[Detection of Gamma-Ray Irradiation History of Dried Plant-Based Foods via the Analysis of 5,6-Dihydrothymidine].

Irradiation is widely used worldwide to sterilize and kill insects in food, and prevent the germination of agricultural products. However, in Japan, food irradiation is prohibited except to prevent potato sprouting. Herein, 5,6-dihydrothymidine (DHdThd) residue-a damaged nucleoside generated from the thymidine (dThd) residue in DNA contained in food upon irradiation-was used as a detection indicator. Eight dried plant-based food samples were gamma ray-irradiated in the range from 3.2 to 8.3 kGy. Subsequently, DNA was extracted from the irradiated sample and digested into nucleosides by the three enzymes, and the test solution was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Evidently, in all samples, the concentration ratio of DHdThd to dThd in the test solution (DHdThd/dThd) was dependent on the irradiation dose; moreover, during storage under frozen conditions for at least 890 d post-irradiation, this concentration ratio was equal to that immediately after irradiation. The irradiation histories of the eight types of dried plant-based food samples were correctly detected.

Surface-enhanced Raman scattering of DNA bases using frozen silver nanoparticle dispersion as a platform.

Raman spectroscopy is a powerful method to characterize molecules in various media. Although surface-enhanced Raman scattering (SERS) is often employed to compensate for the intrinsically poor sensitivity of Raman spectroscopy, there remain serious tasks, such as simple preparations of SERS substrates, sensitivity control, and reproducible measurements. Here, we propose freezing as an efficient way to overcome these problems in SERS measurements using DNA bases as model targets. Solutes are expelled from ice crystals and concentrated in the liquid phase upon freezing. Silver nanoparticles (AgNPs) are also concentrated in the liquid phase to aggregate with Raman target analytes. The SERS signal intensity is maximized when the AgNP concentration exceeds the critical aggregation value. Freezing allows up to 5000 times enhancements of the SERS signal. Thus, an efficient SERS platform is prepared by simple freezing. The simultaneous detection of four DNA bases effectively eliminates variations of signal intensities and allows the reliable determination of concentration ratios.

Quantitative LC-MS/MS analysis of 5-hydroxymethyl-2'-deoxyuridine to monitor the biological activity of J-binding protein.

Base J replaces 1% of thymine in most kinetoplastid flagellates, and is implicated in transcription regulation. Base J is synthesized in two steps: first, a thymine base in DNA is converted to 5-hydroxymethyluracil by J-binding proteins (JBP1, JBP2); secondly, a glucosyl transferase glycosylates the 5-hydroxymethyluracil to form base J. Here, we present a highly sensitive and selective LC-MS/MS method to quantify the in vitro JBP1 activity on synthetic oligonucleotide substrates. The method demonstrated successful to support biochemical studies of JBPs and can be used as a template for additional JBP activity studies or for inhibitor screening in the future.

Determination of genomic N3-methylthymidine in human cancer cells treated with nitrosamines using capillary electrophoresis with laser-induced fluorescence.

Methylating substances alter DNA by forming N3-methylthymidine (N3mT), a mutagenic base modification. To develop a sensitive analytical method for the detection of N3mT in DNA based on capillary electrophoresis with laser-induced fluorescence detection (CE-LIF), we synthesized the N3mT-3'-phosphate as a chemical standard. The limit of detection was 1.9 amol of N3mT, which corresponds to one molecule of N3mT per 1000 normal nucleotides or 0.1%. With this method, we demonstrated that the carcinogenic nitrosamine N'-nitrosonornicotine (NNN) induced N3mT in the human lung cancer cell line A549. Treatment with NNN also caused an elevated degree of 5-hydroxymethylcytidine (5hmdC) in DNA, while the methylation degree (i.e. 5-methylcytidine; 5mdC) stayed constant. According to our data, NNN could, via yet unknown mechanisms, play a role in the formation of N3mT as well as 5hmdC. In this study we have developed a new sensitive analytical method using CE-LIF for the simultaneous detection of the three DNA modifications, 5mdC, 5hmdC and N3mT.

Drawbacks of the use of cotrimoxazole in foreign-body infections.

INTRODUCTION: The anti-staphylococcal efficacy of cotrimoxazole in the setting of difficult-to-treat infections seems to be compromised by large amounts of pus and devitalized tissue, and, therefore, high levels of thymidine. Our objective was to evaluate the activity of cotrimoxazole against a staphylococcal foreign-body infection experimental model, which also yields significant quantities of thymidine. MATERIAL AND METHODS: We used a rat tissue-cage model of infection (with high inherent thymidine levels) caused by a strain of methicillin-susceptible Staphylococcus aureus (MSSA; ATCC 29213). MIC values were determined (microdilution method) and compared in the presence or absence of tissue-cage fluid samples. RESULTS: The inefficacy of cotrimoxazole was found to be similar to that of the control group. The MIC of cotrimoxazole was 4-8 fold higher in the presence of rat tissue-cage fluid. CONCLUSIONS: The inefficacy of cotrimoxazole in our foreign-body infection model by MSSA, and the probable negative impact of the presence of thymidine on its efficacy, challenge the use of this drug in acute phases of foreign-body infections. It should be reserved as an alternative treatment when the infection is more controlled.

Rapid and Reliable Method for Determining Irradiation Histories of Ground Beef and Prawns by Measuring 5,6-Dihydrothymidine.

A rapid and reliable method for determining irradiation histories of ground beef and prawns was developed on the basis of a method for determining the irradiation history of beef liver by liquid chromatography-tandem mass spectrometry (LC-MS/MS) of 5,6-dihydrothymidine (DHdThd). Improvements in the method included the following: (1) 50% ethanol precipitation in the DNA extraction step was conducted before the RNase step, (2) snake venom phosphodiesterase I was used for DNA digestion to boost liberation of DHdThd, and (3) a matrix-matched calibration curve was used for determining DHdThd by LC-MS/MS analysis. This method successfully determined irradiation histories of ground beef and prawns. Furthermore, a close correlation between the formation of DHdThd and 2-alkylcyclobutanones, which are an established index of irradiation histories, was observed in ground beef. DHdThd in DNA could be a promising candidate for a new index of irradiation histories of various foods.

Determination of irradiation histories of raw beef livers using liquid chromatography-tandem mass spectrometry of 5,6-dihydrothymidine.

A method for detecting irradiation histories of raw beef livers was developed by measuring 5,6-dihydrothymidine (DHdThd) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Liver DNA was extracted using phenol-chloroform extraction followed by precipitation in 50% ethanol. DNA was then enzymatically digested and nucleosides were purified using an OASIS MCX column. DHdThd and thymidine (dThd) contents of resulting test solutions were analyzed using LC-MS/MS. DHdThd was detected specifically after γ-irradiation. Concentration ratios of DHdThd to dThd in the test solutions increased dose-dependently after irradiation at 1.0-11.3kGy, which included the practical dose for sterilization of 2-7kGy. Dose-response curves from beef livers of individual animals almost overlapped. Thus, this method is a candidate for the detection of irradiation histories of foods from which DNA can be extracted.

A simple histological technique to improve immunostaining when using DNA denaturation for BrdU labelling.

The typical immunohistochemistry technique used to reveal 5-bromo-2'-deoxyuridine (BrdU) incorporation requires denaturation of the DNA by heat and acid to permeabilize the cell nucleus. This treatment can damage tissue and reduce the antigenicity of several proteins, which then leads to weak immunostaining and/or false negatives. We show that an overnight post-fixation step following immunohistochemistry for antigens of interest protects immunostaining during the acid/heat denaturation treatment for subsequent BrdU staining. We used this technique to study the differentiation of recently divided oligodendrocyte progenitor cells in NG2CreER:EYFP reporter mice. We used a GFP anti-EYFP antibody to maximize visualization of the EYFP-containing oligodendrocyte progenitor cells, Olig1, and GST-pi to confirm the cell phenotype. Immunostaining for GFP, Olig1, and GST-pi is reduced by DNA denaturation. We found that incorporating a post-fixation step after double immunostaining for GFP/Olig1 and GFP/GST-pi prior to DNA denaturation prevented the fading and false negatives associated with this treatment. This simple addition to BrdU immunohistochemistry protocols extends the range of proteins that can be detected in combination with BrdU, along with the number of antibodies that can be used successfully in the study of cell proliferation.

Detection and simultaneous quantification of three smoking-related ethylthymidine adducts in human salivary DNA by liquid chromatography tandem mass spectrometry.

Smoking cigarette increases levels of certain ethylated DNA adducts in certain tissues and urine. Cigarette smoking is a major risk factor of various cancers and DNA ethylation is involved in smoking-related carcinogenesis. Among the ethylated DNA adducts, O(2)-ethylthymidine (O(2)-edT) and the promutagenic O(4)-ethylthymidine (O(4)-edT) are poorly repaired and they can accumulate in vivo. Using an accurate, highly sensitive, and quantitative assay based on stable isotope dilution nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS), O(2)-edT, N(3)-edT (N(3)-ethylthymidine), and O(4)-edT adducts in human salivary DNA were simultaneous detected and quantified. Saliva is easily accessible and available and it can be a potential target in searching for noninvasive biomarkers. Under the highly selected reaction monitoring (H-SRM) mode, salivary samples from 20 smokers and 13 nonsmokers were analyzed. Starting with 50 µg of DNA isolated from about 3.5 mL of saliva, levels of O(2)-edT, N(3)-edT, and O(4)-edT in 20 smokers' salivary DNA samples were 5.3±6.2, 4.5±5.7, 4.2±8.0 in 10(8) normal nucleotides, respectively, while those in 13 nonsmokers were non-detectable. In addition, statistically significant correlations (p<0.0001) were observed between levels of O(2)-edT and N(3)-edT (γ=0.7388), between levels of O(2)-edT and O(4)-edT (γ=0.8839), and between levels of N(3)-edT, and O(4)-edT (γ=0.7835). To the best of our knowledge, this is the first report of detection and quantification of these three ethylthymidine adducts in human salivary DNA, which might be potential biomarkers for exposure to ethylating agents and possibly for cancer risk assessment.

Synthesis of a probe for monitoring HSV1-tk reporter gene expression using chemical exchange saturation transfer MRI.

In experiments involving transgenic animals or animals treated with transgenic cells, it is important to have a method to monitor the expression of the relevant genes longitudinally and noninvasively. An MRI-based reporter gene enables monitoring of gene expression in the deep tissues of living subjects. This information can be co-registered with detailed high-resolution anatomical and functional information. We describe here the synthesis of the reporter probe, 5-methyl-5,6-dihydrothymidine (5-MDHT), which can be used for imaging of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene expression in rodents by MRI. The protocol also includes data acquisition and data processing routines customized for chemical exchange saturation transfer (CEST) contrast mechanisms. The dihydropyrimidine 5-MDHT is synthesized through a catalytic hydrogenation of the 5,6-double bond of thymidine to yield 5,6-dihydrothymidine, which is methylated on the C-5 position of the resulting saturated pyrimidine ring. The synthesis of 5-MDHT can be completed within 5 d, and the compound is stable for more than 1 year.

Replication across regioisomeric ethylated thymidine lesions by purified DNA polymerases.

Causal links exist between smoking cigarettes and cancer development. Some genotoxic agents in cigarette smoke are capable of alkylating nucleobases in DNA, and higher levels of ethylated DNA lesions were observed in smokers than in nonsmokers. In this study, we examined comprehensively how the regioisomeric O(2)-, N3-, and O(4)-ethylthymidine (O(2)-, N3-, and O(4)-EtdT, respectively) perturb DNA replication mediated by purified human DNA polymerases (hPols) η, κ, and ι, yeast DNA polymerase ζ (yPol ζ), and the exonuclease-free Klenow fragment (Kf(-)) of Escherichia coli DNA polymerase I. Our results showed that hPol η and Kf(-) could bypass all three lesions and generate full-length replication products, whereas hPol ι stalled after inserting a single nucleotide opposite the lesions. Bypass conducted by hPol κ and yPol ζ differed markedly among the three lesions. Consistent with its known ability to efficiently bypass the minor groove N(2)-substituted 2'-deoxyguanosine lesions, hPol κ was able to bypass O(2)-EtdT, though it experienced great difficulty in bypassing N3-EtdT and O(4)-EtdT. yPol ζ was only modestly blocked by O(4)-EtdT, but the polymerase was strongly hindered by O(2)-EtdT and N3-EtdT. LC-MS/MS analysis of the replication products revealed that DNA synthesis opposite O(4)-EtdT was highly error-prone, with dGMP being preferentially inserted, while the presence of O(2)-EtdT and N3-EtdT in template DNA directed substantial frequencies of misincorporation of dGMP and, for hPol ι and Kf(-), dTMP. Thus, our results suggested that O(2)-EtdT and N3-EtdT may also contribute to the AT → TA and AT → GC mutations observed in cells and tissues of animals exposed to ethylating agents.

Quantitation of pyridyloxobutyl-DNA adducts in tissues of rats treated chronically with (R)- or (S)-N'-nitrosonornicotine (NNN) in a carcinogenicity study.

We quantified DNA adducts resulting from 2'-hydroxylation of enantiomers of the tobacco-specific nitrosamine N'-nitrosonornicotine (NNN) in tissues of male F-344 rats after 10, 30, 50, and 70 weeks of treatment with 14 ppm in the drinking water. These rats were in subgroups of a carcinogenicity study in which (S)-NNN was highly tumorigenic in the oral cavity and esophagus, while (R)-NNN was relatively weakly active. DNA adducts were quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry in six tissues: oral mucosa, esophageal mucosa, nasal respiratory mucosa, nasal olfactory mucosa, liver, and lung. O²-[4-(3-Pyridyl)-4-oxobut-1-yl]thymidine (O²-POB-dThd, 7) and 7-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine (7-POB-dGuo, 8), the latter as 7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine (7-POB-Gua, 11), were detected at each time point in each tissue. In the target tissues for carcinogenicity, oral mucosa and esophageal mucosa, levels of 7-POB-Gua (11) and O²-POB-dThd (7) were similar, or 11 predominated, while in all other tissues at all time points for both enantiomers, 7 was clearly present in greater amounts than 11. Total measured DNA adduct levels in esophageal mucosa and oral mucosa were higher in rats treated with (S)-NNN than (R)-NNN. The highest adduct levels were found in the nasal respiratory mucosa. DNA adducts generally persisted in all tissues without any sign of substantial decreases throughout the 70 week time course. The results of this study suggest that inefficient repair of 7-POB-dGuo (8) in the rat oral cavity and esophagus may be important in carcinogenesis by NNN and support the development of these DNA adducts as potential biomarkers of NNN metabolic activation in people who use tobacco products.

Quantitative assessment of Tet-induced oxidation products of 5-methylcytosine in cellular and tissue DNA.

Recent studies showed that Ten-eleven translocation (Tet) family dioxygenases can oxidize 5-methyl-2'-deoxycytidine (5-mdC) in DNA to yield the 5-hydroxymethyl, 5-formyl and 5-carboxyl derivatives of 2'-deoxycytidine (5-HmdC, 5-FodC and 5-CadC). 5-HmdC in DNA may be enzymatically deaminated to yield 5-hydroxymethyl-2'-deoxyuridine (5-HmdU). After their formation at CpG dinucleotide sites, these oxidized pyrimidine nucleosides, particularly 5-FodC, 5-CadC, and 5-HmdU, may be cleaved from DNA by thymine DNA glycosylase, and subsequent action of base-excision repair machinery restores unmethylated cytosine. These processes are proposed to be important in active DNA cytosine demethylation in mammals. Here we used a reversed-phase HPLC coupled with tandem mass spectrometry (LC-MS/MS/MS) method, along with the use of stable isotope-labeled standards, for accurate measurements of 5-HmdC, 5-FodC, 5-CadC and 5-HmdU in genomic DNA of cultured human cells and multiple mammalian tissues. We found that overexpression of the catalytic domain of human Tet1 led to marked increases in the levels of 5-HmdC, 5-FodC and 5-CadC, but only a modest increase in 5-HmdU, in genomic DNA of HEK293T cells. Moreover, 5-HmdC is present at a level that is approximately 2-3 and 3-4 orders of magnitude greater than 5-FodC and 5-CadC, respectively, and 35-400 times greater than 5-HmdU in the mouse brain and skin, and human brain. The robust analytical method built a solid foundation for dissecting the molecular mechanisms of active cytosine demethylation, for measuring these 5-mdC derivatives and assessing their involvement in epigenetic regulation in other organisms and for examining whether these 5-mdC derivatives can be used as biomarkers for human diseases.

Comparison of supercritical fluid chromatography and reverse phase liquid chromatography for the impurity profiling of the antiretroviral drugs lamivudine/BMS-986001/efavirenz in a combination tablet.

Dual and triple combinations of antiretroviral drugs are a cornerstone of human immunodeficiency virus type 1 (HIV-1) treatment. Supercritical fluid chromatography (SFC) and reverse phase liquid chromatography (RPLC) methods have been developed for the impurity profiling of a prototype combination tablet containing three such drugs: lamivudine, BMS-986001 and efavirenz. Separation by SFC was achieved using a Princeton 2-ethyl pyridine stationary phase and a mobile phase B consisting of methanol with 10 mM ammonium acetate and 0.1% isopropyl amine. This combination of mobile phase additives was required for both the separation of minor components and to minimize peak tailing of the active pharmaceutical ingredients (APIs). Separation by RPLC was achieved using a Discovery HSF5 stationary phase and a mobile phase consisting of 10 mM ammonium acetate, pH 5.5 and methanol. Mobile phase gradient elution was employed in each case to elute components with a wide range of polarities. Both these methods were found to have advantages and disadvantages. Out of the three APIs and 13 possible impurity/degradation products selected, all were resolved by RPLC. By SFC, 15 peaks were resolved with one co-eluting pair and a high degree of orthogonality was achieved relative to RPLC. A more even distribution of peaks across the separation space, a non-sloping baseline and fewer system peaks were significant advantages associated with the SFC method. Particular attention had to be paid to optimizing the reverse phase diluent strength/initial mobile phase composition to avoid distortion of the peak shapes for early eluting components. This was not an issue with SFC, as the diluent of choice (methanol) was also the solvent of choice (in combination with ≤20% water) for the dissolution of the triple combination tablet. As with RPLC, SFC was found to exhibit the required sensitivity for successful quantitation of potential impurities/degradation products at the 0.05-0.1 area% level.

Validation of a HPLC method for the measurement of erythrocyte encapsulated thymidine phosphorylase (EE-TP) activity.

A sensitive and simple reverse-phase high performance liquid chromatographic (HPLC) assay has been validated for the determination of thymine as a measure of thymidine phosphorylase activity encapsulated in erythrocytes (EE-TP), a formulation which is under clinical development as an enzyme replacement therapy for the treatment of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). Diluted erythrocyte lysates were incubated in 100mM sodium phosphate buffer and 10mM thymidine at 37°C for 10min and the reaction stopped with 40% trichloroacetic acid. Following centrifugation, the supernatant was washed with water saturated diethyl ether, and injected onto a Spherisorb C(18) column (125mm×4.6mm, 5µm), with a mobile phase (40mM ammonium acetate, 5mM tetrabutyl ammonium hydrogen sulphate, pH 2.70) delivered at a flow rate of 1.0ml/min and run time of 8min. Ultraviolet detection (UV) was employed at 254nm. The method was linear in the range of 5-500nmol/ml (r(2)=0.992), specific with intra- and inter-day precisions of <9.6 and accuracies within ±20%. Limits of detection and quantification were 1.2nmol/ml and 10nmol/ml, respectively. The method was applied to quantify thymidine phosphorylase activity in samples of in-process controls and batches of EE-TP manufactured for clinical use.

Detection of rapidly exchanging compounds using on-resonance frequency-labeled exchange (FLEX) transfer.

Frequency-labeled exchange transfer is a promising MRI technique for labeling and detecting exchanging protons of low-concentration solutes through the water signal. Early frequency-labeled exchange studies have used off-resonance excitation-based labeling schemes that are well suited to study rapidly exchanging protons or molecules far from the water resonance (e.g., water in paramagnetic contrast agents) or slowly exchanging protons close to the water resonance (e.g., some amide protons). However, off-resonance labeling is not efficient for rapidly exchanging protons close to water. Here, we show that a new frequency-labeled exchange labeling scheme with excitation pulses applied on the water resonance gives much higher exchange contrast for rapidly exchanging protons resonating close to the water resonance frequency. This labeling scheme is particularly suited for studying rapidly exchanging hydroxyl, amine, and imino protons in diamagnetic chemical exchange saturation transfer agents.

Analysis of ethylated thymidine adducts in human leukocyte DNA by stable isotope dilution nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry.

Studies showed that levels of ethylated DNA adducts in certain tissues and urine are higher in smokers than in nonsmokers. Because cigarette smoking is a major risk factor of various cancers, DNA ethylation might play an important role in cigarette smoke-induced cancer formation. Among the ethylated DNA adducts, O(2)-ethylthymidine (O(2)-edT) and O(4)-ethylthymidine (O(4)-edT) are poorly repaired and are accumulated in the body. In addition, O(4)-edT possesses promutagenic properties. In this study, we have developed a highly sensitive, accurate, and quantitative assay for simultaneous detection and quantification of O(2)-edT, N(3)-edT (N(3)-ethylthymidine), and O(4)-edT adducts by isotope dilution nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS). Under the highly selected reaction monitoring (H-SRM) mode, the detection limit of O(2)-edT, N(3)-edT, and O(4)-edT injected on-column was 5.0, 10, and 10 fg, respectively. The quantification limit for the entire assay was 50, 100, and 100 fg of O(2)-edT, N(3)-edT, and O(4)-edT, respectively, corresponding to 1.1, 2.3, and 2.3 adducts in 10(9) normal nucleotides, respectively, starting with 50 µg of DNA (from 1.5-2.0 mL of blood). Levels of O(2)-edT, N(3)-edT, and O(4)-edT in 20 smokers' leukocyte DNA were 44.8 ± 52.0, 41.1 ± 43.8, 48.3 ± 53.9 in 10(8) normal nucleotides, while those in 20 nonsmokers were 0.19 ± 0.87, 4.1 ± 13.3, and 1.0 ± 2.9, respectively. Levels of O(2)-edT, N(3)-edT, and O(4)-edT in human leukocyte DNA are all significantly higher in smokers than in nonsmokers, with pvalues of 0.0004, 0.0009, and 0.0004, respectively. Furthermore, levels of O(2)-edT show a statistically significant association (γ = 0.4789, p = 0.0327) with the smoking index in smokers. In the 40 leukocyte DNA samples, the extremely significant statistical correlations (p < 0.0001) are observed between levels of O(2)-edT and O(4)-edT (γ = 0.9896), between levels of O(2)-edT and N(3)-edT (γ = 0.9840), and between levels of N(3)-edT and O(4)-edT (γ = 0.9901). To our knowledge, this is the first mass spectrometry-based assay for ethylated thymidine adducts. Using this assay, the three ethylated thymidine adducts were detected and quantified for the first time. Therefore, this highly sensitive, specific, and accurate assay should be clinically feasible for simultaneous quantification of the three ethylated thymidine adducts as potential biomarkers for exposure to ethylating agents and for cancer risk assessment.

Characterization of a ViI-like phage specific to Escherichia coli O157:H7.

Phage vB_EcoM_CBA120 (CBA120), isolated against Escherichia coli O157:H7 from a cattle feedlot, is morphologically very similar to the classic phage ViI of Salmonella enterica serovar Typhi. Until recently, little was known genetically or physiologically about the ViI-like phages, and none targeting E. coli have been described in the literature. The genome of CBA120 has been fully sequenced and is highly similar to those of both ViI and the Shigella phage AG3. The core set of structural and replication-related proteins of CBA120 are homologous to those from T-even phages, but generally are more closely related to those from T4-like phages of Vibrio, Aeromonas and cyanobacteria than those of the Enterobacteriaceae. The baseplate and method of adhesion to the host are, however, very different from those of either T4 or the cyanophages. None of the outer baseplate proteins are conserved. Instead of T4's long and short tail fibers, CBA120, like ViI, encodes tail spikes related to those normally seen on podoviruses. The 158 kb genome, like that of T4, is circularly permuted and terminally redundant, but unlike T4 CBA120 does not substitute hmdCyt for cytosine in its DNA. However, in contrast to other coliphages, CBA120 and related coliphages we have isolated cannot incorporate 3H-thymidine (3H-dThd) into their DNA. Protein sequence comparisons cluster the putative "thymidylate synthase" of CBA120, ViI and AG3 much more closely with those of Delftia phage φW-14, Bacillus subtilis phage SPO1, and Pseudomonas phage YuA, all known to produce and incorporate hydroxymethyluracil (hmdUra).

Attenuation of oxidative stress, inflammation and early markers of tumor promotion by caffeic acid in Fe-NTA exposed kidneys of Wistar rats.

Iron nitrilotriacetate (Fe-NTA), a chief environmental pollutant, is known for its extensive toxic manifestations on renal system. In the present study, caffeic acid, one of the most frequently occurring phenolic acids in fruits, grains, and dietary supplements was evaluated for its shielding effect against the Fe-NTA-induced oxidative, inflammatory, and pathological damage in kidney. Fe-NTA was administered (9 mg Fe/kg body weight) intraperitoneally to the Wistar male rats on 20th day while caffeic acid was administered orally (20 and 40 mg/kg body weight) before administration of Fe-NTA. The intraperitoneal administration of Fe-NTA-enhanced lipid peroxidation, xanthine oxidase, and hydrogen peroxide generation with reduction in renal glutathione content, antioxidant enzymes, viz., catalase, glutathione peroxidase, and glutathione reductase. A sharp elevation in the levels of myloperoxidase, blood urea nitrogen (BUN), and serum creatinine has also been observed. Tumor promotion markers viz., ornithine decarboxylase (ODC) and [(3)H] thymidine incorporation into renal DNA were also significantly increased. Treatment of rats orally with caffeic acid (20 and 40 mg/kg body weight) resulted in a significant decrease in xanthine oxidase (P < 0.001), lipid peroxidation (P < 0.001), γ-glutamyl transpeptidase (P < 0.01), and H(2)O(2) (P < 0.01). There was significant recovery of renal glutathione content (P < 0.001) and antioxidant enzymes (P < 0.001). There was also a reversal in the enhancement of renal ODC activity, DNA synthesis, BUN, and serum creatinine (P < 0.001). All these changes were supported by histological observations. The results indicate that caffeic acid may be beneficial in ameliorating the Fe-NTA-induced oxidative damage and tumor promotion in the kidney of rats.

Growth of saprotrophic fungi and bacteria in soil.

Bacterial and fungal growth rate measurements are sensitive variables to detect changes in environmental conditions. However, while considerable progress has been made in methods to assess the species composition and biomass of fungi and bacteria, information about growth rates remains surprisingly rudimentary. We review the recent history of approaches to assess bacterial and fungal growth rates, leading up to current methods, especially focusing on leucine/thymidine incorporation to estimate bacterial growth and acetate incorporation into ergosterol to estimate fungal growth. We present the underlying assumptions for these methods, compare estimates of turnover times for fungi and bacteria based on them, and discuss issues, including for example elusive conversion factors. We review what the application of fungal and bacterial growth rate methods has revealed regarding the influence of the environmental factors of temperature, moisture (including drying/rewetting), pH, as well as the influence of substrate additions, the presence of plants and toxins. We highlight experiments exploring the competitive and facilitative interaction between bacteria and fungi enabled using growth rate methods. Finally, we predict that growth methods will be an important complement to molecular approaches to elucidate fungal and bacterial ecology, and we identify methodological concerns and how they should be addressed.