adapted to different salt concentrations (from 171 to 1026mm NaCl), a continuous decrease in the RNA content with increasing salt concentrations up to 684mm NaCl was found, whereas the lowest DNA content was measured around 342mm NaCl, i.e., the salinity at which maximal growth occurred. With the uracil and thymidien incorporation technique, maxima in DNA and RNA synthesis were detected in control cells. Comparing these rates with nucleic acid synthesis rates calculated from the contents of DNA and RNA and the growth rates indicated that adaptation to 1026mm NaCl seemed to lead to an increased RNA turnover inSynechocystis. Analysis of protein synthesis with35S-methionine labeling showed alterations in salt-adapated cells ofSynechocystis. At least three proteins (20.5, 25.8, and 35.8 kDa) were synthesized with highest rates at salinities leading to maximal growth, the synthesis of nine proteins (12.5, 16.9, 19.2, 22.2, 24.7, 28.5, 30.5, 50.3, and 63.5 kDa) increased and that of several other proteins decreased with increasing salinity; but only three proteins (12.5, 22.2, and 30.5 kDa) accumulated under these conditions. The adaptation ofSynechocystis to enhanced salt concentrations led also to increased contents of glucosylglycerol, glycogen, and significant amounts of K+ as well as Na+ ions.

Origin of the Hormone Action Gordon Research Conference

Like a pearl, the Hormone Action Conference began as a result of an irritation. In the summer of 1967, I was invited to give the first talk (on methods to measure protein and nucleic acid synthesis rates, what was called “molecular biology” in those days) at a Gordon Conference that I won't specify for obvious reasons. As the week progressed, I found that the program was not one very close to my own interests and experience; in short, I got pretty bored. So my mind wandered back to a Gatlinberg Conference on effects of hormones a few years earlier (organized, I think, by Frank Kenney), and I wondered why something like that wasn't offered on the Gordon Conference program. Certainly that would be much more interesting, at least from my point of view. Then as now it was customary for the Executive Director of the GRCs to visit each meeting site each week. In contrast to popular belief, Alex Cruickshank has not been Executive Director since the Conferences first started in 1931. In fact, it was Alex's predec...

Microbiology of a high energy beach sediment: evidence for an active and growing community

Microbial biomass, heterotrophic activity, and growth rates were measured in sandy sedirnents from 2 very different beaches in Nova Scotia, Canada: a protected beach inside Halifax Harbor (HH), and an exposed, high energy beach along the coast (LT). SEM of sand grains revealed that the microbial cells from HH were predominantly cocci whereas the cells from LT were mostly rods. HH sand contained 3.8 times as many cells (direct count) and 6.9 times as much biomass (ATP) as did LT sand. Likewise, the heterotrophic uptake, rates of nucleic acid synthesis, and the rate of carbon production were all several times higher in HH sand than in LT sand. However, on a biomass-specific basis, both sediments had similar activity. Moreover, specific growth rates (5.18 d-') were identical. The data indicate that size and total activity of the microbial population depend on the location of the sediment but that growth rate and per cell activity are apparently not affected by abrasion and shear stress.

Radiation Stimulation During the Early Stationary Growth Phase in Synechococcus Lividus and Its Correlation With Photooxidative Stress Occurring Before the Stationary Phase

The effects of chronic gamma radiation at dose rates ranging from 0.058 mGy d-1 on growth rate calculated during the early stationary phase were studied. A stimulatory effect occurred for all doses and for all phases of the cells selected for use in the inoculation of the medium. During the same period, the rate of nucleic acid synthesis was increased in irradiated cultures compared to control cultures. The stimulating effect always occurred in cultures irradiated from the inoculation to the eighteenth day only. This result led us to conclude that the stimulation mechanism depended upon the events occurring at the end of the exponential phase in the deceleration period. Studies on cell metabolism showed that cells presented features of photooxidative stress in this period. Increases in superoxide dismutase, glutathione reductase and glucose-6-phosphate dehydrogenase were observed in irradiated cultures. It was assumed that irradiation at very low doses could help cells to better defend against photooxidative stress by increasing oxidants that activate the glucose metabolism and C5-sugars production and nucleic acid synthesis.

Microbial growth rates in a cold-core gulf stream eddy of the northwestern Sargasso sea

Horizontal spatial variations of microbial and bacterial abundance and biomass, and rates of nucleic acid synthesis in surface waters across cold-core Gulf Stream eddy P were investigated in April 1982. The age of eddy P was estimated at 8 to 10 months. Chlorophyll a reached 0.38 μg l −1 in an area of high chlorophyll a fluorescence located in 18 to 19°C waters. Bacteria were mostly (98%) free-floating, and microbial-ATP biomass correlated with chlorophyll a fluorescence across eddy P. Rates of microbial metabolism and growth, and bacterial production were not proportional across areas of high chlorophyll a fluorescence. Estimates of microbial growth rates (δ), were 0.3 to 0.6 h −1 in the mixed surface waters of eddy P and 0.06 to 0.08 h −1 in adjacent waters of the northwestern Sargasso Sea. Bacterial production ranged from 0.12 to 1.41 μgC l −1 d −1, and production: biomass ratios (turnover times) were 0.024 to 0.27 h −1 in the eddy and ca. 0.002 h −1 in the Sargasso Sea. Because microbial metabolism (RNA synthesis) and growth (DNA synthesis) across eddy P were not closely coupled, although intertwined with diurnal variations, we conclude that the microbial communities, based on the experimental time scale, were in a state of unbalanced growth.

Fine-scale vertical distributions of microbial biomass and activity in California Borderland sediments

Vertical distributions of total organic carbon (TOC), porosity, adenosine triphosphate (ATP), and rates of nucleic acid synthesis were measured at millimeter depth intervals in basin sediments from the California Borderland. Within the upper 5 mm, large gradients were measured in sediment microbial biomass (ATP) and nucleic acid synthesis rates, especially deoxyribonucleic acid (DNA) synthesis. A subsurface secondary maximum in specific growth rate (extrapolated from DNA synthesis and ATP biomass measurements) was also observed and may be associated with the onset of sulfate reduction. Carbon production rates integrated over the top 40 mm of the sediment profile in San Pedro Basin (160 to 220 mg carbon m −2 day −1) suggest that the central portion of the basin receives larger inputs of organic carbon than would be predicted from surface water primary production rates and a simple one-dimensional vertical settling model. Lateral transport of carbon from the shallower portions of the basin to the center is the most likely source of this carbon.

Laboratory Calibrations of the [ 3 H]Adenine Technique for Measuring Rates of RNA and DNA Synthesis in Marine Microorganisms

The nucleic acid synthesis rates of several marine phytoplankton and bacteria grown in chemostat and batch cultures were measured by using [H]adenine. The [H]adenine synthesis rates showed excellent agreement with the known rates of synthesis estimated from chemical RNA and DNA data. Under certain conditions, RNA turnover and ATP pool compartmentalization produce inaccuracies in synthesis measurements made with [H]adenine. However, accurate measurements of the rates of microbial RNA and DNA synthesis can be made in any environmental situation provided a few simple precautions are observed. First, time course experiments are recommended. Second, experiments should be conducted for periods long enough to avoid problems arising from disequilibria of internal ATP pools. Finally, exogenous [H]adenine should remain in the medium over the length of the time course.

Microbial RNA and DNA synthesis in marine sediments

A technique for measuring rates of RNA and DNA synthesis in sedimentary microbial communities has been adapted from methods developed for marine and freshwater microplankton research. The procedure measures the uptake, incorporation and turnover of exogenous [2, 3H]-adenine by benthic microbial populations. With minor modification, it is applicable to a wide range of sediment types. Measurement of nucleic acid synthesis rates are reported from selected benthic marine environments, including coral reef sediments (Kaneohe Bay, Oahu, Hawaii), intertidal beach sands (Oahu and southern California) and California borderland basin sediment (San Pedro Basin), and comparisons are made to selected water-column microbial communities. Biomass-specific rates of nucleic acid synthesis in sediment microbial communities were comparable to those observed in water-column assemblages (i.e., 0.02 to 2.0 pmol deoxyadenine incorporated into DNA [ng ATP]-1 h-1 and 0.2 to 8.9 pmol adenine incorporated into RNA [ng ATP]-1 h-1). DNA synthesis rates were used to calculate carbon production estimates ranging from 2 μg C cm-3 h-1 in San Pedro Basin sediment (880 m water depth) to 807 μg C cm-3 h-1 in coral reef sediment from the Kaneohe Bay. Microbial community specific growth rate, μ(d-1), estimated from DNA synthesis rates in surface sediments ranged from 0.1 in San Pedro Basin to 4.2 in Scripps Beach (La Jolla, California) intertidal sand.

Estimates of bacterial growth from changes in uptake rates and biomass.

Rates of nucleic acid synthesis have been used to examine microbiol growth in natural waters. These rates are calculated from the incorporation of [3H]adenine and [3H]thymidine for RNA and DNA syntheses, respectively. Several additional biochemical parameters must be measured or taken from the literature to estimate growth rates from the incorporation of the tritiated compounds. We propose a simple method of estimating a conversion factor which obviates measuring these biochemical parameters. The change in bacterial abundance and incorporation rates of [3H]thymidine was measured in samples from three environments. The incorporation of exogenous [3H]thymidine was closely coupled with growth and cell division as estimated from the increase in bacterial biomass. Analysis of the changes in incorporation rates and initial bacterial abundance yielded a conversion factor for calculating bacterial production rates from incorporation rates. Furthermore, the growth rate of only those bacteria incorporating the compound can be estimated. The data analysis and experimental design can be used to estimate the proportion of nondividing cells and to examine changes in cell volumes.

Relationship between ribo- and deoxyribonucleotide concentrations and biological parameters in cultured chinese hamster ovary cells

The relationship between ribo- and deoxyribonucleotide concentrations, growth rate and cell viability has been studied. Nucleotide pools were manipulated using mycophenolic acid, pyrazofurin, phosphonoacetyl- l-aspartate (PALA) or thymidine. Growth rate, cell viability, nigrosin exclusion, progression through the cell cycle, and the rates of nucleic acid synthesis were measured. The observed qualitative relationships between nucleotide concentrations and growth rate can be rationalized in terms of the effects of changes in substrate availability on nucleic acid synthesis. However, there is no discernible relationship between changes in nucleotide concentrations and the loss of cell viability. More detailed studies will be required to elucidate each step in the complex process leading from the initial changes in nucleotide concentrations to cell death.

Improved tumour yields by means of a TPA-DMBA-TPA variation of the Berenrlum-Mottram experiment on the back skin of NMRI mice. : The effect of stationary hyperplasia without inflammation

Application of the phorbol ester TPA to the back skin of NMRI mice 14 times within a period of 7 weeks causes a stationary hyperplasia, with a corresponding increase in the labelling index of the basal cells from 1% to 14%. By initiation of skin, which has been pretreated with TPA in this way, with the carcinogen DMBA, followed by continued treatment with TPA (initiation-promotion corresponding to the classical Berenblum-Mottram experiment) the tumour yield (papillomas, carcinomas) is very much higher than that obtained using the scheme of the normal Berenblum-Mottram experiment. The preliminary induction of a stationary hyperplasia with high rates of nucleic acid synthesis must be considered an important co-factor in epidermal carcinogenesis.

Nucleic acid synthesis and effect of glucose on its kinetics in cotyledons ofChenopodium rubrum l. during photoperiodic induction

In cotyledons ofChenopodium rubrum L. polydisperse RNA is synthesized in the region of the low molecular weight RNAs during photoperiodic induction. After short-time labelling the rate of 4s RNA synthesis was always higher in induced plants than in plants having obtained a light-break in the middle of the dark period. When glucose was added to the nutrient medium during the dark period of a single photoperiodic cycle the rate of nucleic acid (NA) synthesis was higher in non-induced plants than in induced ones at the termination of the dark period. In plants induced by two cycles in the absence of glucose the rate of NA synthesis at the termination of the second dark period was higher in induced than in non-induced plants. This difference is due to the differential kinetics of NA synthesis during darkness. In plants induced in the presence of glucose the peak of the rhythm in NA synthesis was advanced by 4 h relative to that found in plants induced in the absence of sugar. Thus, the termination of the dark period coincided with the negative slope of the oscillation in plants induced in the presence of glucose, while in plants having obtained a light-break NA synthesis decreased only slightly after having attained its peak. In plants induced in the absence of glucose the termination of the dark period coincided with the peak in the rhythm in NA synthesis. The rhythm in NA synthesis of the cotyledons during the dark period of an inductive cycle is out of phase with the rhythm in flower initiation.

[Effect of dexamethasone hemisulfate on nucleic acid synthesis in human granulocytes].

Dexamethasone hemisulphate (Dexa-Scheroson®) concentrations of 100 <i>μ</i>/ml culture medium produces a marked inhibition of the incorporation of ¹⁴C-adenine into the trichloroacetic acid precipitate and nucleic acids of granulocytes. Exact analysis of the precursor pool and measurement of the rate of nucleic acid synthesis showed that this inhibition of incorporation by the dexamethasone ester was due to a reduction in the formation of fresh nucleic acids. Fractionation of these extracts in linear sucrose gradients revealed a remarkable diminution in the radioactivity of the fractions 18 S and 4 S. We believe the cause of this change in metabolism could be that a false ribosomal RNA precursor is formed which does not contain the 18S portion. Alternatively, one might postulate an increased instability of this component. Such changes could lead to a reduction in existing ribosomes and in protein synthesis. In this case, the diminished turnover of transfer nucleic acids, perhaps corresponding to the component sedimenting at 4S, could be interpreted as a further rational biological reaction.

Sources of the Nucleotide Pools in Escherichia coli after Infection with Bacteriophage T2

Abstract It has long been known that infection of Escherichia coli with bacteriophage T2 causes degradation of bacterial deoxyribonucleic acid and that 70 to 80% of the degradation products appear in viral DNA within 30 min after infection. It is reasonable to assume that exonucleases participate in this process and catalyze the production of mononucleotide intermediates. To study this system in more detail, the amounts of free mononucleotides present in the cells were measured at various times after infection with phage T2. The extent of transferal of labeled nucleotides from bacterial nucleic acids to the free mononucleotide pools was also studied. Only a gradual and small increase in the total amounts of deoxyribonucleotides in the pools was observed after infection, in spite of drastic changes both in the rate of nucleic acid synthesis and in the pathways of nucleic acid metabolism that occur at this time. This suggests that the intracellular concentrations of these intermediates are precisely controlled. By labeling the bacterial nucleic acid with 32P-orthophosphate and subsequently infecting the cells with T2 phage, the amount of nucleic acid-derived nucleotides present in the deoxyribonucleotide pools was found to increase rapidly 4 min after infection and decline precipitously 10 to 15 min after infection. At 7 to 8 min after infection, 50 to 75% of the nucleotides entering these pools came from the bacterial nucleic acids. Of the known bacterial and phage-induced exonucleases, only T2 exonuclease A exhibits sufficient activity in vitro to readily account for the rate of nucleotide production that is observed. The time of appearance of this enzymatic activity is correlated with the time of increase of nucleic acid-derived nucleotides in the pools. It is paradoxical that, 10 to 15 min after infection, a rapid decline of labeling of the pools occurs at a time when half of the bacterial DNA remains. Knowledge of the relative specific activities of the deoxycytidylate and thymidylate pools makes it possible to estimate the rate of their interconversion in vivo by the pathway involving the phage-induced enzymes deoxycytidylate deaminase and thymidylate synthetase. The data indicate that, at 9 min after infection, approximately 15% of the deoxycytidylate derived from bacterial DNA is converted to thymidylate via this pathway. The high specific activity of the deoxyadenylate pool suggests that some of this compound may have been derived from a deoxyguanosine nucleotide. A small but reproducibly measurable amount of nucleic acid-derived phosphate was found in the nucleotide pools of uninfected cells. This suggests that a slow turnover of stable ribonucleic acid, or perhaps both RNA and DNA, occurs in rapidly growing E. coli.

Kinetic studies on the relationship of ribonucleotide precursor pools and ribonucleic acid synthesis

A study has been made of the kinetics of flow of [ 14C]uracil through the intracellular pools of pyrimidine ribonucleotides of Bacillus subtilis. The rate at which these pools fill is slow as compared to that predicted from the measured sizes of these pools and the rate of nucleic acid synthesis. These results may be a consequence of the continued entry of uracil derivatives into the pools from messenger RNA turnover, or alternatively due to an internal compartmentalization of the pool components. The uracil derivatives (UMP, UDP, UTP, UDPG), which make up about 75% of the total pool size, are rapidly equilibrated with one another; this is seen in the fact that one can only trace a sequential flow of radioactive uracil through them in experiments at lowered temperatures. On the other hand, the kinetics of uracil flow through the soluble pools is the same as that through messenger, which is consistent with the equilibration of the two through messenger turnover and recycling of isotope from the pools into the messenger. By measuring the rate of [ 14C]uracil entry into the pools, and the size of the pools, an estimate of the size of that fraction of the RNA of the cells that is turning over is obtained (7%), which is in agreement with the value obtained with the aid of actinomycin D.

Effects of actinomycin D and 2-mercapto-1-(β-4-pyridethyl) benzimidazole on the incorporation of [ 3H]uridine by chick embryo cells

1. The incorporation of [ 3H]uridine into RNA and RNA precursor fractions of chick embryo cells was investigated. 2. Actinomycin D inhibited the incorporation of [ 3H]uridine into RNA and also led to a decreased rate of incorporation into the nucleic acid precursor fraction. 3. 2-Mercapto-1-(β-4-pyridethyl)benzimidazole competitively inhibited the incorporation of [ 3H]uridine into the precursor fraction and consequently decreased the apparent rate of nucleic acid synthesis. 4. The mechanisms of [ 3H]uridine incorporation into chick cells and of these inhibitions are discussed.

The Effect of Antigen on the Rate of Guanosine-8-C14 Incorporation by Lymphocytes

Summary Lymphocyte cultures from PPD-hypersensitive human donors have been shown to develop an increase in the rate of nucleic acid synthesis upon stimulation with PPD. This increase, as reflected by the rate of incorporation of guanosine-8-C14 into a trichloroacetic acid-insoluble fraction, occurs between the 5th and the end of the 13th day of culture with a peak at 8 days. Cells from weakly-sensitive donors have a peak of incorporation at approximately 3 days. Lymphocyte cultures from ragweed-sensitive human donors, demonstrating no delayed hypersensitivity reaction, developed a slight increase in the rate of nucleic acid synthesis upon stimulation with ragweed extract. The peak observed was at day 6 and the rate of incorporation fell to normal levels by day 8. The reaction appears not to be influenced by antibody or antibody-producing cells.

Studies on cyclophosphamide-induced tolerance to sheep erythrocytes.

Complete immunological tolerance to sheep cells can be induced in mice when cyclophosphamide is injected together with sheep cells or up to 72 hr before or 48 hr after the antigen. As is true for radiation-induced immune suppression, the drug is most effective when given in the 24 hr prior to antigen. Complete cyclophosphamide-induced immunological suppression requires large doses of sheep cells (6.2 x 10(9) cells), presumably to enable antigen to reach sequestered receptor sites. The cyclophosphamide tolerance system has been analyzed with the Jerne technique to determine plaque-forming cells and with isotopic methods to measure rates of nucleic acid synthesis. This drug suppression has been found to consist of two components. The first is nonspecific injury to the lymphoid system caused by the cytotoxic drug and is related to the proportion of spleen cells killed. The second is antigen-specific immunological tolerance and appears to correlate with profound depression of deoxyribonucleic acid synthesis in the surviving cells. This tolerance is thought to be most consistent with a mechanism in which antigenic stimulation in the presence of cyclophosphamide-inhibited DNA synthesis and mitosis leads to the elimination or death of the specific immunological clone. Tolerance induction with cyclophosphamide is associated with loss of the 19S hemolysin plaques which are seen in nonstimulated mouse spleen, implicating these cells in immune responsiveness. The ability to induce tolerance is lost on the 3rd postantigen day at the end of a 24-hr period in which 19S cells have increased 8-fold and 7S cells 200-fold. The data suggest that loss of sensitivity is due to the emergence on day 3 of drug-resistant plaque-forming cells, particularly those of the 19S variety. In the succeeding days after antigen injection there is a progressive increase in the resistance of plaque-forming cells to cyclophosphamide administration.