Effect of 5-fluoro-2'-deoxyuridine on [h]thymidine incorporation by bacterioplankton in the waters of southwest Florida

The effect of 5-fluoro-2'-deoxyuridine (FdUrd) on [methyl-H] thymidine incorporation by bacterioplankton populations in subtropical freshwater, estuarine, and oceanic environments was examined. In estuarine waters, intracellular isotope dilution was inhibited by FdUrd, which enabled us to estimate both intracellular and extracellular isotope dilution. In 2 of 10 cases, extracellular isotope dilution was significant. At low concentrations of [methyl-H]thymidine or [6-H]thymidine, FdUrd completely inhibited incorporation of radioactivity into protein and RNA. At high concentrations of [H]thymidine, however, FdUrd had little effect on labeling patterns. The dihydrofolate reductase inhibitors amethopterin and trimethoprim had no effect on macromolecular labeling patterns. These results suggest that thymidylate synthase is not involved in nonspecific labeling and that FdUrd inhibits nonspecific labeling by blocking some other enzyme involved in thymidine catabolism. In oligotrophic oceanic and freshwater samples, FdUrd did not inhibit intracellular isotope dilution or [H]thymidine labeling of protein and RNA, but caused some inhibition of [H]thymidine incorporation into DNA. The ability of FdUrd to inhibit nonspecific macromolecular labeling during [H]thymidine incorporation was significantly correlated (r = 0.84) with total thymidine incorporation (in picomoles per liter per hour). The results are discussed in terms of applications of FdUrd to routine bacterial production measurements and the general assumptions of [H]thymidine incorporation.     

Selected Nucleic Acid Precursors in Studies of Aquatic Microbial Ecology

The use of radiolabeled nucleosides and nucleic acid bases to estimate the rates of RNA and DNA synthesis in naturally occurring microbial assemblages requires numerous assumptions, several of which are evaluated herein. Comparative time series analyses of the uptake and incorporation, labeling specificity, and extent of catabolism of [2-³H]adenine, [methyl-³H]thymidine, and [5-³H]uridine were performed with pure bacterial and algal cultures, as well as with environmental samples. [³H]thymidine yielded the most variable results, especially with regard to the extent of nonspecific macromolecular labeling. The pathways of [³H]thymidine and [³H]adenine metabolism were further evaluated by isotope dilution methods and by comparing incorporation patterns of thymidine labeled at different sites of the molecule. The advantages, uncertainties, and limitations of the use of radiolabeled nucleic acid precursors in studies of aquatic microbial ecology are discussed and a prospectus for future studies presented.     

Underestimation of DNA Synthesis by [3H]Thymidine Incorporation in Marine Bacteria

A direct comparison of [³H]thymidine incorporation with DNA synthesis was made by using an exponentially growing estuarine bacterial isolate and the naturally occurring bacterial populations in a eutrophic subtropical estuary and in oligotrophic offshore waters. Simultaneous measurements of [³H]thymidine incorporation into DNA, fluorometrically determined DNA content, and direct counts were made over time. DNA synthesis estimated from thymidine incorporation values was compared with fluorometrically determined changes in DNA content. Even after isotope dilution, nonspecific macromolecular labeling, and efficiency of DNA recovery were accounted for, [³H]thymidine incorporation consistently underestimated DNA synthesized by six- to eightfold. These results indicate that although the relationship of [³H]thymidine incorporation to DNA synthesis appears consistent, there are significant sources of thymine bases incorporated into DNA which cannot be accounted for by standard [³H]thymidine incorporation and isotope dilution assays.     

Underestimation of DNA synthesis by [h]thymidine incorporation in marine bacteria

A direct comparison of [H]thymidine incorporation with DNA synthesis was made by using an exponentially growing estuarine bacterial isolate and the naturally occurring bacterial populations in a eutrophic subtropical estuary and in oligotrophic offshore waters. Simultaneous measurements of [H]thymidine incorporation into DNA, fluorometrically determined DNA content, and direct counts were made over time. DNA synthesis estimated from thymidine incorporation values was compared with fluorometrically determined changes in DNA content. Even after isotope dilution, nonspecific macromolecular labeling, and efficiency of DNA recovery were accounted for, [H]thymidine incorporation consistently underestimated DNA synthesized by six- to eightfold. These results indicate that although the relationship of [H]thymidine incorporation to DNA synthesis appears consistent, there are significant sources of thymine bases incorporated into DNA which cannot be accounted for by standard [H]thymidine incorporation and isotope dilution assays.     

Spatial and Temporal Variations in Bacterial Macromolecule Labeling with [methyl-3H]Thymidine in a Hypertrophic Lake

The incorporation of [methyl-³H]thymidine into three macromolecular fractions, designated as DNA, RNA, and protein, by bacteria from Hartbeespoort Dam, South Africa, was measured over 1 year by acid-base hydrolysis procedures. Samples were collected at 10 m, which was at least 5 m beneath the euphotic zone. On four occasions, samples were concurrently collected at the surface. Approximately 80% of the label was incorporated into bacterial DNA in surface samples. At 10 m, total incorporation of label into bacterial macromolecules was correlated to bacterial utilization of glucose (r = 0.913, n = 13, P < 0.001). The labeling of DNA, which ranged between 0 and 78% of total macromolecule incorporation, was inversely related to glucose uptake (r = -0.823), total thymidine incorporation (r = -0.737), and euphotic zone algal production (r = -0.732, n = 13, P < 0.005). With decreased DNA labeling, increasing proportions of label were found in the RNA fraction and proteins. Enzymatic digestion followed by chromatographic separation of macromolecule fragments indicated that DNA and proteins were labeled while RNA was not. The RNA fraction may represent labeled lipids or other macromolecules or both. The data demonstrated a close coupling between phytoplankton production and heterotrophic bacterial activity in this hypertrophic lake but also confirmed the need for the routine extraction and purification of DNA during [methyl-³H]thymidine studies of aquatic bacterial production.     

Effect of hypoxia on nucleic acid and protein synthesis in different brain regions

The incorporation of [methyl-³H]thymidine into DNA, of [5-³H]uridine into RNA, and of [1-¹⁴C]leucine into proteins of cerebral hemispheres, cerebellum, and brainstem of guinea pigs after 80 hr of hypoxic treatment was measured. Both in vivo (intraventricular administration of labeled precursors) and in vitro (tissue slices incubation) experiments were performed. The labeling of macromolecules extracted from the various subcellular fractions of the above-mentioned brain regions was also determined. After hypoxic treatment the incorporation of the labeled precursors into DNA, RNA, and proteins was impaired to a different extent in the three brain regions and in the various subcellular fractions examined; DNA and RNA labeling in cerebellar mitochondria and protein labeling in microsomes of the three brain regions examined were particularly affected.     

Catabolism of Tritiated Thymidine by Aquatic Microbial Communities and Incorporation of Tritium into RNA and Protein

The incorporation of tritiated thymidine by five microbial ecosystems and the distribution of tritium into DNA, RNA, and protein were determined. All microbial assemblages tested exhibited significant labeling of RNA and protein (i.e., nonspecific labeling), as determined by differential acid-base hydrolysis. Nonspecific labeling was greatest in sediment samples, for which ≥95% of the tritium was recovered with the RNA and protein fractions. The percentage of tritium recovered in the DNA fraction ranged from 15 to 38% of the total labeled macromolecules recovered. Nonspecific labeling was independent of both incubation time and thymidine concentration over very wide ranges. Four different RNA hydrolysis reagents (KOH, NaOH, piperidine, and enzymes) solubilized tritium from cold trichloroacetic acid precipitates. High-pressure liquid chromatography separation of piperidine hydrolysates followed by measurement of isolated monophosphates confirmed the labeling of RNA and indicated that tritium was recovered primarily in CMP and AMP residues. We also evaluated the specificity of [2-³H]adenine incorporation into adenylate residues in both RNA and DNA in parallel with the [³H]thymidine experiments and compared the degree of nonspecific labeling by [³H]adenine with that derived from [³H]thymidine. Rapid catabolism of tritiated thymidine was evaluated by determining the disappearance of tritiated thymidine from the incubation medium and the appearance of degradation products by high-pressure liquid chromatography separation of the cell-free medium. Degradation product formation, including that of both volatile and nonvolatile compounds, was much greater than the rate of incorporation of tritium into stable macromolecules. The standard degradation pathway for thymidine coupled with utilization of Krebs cycle intermediates for the biosynthesis of amino acids, purines, and pyrimidines readily accounts for the observed nonspecific labeling in environmental samples.     

Experimental Evaluation of Conversion Factors for the [3H]Thymidine Incorporation Assay of Bacterial Secondary Productivity

The relationship between bacterial growth and incorporation of [methyl-³H]thymidine in oligotrophic lake water cultures was investigated. Prescreening, dilution, and addition of organic and inorganic nutrients were treatments used to prevent bacterivory and stimulate bacterial growth. Growth in unmanipulated samples was estimated through separate measurements of grazing losses. Both bacterial number and biovolume growth responses were measured, and incorporation of [³H]thymidine in both total macromolecules and nucleic acids was assayed. The treatments had significant effects on conversion factors used to relate thymidine incorporation to bacterial growth. Cell number-based factors ranged from 1.1 × 10¹⁸ to 38 × 10¹⁸ cells mol of total thymidine incorporation⁻¹ and varied with treatment up to 10-fold for the same initial bacterial assemblage. In contrast, cell biovolume-based conversion factors were similar for two treatment groups across a 16-fold range of [³H]thymidine incorporation rates: 5.54 × 10¹⁷ μm³ mol of total thymidine incorporation⁻¹ and 15.2 × 10¹⁷ μm³ mol of nucleic acid incorporation⁻¹. Much of the variation in cell number-based conversion factors was related to changes in apparent mean cell volume of produced bacteria. Phosphorus addition stimulated [³H]thymidine incorporation more than it increased bacterial growth, which resulted in low conversion factors.     

Measuring bacterial production via rate of incorporation of [3H]thymidine into DNA

Thymidine (TdR) incorporation into DNA as a measure of bacterial production in environmental samples relies on assumptions about what organisms incorporate exogenous thymidine, extent of dilution of labelled thymidine by internal and external pools, and analytical methods for recovery and purification of bacterial DNA. We have examined these assumptions with regard to the feasibility of using [³H]TdR incorporation in the water column and sediments of a blackwater river. The extent of dilution of added [³H]TdR may be determined with isotope dilution plots (Moriarty and Pollard, 1981 and 1982) and these indicate a wide range of degree of participation of added [³H]TdR. Previously described methods for extracting DNA from sediment bacteria may lead to underestimates and we described a more efficient recovery scheme.     

Activity of an Attached and Free-Living Vibrio sp. as Measured by Thymidine Incorporation, p-Iodonitrotetrazolium Reduction, and ATP/DNA Ratios

Three independent techniques, [³H]thymidine incorporation, the reduction rate of p-iodonitrotetrazolium violet (INT) to INT formazan normalized to DNA, and the ratio of ATP to DNA, were adapted to measure the activity of attached and unattached estuarine bacteria. In experiments employing the estuarine isolate Vibrio proteolytica, nutrient concentrations were manipulated by varying the concentration of peptone-yeast extract. In the presence of exogenous nutrients, the activity of free-living cells was greater than that of attached cells as measured by [³H]thymidine incorporation and ATP/DNA ratios. In the absence of peptone-yeast extract, however, the activity of attached cells surpassed that of free-living cells as determined by [³H]thymidine incorporation and INT formazan normalized to DNA. Of the three techniques, [³H]thymidine incorporation was deemed most sensitive for detecting changes in activity resulting from slight differences in nutrient concentration. By this technique, attached cells were much less sensitive to changing nutrient concentrations than were free-living cells. Below a threshold concentration, attached cell activity remained constant, while the activity of unattached cells decreased as a function of decreasing nutrient concentration. The results suggest that loss of cell surface area available for substrate uptake due to attachment may be an important factor in determining the relative activities of attached and free-living cells.     

Comparative studies on S-adenosyl-l-methionine binding sites of protein N-methyltransferases,using 8-azido-S-adenosyl-l-methionine as photoaffinity probe

Employing a photoaffinity labeling procedure with 8-azido-S-adenosyl-l-[methyl-³H]methionine (8-N₃-Ado[methyl-³H]Met), the binding sites for S-adenosyl-l-methionine (AdoMet) of three protein N-methyltransferases [AdoMet:myelin basic protein-arginine N-methyltransferase (EC2.1.1.23); AdoMet:histone-arginin N-methyltransferase (EC2.1.1.23); and AdoMet:cytochromec-lysine N-methyltransferase (EC2.1.1.59)] have been investigated. The incorporation of the photoaffinity label into the enzymes upon UV irradiation was highly specific. In order to define the AdoMet binding sites, the photolabeled enzymes were sequentially digested with trypsin, chymotrypsin, and endoproteinase Glu-C. After each proteolytic digestion, radiolabeled peptide from each enzyme was resolved on HPLC first by gradient elution and further purified by an isocratic elution. Retention times of the purified radiolabeled peptides from the three enzymes from the corresponding proteolysis were significantly different, indicating that their sizes and compositions were different. Amino acid composition analysis of these peptides confirmed further that the AdoMet binding sites of these protein N-methyltransferases are quite different.     

Biosynthesis of the 3-benzylchroman-4-one eucomin in Eucomis bicolor

Feeding experiments have demonstrated the specific incorporation of radioactivity from dl-phenylalanine-[1-¹⁴C], l-phenylalanine-[U-¹⁴C], sodium acetate-[2-¹⁴C] and l-methionine-[methyl-¹⁴C] into the 3-benzylchroman-4-one eucomin in Eucomis bicolor. The labelling patterns indicate that eucomin is biosynthesized by the addition of a carbon atom derived from methionine onto a C₁₅ chalcone-type skeleton. Radioactivity from 2′,4′,4-trihydroxy-6′-methoxychalcone-[methyl-¹⁴C] and 2′,4′-dihydroxy-4,6′-dimethoxychalcone-[6′-methyl-¹⁴C] was incorporated into eucomin, the latter compound being the better precursor, demonstrating the feasibility that 2′-methoxychalcones are biosynthetic precursors of the “homoisoflavonoids”. Possible biosynthetic relationships in this class of compounds are discussed.     

Letter: Methylation of mitochondrial RNA species in the wild-type and poky strains of Neurospora crassa.

Methylation of mitochondrial RNAs in the me-3 and poky f⁺ me-3 strains of Neurospora crassa has been re-examined using procedures based on steadystate labeling with [methyl-³H]methionine and taking the precaution of adding sodium formate to suppress randomization of the methyl-³H label. Under these conditions, the values measured for the mitochondrial ribosomal RNAs are 0·05 to 0·16 methyl group per 100 nucleotides, much lower than had been reported previously and, in addition, there is no longer a significant difference between the me-3 and poky f⁺ me-3 strains (contrast Kuriyama &; Luck, 1974). Control experiments rule out the possibility that the much lower values result from grossly inefficient labeling of intra-mitochondrial methyl groups.     

Further Verification of the Isotope Dilution Approach for Estimating the Degree of Participation of [3H]thymidine in DNA Synthesis in Studies of Aquatic Bacterial Production

The optimal concentration of [³H]thymidine (i.e., the maximal degree of participation in DNA synthesis) as determined by adding increasing amounts of labeled thymidine at the same specific activity was similar to the concentration of thymidine inhibiting the de novo pathway as determined by isotope dilution plots. These experiments provide further verification of the isotope dilution approach for determining the degree of participation of [³H]thymidine in DNA synthesis.     

Inhibition of lectin-induced lymphocyte activation by diamide and other sulfhydryl reagents

Inhibition of lectin-induced lymphocyte activation by five reagents capable of combining with or oxidizing free sulfhydryl groups was examined. Each of the reagents tested was capable of inhibiting [methyl-³H]thymidine or [¹⁴C]uridine incorporation into trichloroacetic acid-insoluble material. Four of these reagents, iodoacetamide and N-ethylmaleimide (alkylating agents) and 5,5′-dithiobis (2-nitrobenzoic acid) and p-hydroxymercuriphenylsulfonic acid (sulfhydryl binding agents), inhibited activation when added to lymphocyte cultures together with lectin or at any time thereafter through 48 hr. In contrast, the sulfhydryl oxidizing agent diazine dicarboxylic acid bis[N,N-dimethylamide] (diamide) was effective only when added within 30–60 min of lectin or when added after 24 hr. This inhibition of lymphocyte activation was not due to decreased intracellular levels of reduced glutathione or to inhibition of binding of lectin to the lymphocyte. These results suggest that maintenance of free sulfhydryl groups is important during the early induction of lymphocyte activation and suggest that an obligatory step or steps in the activation sequence may involve sulfhydryl interactions.     

Effect of undernutrition on DNA and RNA synthesis in subcellular fractions from different regions of the developing rat brain

The effect of undernutrition on the incorporation of [methyl-³H]thymidine into DNA and of 5-[³H]uridine into RNA of cerebral hemispheres, cerebellum, and brain stem was studied in vivo and in vitro in rats. The labeling of DNA from nuclei and mitochondria and of RNA from nuclei, mitochondria, microsomes, and soluble fractions, was also measured in vitro. The results demonstrate that nucleic acid synthesis is impaired and delayed during undernutrition. Specific effects were observed for the different brain regions and subcellular fractions: at 10 days nuclear and mitochondrial DNA and RNA synthesis was impaired, whereas at 30 days only the mitochondrial nucleic acid synthesis was affected.The delay of DNA and RNA labeling, caused by undernutrition, was most evident in the cerebellum, probably due to its intense cell proliferation during postnatal development. The specific sensitivity of mitochondria as compared to other subcellular fractions, may be due to the intense biogenesis and/or turnover of nucleic acids in brain mitochondria not only during postnatal development, but also in the adult animal.     

Adherence of Pseudomonas aeruginosa to inanimate polymers including biomaterials

Cells of Pseudomonas aeruginosa were adhered to polymethyl methacrylate, polyvinyl acetate, polyvinyl chloride, polyhydroxyethyl methacrylate, mixed-acrylic, silicone, and natural latex materials. Planktonic bacteria and bacteria that adhered to the test materials were compared for their uptake of either L-[3,4,5-³H] leucine or [methyl-³H] thymidine during growth in a minimal medium. Leucine incorporation was reduced and thymidine uptake was negligible in adherent bacteria for up to 8 h following primary attachment by which time cells in the planktonic state showed active uptake of both substrates. These reduced uptake periods correlated with lag phases of growth of adherent cells as determined with a sonication-release plate count procedure and analyses of adenosine triphosphate (ATP). The extent of the lag phase of the adherent populations was dependent on initial densities of adhered cells and the nature of the substratum. Received 02 December 1998/ Accepted in revised form 25 April 1999     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of [³H]uridine incorporation into RNA and [³H]leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10⁻²¹ M). Insulin stimulated the rate of [³H]thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100–1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [³H]thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of [³H]uridine, [³H]thymidine and [³H]leucine into their respective precursor pools is not responsible for the apparent stimulatation of RNA, DNA and protein synthesis.     

Biosynthesis of N-methyl-l-glucosamine from d-glucose by Streptomyces griseus

Biosynthesis of $\text{N-}\text{methyl}\text{-}\text{l}\text{-}\text{glucosamine}$ moiety of streptomycin from d-glucose by Streptomyces griseus was studied. A mixture of d-[1-¹⁴C]glucose and d-[6-³H]glucose was given to the culture of S. griseus. The ³H/¹⁴C ratio found in $\text{N-}\text{methyl}\text{-}\text{d}\text{-}\text{glucosamine}$ further supports a mechanism that the conversion of d-glucose to l-hexose is carried out without scission of carbon skeleton. When d-[1-¹⁴C]glucose and d-[3-³H]glucose were used, the fall of ³H/¹⁴C ratio in $\text{N-}\text{methyl}\text{-}\text{l}\text{-}\text{glucosamine}$ showed that the hydrogen atom at C-3 plays a rôle in such a transformation.     

Biosynthesis of internally branched monomethylalkanes in the cockroach Periplaneta fuliginosa.

Sodium [1-¹⁴C]acetate, sodium [1-¹⁴C]propionate, sodium [2-¹⁴C]propionate, sodium [3-¹⁴C]propionate and sodium [methyl-¹⁴C]methylmalonate were readily incorporated into the cuticular hydrocarbons of nymphal stages of the cockroach Periplaneta fuliginosa both in vivo and in vitro, whereas no incorporation of [methyl-¹⁴C]methionine was observed. The alkanes of the nymphal stages of this insect are 25+% n-alkanes, 14% 3-methylalkanes, and 59+% internally branched monomethylalkanes, principally 13-methylpentacosane. Sodium [1-¹⁴C]acetate was incorporated into each class of alkane at about its percentage composition. In contrast, labeled sodium propionate and sodium methylmalonate were preferentially incorporated into the branched fractions. Radio-gas-liquid chromatography showed that sodium [1-¹⁴C]propionate was incorporated almost exclusively into 3-methyltricosane and 13-methylpentacosane, whereas sodium [1-¹⁴C]acetate was incorporated into each glc peak at about its percentage composition. These data suggest that propionate, incorporated during chain elongation, serves as the branching methyl group donor for both the 3-methyl and the internally branched monomethylalkanes in insects. The location of hydrocarbon synthesis in P. fuliginosa was studied using an in vitro tissue slice system. Excised cuticle slices, with adhering fat body tissue removed, gave good incorporation of labeled substrates into the hydrocarbon fraction. No hydrocarbon synthesis was observed in fat body preparations.