Microbial biomass and activities associated with subsurface environments contaminated with chlorinated hydrocarbons

Soil microcosms and enrichment cultures from subsurface sediments and groundwaters contaminated with trichloroethylene (TCE) were examined. Total lipids, [I‐‘⁴C]acetate incorporation into lipids, and [Me‐³H]thymidine incorporation into DNA were determined in these subsurface environments. In heavily TCE‐contam‐inated zones (greater than 500 mg/L) radioisotopes were not incorporated into lipids or DNA. Radioisotope incorporation occurred in sediments both above and below the TCE plume. Phospholipid fatty acids (PLFA) were not detected, i.e., less than 0.5 pmol/L in heavily contaminated groundwater samples. In less contaminated waters, extracted PLFA concentrations were greater than 100 pmollL and microbial isolates were readily obtained. Degradation of 30–100 mg/L TCE was observed when sediments were amended with a variety of energy sources. Microorganisms in these subsurface sediments have adapted to degrade TCE at concentrations greater than 50 mg/L.     

INCORPORATION OF TRITIATED THYMIDINE DURING VARIOUS STAGES OF LEAF DEVELOPMENT OF XANTHIUM PENNSYLVANICUM

³H-thymidine was incorporated into leaf tissue of Xanthium pennsylvanicum during the stage of active cell division, during cellular differentiation, and into mature cells. Incorporation into nuclear DNA was high in the early stages of development. No nuclear incorporation was found after cessation of cell division. However, significant incorporation could be demonstrated in cytoplasm of differentiating and mature cells. Depending upon the time of growth in the radioisotope and the time of growth after treatment, ³H-thymidine, or its metabolized fraction, was incorporated into the secondary wall depositions of epidermal cells, mesophyll parenchyma cells, xylem cells, and chloroplasts. Autoradiographic technique and liquid scintillation spectrometry were used in these studies. The significance of ³H-thymidine incorporation into various organelles is discussed in relation to cell metabolism and its regulation during leaf development.     

Incorporation of3H-thymidine into DNA and the activity of alkaline phosphatase in zinc-deficient fetal rat brains

The incorporation of³H-thymidine into DNA in the brains of the 17-day and 20-day old rat fetuses was significantly reduced by maternal zinc restriction during pregnancy. The activity of the enzyme thymidine kinase (EC 2.7.1.21) was similarly reduced in the zine-deprived fetal brains on days 14 and 20 of gestation, but not on day 17. Fetal brain alkaline phosphatase (EC 3.1.3.1) was significantly depressed by maternal zinc deprivation on days 17 and 20 of pregnancy. The data suggest an association between thymidine kinase and the reduced incorporation of³H-thymidine into DNA in the brains of 20-day old fetuses but not in animals on day 17. Alkaline phosphatase was however depressed at this stage. The suggestion is made that because of the complexity of brain development, future biochemical studies in this area should concern specific structures in the brain at particular critical stages during neurogenesis.     

Chlorsulfuron inhibition of cell cycle progression and the recovery of G1 arrested cells by Ile and Val

Cultured pea root tips were treated with 14 nM chlorsulfuron (CS) for up to 60 h. The progression of cells from G₁ into S was monitored by measuring³H-thymidine (³H-Thy) incorporation into DNA. Chlorsulfuron treatment decreased the amount of³H-Thy incorporated; however, this amount never reached zero. Short-term labeling experiments indicate that the cells are arrested in a narrow band within G₁. Cell progression recovered from the CS treatment when roots were transferred to either White's medium or White's supplemented with isoleucine (Ile) and valine (Val). Lag time before recovery began in the White's or White's plus Ile and Val medium was 12 and 4 h, respectively. The initial slope of the recovery curves was similar irrespective of the type of recovery media. Increasing the duration of CS treatment did not change the length of the lag or initial slopes of the recovery curves. The level of³H-Thy incorporation decreased with increasing duration of CS treatment for root segments transferred to Ile and Val recovery medium.     

The effect of tritiated thymidine on human chromosomes

Summary The effect of low dosages of³H-thymidine on human chromosomes was investigated. Two established human cell lines, amnion and HeLa cells, were treated for various periods of time 0.125, 0.25, 0.50 or 0.75 μCi/ml of this agent. Chromatid-type aberrations were observed, occurring only if³H-thymidine was incorporated into DNA during the S phase of the cell cycle. The higher the dosage and the longer the³H-thymidine acted on chromatin, the greater the number of aberrations induced. Chromosomes were most sensitive to the effects of³H-thymidine during the early S phase of the cell cycle. This work was supported by the Medical Research Council, Canada Grant MA 1639.     

Studies on follicular growth in the immature rat and hamster: Effect of a single injection of gonadotropin or estrogen on the rate of3H-thymidine incorporation into ovarian DNAin vitro

Initiation of follicular growth by specific hormonal stimuli in ovaries of immature rats and hamsters was studied by determining the rate of incorporation of³H-thymidine into ovarian DNAin vitro. Incorporation was considered as an index of DNA synthesis and cell multiplication. A single injection of pregnant mare serum gonadotropin could thus maximally stimulate by 18 hr³H-thymidine incorporation into DNA of the ovary of immature hamsters. Neutralization of pregnant mare serum gonadotropin by an antiserum to ovine follicle stimulating hormone only during the initial 8–10 hr and not later could inhibit the increase in³H-thymidine incorporationin vitro observed at 18 hr, suggesting that the continued presence of gonadotropin stimulus was not necessary for this response. The other indices of follicular growth monitored such as ovarian weight, serum estradiol and uterine weight showed discernible increase at periods only after the above initial event. A single injection of estrogen (diethyl stilbesterol or estradiol-l7β) could similarly cause 18 hr later, a stimulation in the rate of incorporation of³H-thymidine into DNAin vitro in ovaries of immature rats. The presence of endogenous gonadotropins, however, was obligatory for observing this response to estrogen. Evidence in support of the above was two-fold: (i) administration of antiserum to follicle stimulating hormone or luteinizing hormone along with estrogen completely inhibited the increase in³H-thymidine incorporation into ovarian DNAin vitro; (ii) a radioimmunological measurement revealed following estrogen treatment, the presence of a higher concentration of endogenous follicle stimulating hormone in the ovary. Finally, administration of varying doses of ovine follicle stimulating hormone along with a constant dose of estrogen to immature rats produced a dose-dependent increment in the incorporation of³H-thymidine into ovarian DNAin vitro. These observations suggested the potentiality of this system for developing a sensitive bioassay for follicle stimulating hormone.     

Ovarian maturation in Leucophaea maderae: Juvenile hormone regulation of thymidine uptake into follicle cell DNA

Our research demonstrates that juvenile hormone (JH I) stimulates thymidine incorporation into ovarian follicle cell DNA in the ovoviviparous cockroach, Leucophaea maderae.A rapid, quantitative method for monitoring ³H-thymidine incorporation into ovarian DNA, in vitro, is described. Cultured ovarian tissue from L. maderae incorporates ³H-thymidine into DNA at a linear rate between 16 and 120 min; analysis of the incorporated label revealed at least 98% of it to be in DNA.Using L. maderae females that had been mated 7 days after adult emergence, we monitored the following biochemical phenomena during the 18–22 day period of terminal oöcyte growth: (1) ³H-thymidine incorporation into ovarian DNA: (2) general protein synthesis in fat body; and (3) specific fat body vitellogenin synthesis.Decapitation of mated females with maturing oöcytes arrested both ovarian DNA synthesis and fat body vitellogenin synthesis. Substantial restoration of both types of synthesis was induced by injection of JH I. The resumption of thymidine incorporation into DNA was localized in the follicular epithelium of the terminal oöcyte.In decapitated virgin females, injection of JH I stimulated oöcyte growth and ³H-thymidine incorporation into ovarian DNA. Dose and time response curves indicate that peak stimulation of ovarian DNA synthesis occurred between 72 and 96 hr after administration of a single optimal dose of 25 μg JH I. The concurrent manifestation of ³H-thymidine uptake into ovarian DNA and activity within the fat body indicates that a similar hormonal mode of action may be operative with respect to both tissue types in virgin females.     

Use of the [14C]Leucine Incorporation Technique To Measure Bacterial Production in River Sediments and the Epiphyton

Bacterial production is a key parameter for the understanding of carbon cycling in aquatic ecosystems, yet it remains difficult to measure in many aquatic habitats. We therefore tested the applicability of the [¹⁴C]leucine incorporation technique for the measurement of bulk bacterial production in various habitats of a lowland river ecosystem. To evaluate the method, we determined (i) extraction efficiencies of bacterial protein from the sediments, (ii) substrate saturation of leucine in sediments, the biofilms on aquatic plants (epiphyton), and the pelagic zone, (iii) bacterial activities at different leucine concentrations, (iv) specificity of leucine uptake by bacteria, and (v) the effect of the incubation technique (perfused-core incubation versus slurry incubation) on leucine incorporation into protein. Bacterial protein was best extracted from sediments and precipitated by hot trichloroacetic acid treatment following ultrasonication. For epiphyton, an alkaline-extraction procedure was most efficient. Leucine incorporation saturation occurred at 1 μM in epiphyton and 100 nM in the pelagic zone. Saturation curves in sediments were difficult to model but showed the first level of leucine saturation at 50 μM. Increased uptake at higher leucine concentrations could be partly attributed to eukaryotes. Addition of micromolar concentrations of leucine did not enhance bacterial electron transport activity or DNA replication activity. Similar rates of leucine incorporation into protein calculated for whole sediment cores were observed after slurry and perfused-core incubations, but the rates exhibited strong vertical gradients after the core incubation. We conclude that the leucine incorporation method can measure bacterial production in a wide range of aquatic habitats, including fluvial sediments, if substrate saturation and isotope dilution are determined.     

The uptake of3H-thymidine and its transport inDatura stramonium L.

Exogenous³H-thymidine is absorbed by the primary root of young plants ofDatura stramonium L. and gradually translocated into shoots: following a 3 to 72-h application of³H-thymidine the radioactivity was revealed, using the autoradiographic technique, especially in the region of primary and secondary meristems and in proximity to vascular bundles of the primary root, stem, hypocotyl and leaves. These regions may be considered as the sites of active DNA synthesis. The intensity of incorporation was dependent on the time of plant incubation in labelled thymidine.     

Spores of microorganisms

Preexisting¹⁴C-DAP in vegetative cells ofBacillus cereus is not incorporated into the spores, but is released into the medium after sporogenesis is complete. Exogenous¹⁴C-DAP added to the medium before sporulation is incorporated intensively into the sporangia and practically all of it is taken up by the spores. During sporogenesis, two periods of increased incorporation of¹⁴C into hot TCA-precipitate of cells are found after adding¹⁴C-DAP— one before formation of the spores, when¹⁴C-lysine formed by decarboxylation is incorporated together with¹⁴C-DAP, and one during the “whitening” phase, when any¹⁴C-lysine is no longer incorporated. The incorporation of exogenous¹⁴C-lysine into the sporangial proteins is also markedly elevated during the presporulation phase and at the outset of sporogenesis.     

DNA synthesis in Hela cells at low temperature

It has been proved that ³H-thymidine is incorporated into DNA of HeLa cells cultured at 4 °C and its labelling distribution in DNA is homogeneous. This incorporation of ³H-thymidine increased with the duration of incubation and only 30% of the cell population was labelled after 12 h. When synchronous cell populations were used, the rate and extent of DNA synthesis at 4 °C was proportional to the relative number of cells in S phase at that temperature. Thus, cellular labelling at 4 °C does not result from a non-specific absorption phenomenon, but indicates a DNA synthesis process.     

Effect of 2,3-Dinitrilo-1,4-dithia-9,10-anthraquinone onMycobacterium smegmatis

2,3-Dinitrilo-1,4-dithia-9,10-anthraquinone (DDA) is an effective inhibitor of respiration of intact cells ofMycobacterium smegmatis in the presence of glucose, glycerol, pyruvate, acetate and other citric acid cycle intermediates or substrates associate d with this cycle (glutamate, asparagine). DDA inhibits the incorporation of both¹⁴C-leucine and¹⁴C-adenine into appropriate macromolecules ofM. smegmatis (TCA-precipitable fractions), and causes a drop in the incorporated activity ofU-¹⁴C-glycine or its degradation products in all the cell fractions studied (lipids, RNA, DNA, proteins). DDA suppresses the growth ofM. smegmatis probably through an interference with the cell energy-carbon metabolism.     

H-thymidine incorporation into nuclear DNA of leaf cells

The incorporation of ³H-thymidine into nuclear DNA of leaf cells of Nanthium pennsylvanicum was studied as a function of concentration and specific activity of the radioisotope. From the assessment of the average number of grains per nucleus and the percent of labeled nuclei, it was concluded that the incorporation was a linear function of concentration of the exogenous radioisotopic solution and a logarithmic function of the incubation time. Ten microcuries per milliliter on the average yielded 20% of labeled nuclei with 18 grains per nucleus. Seven-fold increase in concentration only doubled the amount of ³H-thymidine incorporated. The lamina regions near the vein incorporated a significantly greater amount of the radioisotope than the lamina region at some distance from the vein. The specific activities of 2, 3.35, 6.7 and 15.3 c/mmole had no effect upon the amount of ³H-thymidine incorporated, if the amount of microcuries of the incubation solution was the same in each activity. Considering the total number of molecules, the estimated rates of incorporation indicated that at the activity of 2 c/mmole, the system operated with about 7 times higher rates as compared with the activity of 15.3 c/mmole.     

Metabolism ofAedes aegypti cells grown in vitro. 1. Incorporation of3H-uridine and14C-leucine

Summary Metabolic activity ofA. aegypti cells grown in vitro has been studied by incorporation of³H-uridine and¹⁴C-leucine. “Chase” experiments with unlabeled precursors, and the use of actinomycin D and puromycin, showed that³H-uridine was incorporated into cellular RNA, and that¹⁴C-leucine was incorporated into protein of these cells. Incorporation of³H-uridine was inhibited when actinomycin D was used at a concentration of 10 μg/ml, and¹⁴C-leucine incorporation was inhibited to the same extent by puromycin at a concentration of 100 μg/ml medium. Contribution No. 148.     

Incorporation of extracellular 8-oxodG into DNA and RNA requires purine nucleoside phosphorylase in MCF-7 cells

7,8-Dihydro-8-oxo-2′-deoxyguanosine (8-oxodG) is a well-known marker of oxidative stress. We report a mechanistic analysis of several pathways by which 8-oxodG is converted to nucleotide triphosphates and incorporated into both DNA and RNA. Exposure of MCF-7 cells to [¹⁴C]8-oxodG combined with specific inhibitors of several nucleotide salvage enzymes followed with accelerator mass spectrometry provided precise quantitation of the resulting radiocarbon-labeled species. Concentrations of exogenously dosed nucleobase in RNA reached one per 10⁶ nucleotides, 5–6-fold higher than the maximum observed in DNA. Radiocarbon incorporation into DNA and RNA was abrogated by Immucillin H, an inhibitor of human purine nucleoside phosphorylase (PNP). Inhibition of ribonucleotide reductase (RR) decreased the radiocarbon content of the DNA, but not in RNA, indicating an important role for RR in the formation of 8-oxodG-derived deoxyribonucleotides. Inhibition of deoxycytidine kinase had little effect on radiocarbon incorporation in DNA, which is in contrast to the known ability of mammalian cells to phosphorylate dG. Our data indicate that PNP and RR enable nucleotide salvage of 8-oxodG in MCF-7 cells, a previously unrecognized mechanism that may contribute to mutagenesis and carcinogenesis.     

The time course of xylem differentiation and its relation to deoxyribonucleic Acid synthesis in cultured coleus stem segments

The relationship between DNA synthesis and wound xylem differentiation was investigated in cultured stem segments of Coleus blumei. The addition of 50 micrograms of indoleacetic acid per liter to the culture medium resulted in a 400 to 500% increase in the number of wound vessel members formed in 7 days. However, the time course of wound vessel member formation was similar in segments cultured in the presence and absence of auxin. In either case, no wound vessel members appeared before the 3rd day of culture, while the majority of wound vessel members appeared on the 4th and 5th days of culture. ³H-Thymidine incorporation into DNA was used to measure changes in the DNA synthetic activity of the tissues during the culture period. Comparatively little ³H-thymidine incorporation occurred during the 1st day of culture. Maximum ³H-thymidine incorporation was observed on the 2nd day of culture, 2 days before the peak period of xylem differentiation. The rate of incorporation of ³H-thymidine into DNA decreased with increasing time in culture after the 2nd day. Auxin at 50 micrograms per liter had no effect on the time course of ³H-thymidine incorporation, although somewhat more ³H-thymidine was incorporated into DNA throughout the culture period in the presence of auxin. The magnitude of this effect was small when compared to the effect of auxin on xylem differentiation. The antimetabolite 5-fluorodeoxyuridine was shown to block DNA synthesis in the cultured stem segments. When the tissues were isolated on media containing 10⁻⁶m 5-fluorodeoxyuridine, wound vessel member differentiation was inhibited by approximately 80%, in both the presence and absence of auxin. Thymidine at 10⁻⁵m completely overcame the 5-fluorodeoxyuridine inhibition of wound vessel member formation. 5-Fluorodeoxyuridine was effective in blocking xylogenesis only when this substance was supplied to the tissues during the early part of the culture period. 5-Fluorodeoxyuridine had no effect on xylem differentiation when it was applied after the 3rd day of culture.     

Measurement of bacterial growth rates on the rhizoplane using 3H-thymidine incorporation into DNA

Bacterial growth rates on the rhizoplane of rape seedlings grown in sand were determined using ³H-thymidine incorporation into DNA. Axenic roots incorporated thymidine into DNA, which had to be subtracted from values for roots with associated bacteria. Thymidine incorporation into rhizoplane bacterial DNA ranged between 0.6 and 1.4 pmol thymidine h^–1 root^–1 for 6 to 26-day-old plants. Using a conversion factor, the turnover time of bacteria was calculated to decrease from 9.2 h for 6-day-old plants to 160h for 26-day-old plants. A similar value was found for rhizosphere bacteria of plants grown for 26 days in natural soil.     

Einfluss von hydroxyharnstoff auf die DNA-synthese und keimung der samen vonAgrostemma githago sowieVaccaria pyramidata

Application of hydroxyurea to intact seeds ofAgrostemma githago as well asVaccaria pyramidata causes a strong inhibition of radicular³H-thymidine incorporation into DNA, but germination rate is only insignificantly reduced. The seedling development, however, is strongly affected. Consequently, DNA synthesis is not a prerequisite for the radicle protrusion.     

DNA SYNTHESIS,CELL DIVISION,AND CELL DIFFERENTIATION DURING LEAF DEVELOPMENT OF XANTHIUM PENNSYLVANICUM

Leaf growth consists of two basic processes, cell division and cell enlargement. DNA synthesis is an integral part of cell division and can be studied with autoradiographic techniques and incorporation of some labeled precursor. Studies were made on the synthesis of nuclear DNA through incorporation of ³H-thymidine in various parts of the lamina during the entire course of leaf development of Xanthium pennsylvanicum. The time course analysis of DNA synthesis was correlated with cell division and rates of cell enlargement. Significant differences in ³H-thymidine incorporation were found in various parts of the lamina. Cell division and DNA synthesis were highest in the early stages of development. Since no ³H-thymidine was incorporated after cessation of cell division (LPI 2.8) in the leaf lamina, it appears that DNA synthesis is not needed for enlargement and differentiation of Xanthium cells. Rates of cell enlargement were negligible in the early development and reached their maximum after cessation of mitoses, between plastochron ages (LPI) 3 and 4. Cells matured between LPI's 5 and 6. Enzymatic activity was correlated with cell division and cell differentiation at various stages of leaf development.     

Enzymatic synthesis of 5-bromo-2’-deoxyuridine-2-14C and of 5-iodo-2’-deoxyuridine-2-14C and their incorporation into deoxyribonucleic acid (Allium cepa)

5-Bromo-2’-deoxyuridine-2-¹⁴C was prepared from 5-bromouracil-2-¹⁴C and 2’-de-oxyguanosine using trans-N-deoxyribosylase fromLactobacillus helveticus and incorporated into DNA ofAllium cepa roots. After isolating the DNA and hydrolyzing it enzymatically to deoxynucleoside-5’-phosphates a radioactive nucleotide was detected which yielded 5-bromo-2’-deoxyuridine-2-¹⁴C on enzymatic dephosphorylation. The incorporation of 5-iodo-2’-deoxy-uridine-2-¹⁴C was followed only by microautoradiography.