Interactions of DNA with clay minerals and soil colloidal particles and protection against degradation by DNase

Adsorption, desorption, and degradation by nucleases of DNA on four different colloidal fractions from a Brown soil and clay minerals were studied. The adsorption of DNase I and the structures of native DNA, adsorbed and desorbed, were also investigated by Fourier Transform Infrared (FTIR), circular dichroism (CD), and fluorescence spectroscopy, to determine the protection mechanism of DNA molecules by soil colloids and minerals against enzymatic degradation. Kaolinite exhibited the highest adsorption affinity for DNA among the examined soil colloids and clay minerals. In comparison with organomineral complexes (organic clays), DNA was tightly adsorbed by H2O2-treated clays (inorganic clays). FTIR spectra showed that the binding of DNA on kaolinite and inorganic clays changed its conformation from the B-form to the Z-form, whereas montmorillonite and organic clays retained the original B-form of DNA. A structural change from the B- to the C-form in DNA molecules desorbed from kaolinite was observed by CD spectroscopy and confirmed by fluorescence spectroscopy. The presence of soil colloids and minerals provided protection to DNA against degradation by DNase I. The higher level of protection was found with montmorillonite and organic clays compared to kaolinite and inorganic clays. The protection of DNA against nuclease degradation by soil colloids and minerals is apparently not controlled by the adsorption affinity of DNA molecules for the colloids and the conformational change of bound DNA. The higher stability of DNA seemed to be attributed mainly to the presence of organic matter in the system and the adsorption of nucleases on soil colloids and minerals. The information obtained in this study is of fundamental significance for the understanding of the behavior of extracellular DNA in soil environment.     

Biostimulation of PAH degradation with plants containing high concentrations of linoleic acid

Many plant species enhance the biodegradation of polycyclic aromatic hydrocarbons (PAHs), but there is little understanding of the mechanisms by which this occurs. This research identified phytochemicals that stimulate pyrene degradation using crushed roottissues from 43 plants that were screened in soil spiked with 100 ppm pyrene. Among the plants tested, root tissues from Apium graveolens (celery), Raphanus sativus (radish), Solanum tuberosum (potato), and Daucus carota (carrot) were most effective for promoting disappearance of pyrene within 40 days. Experiments with A. graveolens showed that plant culture in soil contaminated with pyrene or benzo[a]pyrene was as effective as addition of crushed root tissues. Comparison of the chemical compositions of the effective plants suggested that linoleic acid was the major substance that stimulated PAH degradation. This hypothesis was supported in experiments examining degradation of pyrene and benzo[a]pyrene in soil amended with linoleate, whereas linolenic and palmitic acids did not stimulate degradation within a 20 day period. Antibiotic inhibitor studies implicated gram positive bacteria as a predominant group responding to linoleic acid. These findings provide insight into the mechanisms by which plants enhance degradation of PAHs, and have practical application for remediation of PAH contaminated soils.     

DNase I activity in pig MII oocytes: implications in transgenesis

Several reliable methods to produce transgenic animals utilize the male genome. After penetration into oocyte, sperm DNA undergoes dramatic conformational changes that could represent a great opportunity for exogenous DNA to be integrated in the zygote genome. Among the enzymes responsible for sperm remodeling, a nuclease could be involved. The presence of a DNase I in oocytes has not been much investigated. To date, an immunolocalization of DNase I has been reported only in rat immature oocytes and the presence of nuclease activities has been shown in avian oocytes. The present study was conducted to verify whether a DNase-I like activity is present in MII mature pig oocytes. To do this, oocyte extracts were assessed for nuclease activity by a plasmid degradation assay and by zymography; these analyses evidenced a 33 kDa, Ca2+/Mg2+ dependent DNase I-like activity that was inhibited by Zn2+. A further identification of DNase I was achieved by Western blot, immunofluorescence and RT-PCR experiments. Moreover, the presence of the enzyme activity was confirmed by the rapid degradation of exogenous DNA microinjected into the ooplasm. Finally, the exogenous DNA transferred to oocyte by spermatozoa during sperm mediated gene transfer in vitro fertilisation protocol seemed to be protected from DNase I degradation and to persist in the ooplasm till 6 h. These results, together with the high efficiency of sperm based transgenesis methods, suggest that the association with spermatozoa protects exogenous DNA from nuclease activities.     

Removal of polycyclic aromatic hydrocarbons from contaminated soil by aqueous DNA solution

An aqueous DNA solution was applied for the extraction of polycyclic aromatic hydrocarbons (PAHs) from a spiked soil. Solubilities of 0.56, 11.78, and 11.24 mg L(-1) for anthracene, phenanthrene, and pyrene, respectively, were achieved after 1 day equilibration in 1% DNA. Using a spiked soil that contained 72 mg kg(-1) anthracene, 102 mg kg(-1) phenanthrene, and 99 mg kg(-1) pyrene, extractions of close to 88, 78, and 94%, respectively, were attained with 5% DNA at a 1:50 soil/extractant ratio. Maximum PAH dissolution occurred after 4-6 h. Comparative tests showed the main advantage of DNA over methyl-beta- and gamma-cyclodextrins and Tween 80 for pyrene removal. An ionic strength of 0.1 M NaCl was found necessary for maximum PAH dissolution and extraction. The performance of hexane regenerated DNA also remained stable after three stages of recycling. These results show the huge potential of DNA as an aqueous washing agent for PAH-contaminated soil.     

Study of the formation of complexes between DNA and esterified dairy proteins

Like with many naturally occurring basic proteins such as histones and lysozymes, plasmid DNA can interact with methylated α-lactalbumin (ALA) and methylated β-lactoglobulin (BLG) forming complexes. The stabilities of these complexes were tested at different pH, temperatures and salt concentrations, and after enzyme digestion with DNase I and pepsin. Incubation at 37°C for long periods (up to 24 h) allowed the interaction of DNA with low concentrations of esterified proteins to take place. High temperature treatment (100°C) for short periods of time enhanced complex formation after 5 min of heating in case of both DNA/methylated ALA and DNA/methylated BLG. The complex of DNA with methylated BLG was more stable than that of DNA and methylated ALA, when the thermal treatment at 100°C was extended to 10 min. Both complexes were formed in larger amounts and were more stable at acid pH (3–6). Generally, at acid pH, the concentration of the stable complex of DNA and methylated BLG was larger than that of the complex between DNA and methylated ALA. These complexes were still quite stable at very acid pH (1–2) but not at all at very basic pH (10–11). Formation and stability of studied complexes of DNA with esterified proteins were generally dependent on salt concentration. Magnesium chloride had the greatest inhibitory effect on the formation and the stability of these complexes while potassium acetate had the least. The inhibitory effect of KCl on both complex formation and stability was observed in the range 0.4–1.0 . The complexes between DNA and esterified milk proteins or lysozyme were more resistant to hydrolysis with DNase I than free non-complexed DNA. Surprisingly, the DNA/methylated ALA complex was more resistant to DNase I digestion than the DNA/methylated BLG or DNA/lysozyme complexes. All the studied proteins were resistant to pepsin when complexed with DNA.     

三种光敏剂介导的光动力对水体中8种多环芳烃的降解作用

为了探究不同光敏剂(姜黄素、核黄素、金丝桃素)介导的光动力对水体中多环芳烃的降解作用,以含有8种多环芳烃的水体为研究对象,在光敏剂浓度为10μM,光照时间为15 min的条件下,采用光动力方法处理并通过同步荧光法测定水体中的各种多环芳烃.结果表明,姜黄素介导的光动力对多环芳烃的降解效果明显优于核黄素和金丝桃素介导的光动...     

Laccase-carrying electrospun fibrous membranes for adsorption and degradation of PAHs in shoal soils

The removal of polycyclic aromatic hydrocarbons (PAHs) from soil is costly and time-consuming. The high hydrophobicity of PAHs makes PAH diffusion from soil particles by hydraulic flow difficult. The phase transfer of PAHs from soil to another available mediator is crucial for PAH removal. This study focuses on the remediation of PAH-contaminated shoal soil, located in Yangtze, China, using three types of laccase-carrying electrospun fibrous membranes (LCEFMs) fabricated via emulsion electrospinning. These LCEFMs were composed of core-shell structural nanofibers (for PAH adsorption), with laccase in the core (for PAH degradation) and pores on the shell (for mass transfer). The LCEFMs with strong adsorptivity extracted the PAHs from the soil particles, resulting in an obvious enhancement of PAH degradation. The removal efficiencies in 6 h for phenanthrene, fluoranthene, benz[a]anthracene and benzo[a]pyrene were greater than 95.1%, 93.2%, 79.1%, and 72.5%, respectively. The removal half-lives were 0.003-1.52 h, much shorter than those by free laccase (17.9-67.9 h) or membrane adsorption (1.25-12.50 h). The third-order reaction kinetics suggested that the superficial adsorption and internal diffusion were the rate-limiting steps of the overall reaction. A synergistic effect between adsorption and degradation was also proposed on the basis of the triple phase distribution and kinetics analyses.     

DNA exposure to buckminsterfullerene (C60): toward DNA stability, reactivity, and replication

Buckminsterfullerene (C(60)) has received great research interest due to its extraordinary properties and increasing applications in manufacturing industry and biomedical technology. We recently reported C(60) could enter bacterial cells and bind to DNA molecules. This study was to further determine how the DNA-C(60) binding affected the thermal stability and enzymatic digestion of DNA molecules, and DNA mutations. Nano-C(60) aggregates and water-soluble fullerenols were synthesized and their impact on DNA biochemical and microbial activity was investigated. Our results revealed that water-soluble fullerenols could bind to lambda DNA and improve DNA stability remarkably against thermal degradation at 70-85 °C in a dose-dependent manner. DNase I and HindIII restriction endonuclease activities were inhibited after interacting with fullerenols at a high dose. Experimental results also showed the different influence of fullerenol and nano-C(60) on their antibacterial mechanisms, where fullerenols contributed considerable impact on cell damage and mutation rate. This preliminary study indicated that the application of fullerenols results in significant changes in the physical structures and biochemical functions of DNA molecules.     

植物甾醇对卷烟主流烟气中PAHs的影响

运用GC-MS法研究了在卷烟烟支中分别加入胆甾醇、麦角甾醇、豆甾醇和β-谷甾醇对主流烟气中14种多环芳烃释放量的影响并探讨了甾醇类物质在卷烟燃吸时产生多环芳烃的机理。结果表明:游离甾醇的总量与主流烟气中多环芳烃的总释放量以及苯并[a]芘的释放量都呈显著正相关,而不同甾醇类物质对主流烟气中不同的多环芳烃含量的影响是有差异性的,胆甾醇显著地影响三环多环芳烃的释放量,β-谷甾醇显著地影响四环多环芳烃的释放量,豆甾醇对于四环、五环、六环多环芳烃的释放量的影响很显著,麦角甾醇对所有PAHs的释放量都有显著的影响。      

Optimization of the peroxy acid treatment of alpha-methylnaphthalene and benzo[a]pyrene in sandy and silty-clay sediments

The majority of polycyclic aromatic hydrocarbons (PAHs) released to the environment come from anthropogenic sources involving the incomplete combustion of organic compounds. Several techniques are available for the degradation of PAHs. Among the abiotic/biotic processes used to degrade PAHs, an alternative strategy utilizing a primary chemical oxidative step to be combined with a biological was created. The degradation of alpha-methylnaphthalene and benzo[a]pyrene using an advanced oxidation process was optimized over a period of 24 h by varying the ratio of acetic acid to hydrogen peroxide, the compounds that form peroxy acids. The optimization process was performed using sandy and silty-clay sediment types. Gas chromatography equipped with a flame ionization detector was used to determine the varied rates of degradation depending on acetic acid:hydrogen peroxide ratios and the characteristics of the sediment sample. Reduction of 20-90% of alpha-methylnaphthalene and benzo[a]pyrene was observed when 2-5 mL of hydrogen peroxide was used, respectively. A peracetic acid solution (e.g., a commercial form of acetic acid and hydrogen peroxide) was used to compare the results from the peroxy acid experiments. In all the experiments, peracetic acid was more reactive than the combination of acetic acid and hydrogen peroxide. Acetic acid, deionized water, and hydrogen peroxide served as controls and demonstrated minimal degradation over the time course study. Therefore, the use of a peroxy acid process to target electron dense pollutants may have a great utility.     

Comparative quantitative prevalence of Mycobacteria and functionally abundant nidA, nahAc, and nagAc dioxygenase genes in coal tar contaminated sediments

The Chattanooga Creek Superfund site is heavily contaminated with metals, pesticides, and coal tar with sediments exhibiting high concentrations of polycyclic aromatic hydrocarbons (PAHs). High molecular weight PAHs are of concern because of their toxicity and recalcitrance in the environment; as such, there is great interest in microbes, such as fast-growing Mycobacterium spp., capable of degradation of these compounds. Real-time quantitative PCR assays were developed targeting multiple dioxygenase genes to assess the ecology and functional diversity of PAH-degrading communities. These assays target the Mycobacterium nidA, beta-proteobacteria nagAc, and gamma-proteobacteria nahAc with the specific goal of testing the hypothesis that Mycobacteria catabolic genes are enriched and may be functionally associated with high molecular weight PAH biodegradation in Chattanooga Creek. Dioxygenase gene abundances were quantitatively compared to naphthalene and pyrene mineralization, and temporal and spatial PAH concentrations. nidA abundances ranged from 5.69 x 10(4) to 4.92 x 10(6) copies per gram sediment; nagAc from 2.42 x 10(3) to 1.21 x 10(7), and nahAc from below detection to 4.01 x 10(6) copies per gram sediment. There was a significantly greater abundance of nidA and nagAc at sites with the greatest concentrations of PAHs. In addition, nidA and nagAc were significantly positively correlated (r = 0.76), indicating a coexistence of organisms carrying these genes. A positive relationship was also observed between nidA and nagAc and pyrene mineralization indicating that these genes serve as biomarkers for pyrene degradation. A 16S rDNA clone library of fast-growing Mycobacteria indicated that the population is very diverse and likely plays an important role in attenuation of high molecular weight PAHs from Chattanooga Creek.     

DNA taken into Bacillus subtilis competent cells by lysed-protoplast transformation is not ssDNA but dsDNA

Competent Bacillus subtilis incorporates whole-genome DNA (4215 kb) from the protoplast lysate of B. subtilis subtilis [Akamatsu, T. and Taguchi, H., Biosci. Biotechnol. Biochem., 65, 823-829 (2001)]. A continuous incorporated DNA is longer than 1500 kb [J. Biosci. Bioeng., 101, 257-262 (2006)]. Whether the incorporated DNA is single-stranded (ssDNA) or double-stranded DNA (dsDNA) has been studied by examining the transforming activity of the incorporated DNA. B. subtilis BEST7027 was used as the donor strain, which has a heterologous region consisting of the 145 kb region of the Synechocystis sp. PCC6803 genome and erm gene. The donor DNA was transferred to a wild-type or a recA recipient strain (AYG2 or SYN9), and protoplast lysate was prepared from the transformants and used as the donor DNA source for the second recipient strain (AU1 or AV1). The intergenote region showed a significant transforming activity. When DNase I was added to both cells collected from the first transformation mixture and the following protoplastization, the result was similar to that obtained without DNase I. All of the observations strongly suggest that the incorporated DNA is dsDNA, and the transformation of competent B. subtilis by DNA in protoplast lysate is different from that by purified DNA taken up conventionally.     

Enhanced concentrations of PAHs in groundwater at a coal tar site

Concentrations of polycyclic aromatic hydrocarbons (PAHs) in groundwater at a coal tar site were elevated by factors ranging from 3 (pyrene) to 50 (indeno[1,2,3-cd]pyrene) over purely dissolved concentrations. Air-groundwater surface tension measurements (70.6 +/- 3 dyn/cm) were not sufficiently different from air-pure water measures (72.2 +/- 0.1 dyn/cm) to ascribe the observed enrichments to either cosolvents or surfactants in the groundwater. Excess pyrene was associated with colloids that passed an ultrafilter at ambient pH but became ultrafilterable when the groundwater pH was lowered to 1. This suggested pyrene association with humic acids. Given the decrease in groundwater total organic carbon (TOC) of 4 mgc/L upon acidification and ultrafiltration, a partition coefficient of 10(5) L/kgc was estimated for this pyrene association. Use of the results for pyrene and scaling for the differences in PAH hydrophobicities enabled good predictions of the observed enrichments of less water-soluble PAHs in the groundwater. This is strong field evidence indicating colloid-facilitated transport of HOCs in groundwater. Assuming that humic-bound PAHs were as mobile as the dissolved PAHs, the fluxes of individual PAHs (e.g., benzo[a]pyrene) from the tar source were as much as 20 times greater than estimates based solely on tarwater partitioning predictions.     

The terminal protein of the linear DNA plasmid pGKL2 shares an N-terminal domain of the plasmid-encoded DNA polymerase

The 36K protein attached at the 5′ end of the linear DNA plasmid pGKL2 from the yeast Kluyveromyces lactis was first purified and characterized. The terminal protein was purified from cells (1 kg wet weight) by ammonium sulphate precipitation and two rounds of centrifugation to equilibrium in CsCl gradients. The pGKL2 was present only in the post-microsomal supernatant. Approximately 10 mg of the purified pGKL2 was recovered and digested with DNase I. The terminal protein (final ca. 0·8 mg) was homogeneous by electrophoresis and we determined the N-terminal amino acid sequence up to ten residues, showing that it existed in the cryptic N-terminal domain of pGKL2-ORF2 (DNA polymerase) sequence.     

New method for testing phototoxicity of polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs), widespread environmental pollutants, were recently reported to show photomutagenesis. As contaminants in the environment are usually exposed to sunlight, a way to evaluate the phototoxic characteristics of pollutants is required. We have previously found that phosphorylation of histone H2AX (gamma-H2AX), which accompanied the induction of DNA double strand breaks (DSBs), was significantly induced by low concentrations of benzo[a]pyrene (10(-9)-10(-7) M) and UVA (0.6 J/cm2) in CHO-K1 cells. Higher concentrations have been required for the detection of DSBs. The aim of the present study is to investigate the applicability of gamma-H2AX in a new phototoxicity assay of PAHs. The human keratinocytes, HaCaT, were treated with four model PAHs (naphthalene, phenanthrene, pyrene, and benzo[a]pyrene, 10(-11)-10(-7) M) and/or UVA (5 J/cm2), and the induction of gamma-H2AX was assessed. Furthermore, DSBs were directly detected using a biased sinusoidal field gel electrophoresis, and the cell viability was examined as a general assay of phototoxicity. The induction of gamma-H2AX was detected in the presence of all the PAHs except naphthalene at concentrations of 10(-9)-10(-7) M, whereas neither DSBs nor cell death could be detected at those concentrations, and higher concentrations were required for the detection. Naphthalene showed no phototoxicity in any of the three different assays. These findings suggest that histone H2AX is a potential moleculartargetfor detecting the phototoxicity of PAHs more sensitively than the detection of cell viability and DSBs.     

Stimulation of pyrene mineralization in freshwater sediments by bacterial and plant bioaugmentation

As a means to study the fate of polycyclic aromatic hydrocarbons (PAHs) in freshwater sediments, pyrene mineralization was examined in microcosms spiked with [14C]pyrene. Some microcosms were planted with reeds (Phragmites australis) and/or inoculated with a pyrene-degrading strain, Mycobacterium sp. 6PY1. Mineralization rates recorded over a 61 d period showed that reeds promoted a significant enhancement of pyrene degradation, which possibly resulted from a root-mediated increase of oxygen diffusion into the sediment layer, as indicated by in situ redox measurements. In inoculated microcosms, mineralization reached a higher level in the absence (8.8%) than in the presence of plants (4.4%). Mineralization activity was accompanied by the release of water-soluble pyrene oxidation products, the most abundant of which was identified as 4,5-diphenanthroic acid. Pyrene was recovered from plant tissues, including stems and leaves, at concentrations ranging between 40 and 240 microg/g of dry mass. Plants also accumulated labeled oxidation products likely derived from microbial degradation. Pyrene-degrading strains were 35-70-fold more abundant in inoculated than in noninoculated microcosms. Most of the pyrene-degrading isolates selected from the indigenous microflora were identified as Mycobacterium austroafricanum strains. Taken together, the results of this study show that plants or PAH-degrading bacteria enhance pollutant removal, but their effects are not necessarily cumulative.     

Polycyclic aromatic hydrocarbons in foods: human exposure through the diet in Catalonia, Spain

The dietary intake of 16 polycyclic aromatic hydrocarbons (PAHs) (naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenz[a,h]anthracene, benzo[g,h,i]perylene, and indeno[1,2,3-c,d]pyrene) by the general population of Catalonia, Spain, was calculated. Concentrations of PAHs in food samples randomly acquired in seven cities of Catalonia from June to August 2000 were measured. Eleven food groups were included in the study. High-performance liquid chromatography was used to analyze PAHs. The dietary intakes of total and carcinogenic PAHs was calculated for five population groups: children, adolescents, male adults, female adults, and seniors. Among the analyzed PAHs, there was a predominance of phenanthrene (16.7 microg/kg) and pyrene (10.7 microg/kg). By food group, the highest levels of total PAHs were detected in cereals (14.5 microg/kg) and in meat and meat products (13.4 microg/kg). The mean estimated dietary intake of the sum of the 16 PAHs was as follows: male adults, 8.4 microg/day; adolescents, 8.2 microg/day; children, 7.4 microg/day; seniors, 6.3 microg/day; female adults, 6.3 microg/day. The calculated daily intake of PAHs would be associated with a 5/106 increase in the risk for the development of cancer in a male adult with a body weight of 70 kg.     

Anaerobic,sulfate-dependent degradation of polycyclic aromatic hydrocarbons in petroleum-contaminated harbor sediment

It has previously been demonstrated that [14C]-labeled polycyclic aromatic hydrocarbons (PAHs) can be oxidized to 14CO2 in anoxic, PAH-contaminated, marine harbor sediments in which sulfate reduction is the terminal electron-accepting process. However, it has not previously been determined whether this degradation of [14C]-PAHs accurately reflects the degradation of the in situ pools of contaminant PAHs. In coal tar-contaminated sediments from Boston Harbor, [14C]-naphthalene was readily oxidized to 14CO2, but, after 95 d of incubation under anaerobic conditions, there was no significant decrease in the detectable pool of in situ naphthalene in these sediments. Therefore, to better evaluate the anaerobic biodegradation of the in situ PAH pools, the concentrations of these contaminants were monitored for ca. 1 year during which the sediments were incubated under conditions that mimicked those found in situ. There was loss of all of the PAHs that were monitored (2-5 ring congeners), including high molecular weight PAHs, such as benzo[a]pyrene, that have not previously been shown to be degraded under anaerobic conditions. There was no significant change in the PAH levels in the sediments amended with molybdate to inhibit sulfate-reducing bacteria or in sediments in which all microorganisms had been killed with glutaraldehyde. In some instances, over half of the detectable pools of in situ 2-3 ring PAHs were degraded. In general, the smaller PAHs were degraded more rapidly than the larger PAHs. A distinct exception in the Boston Harbor sediment was naphthalene which was degraded very slowly at a rate comparable to the larger PAHs. In a similar in situ-like study of fuel-contaminated sediments from Liepaja Harbor, Latvia, there was no decline in PAH levels in samples that were sulfate-depleted. However, when the Latvia sediments were supplemented with sufficient sodium sulfate or gypsum to elevate pore water levels of sulfate to approximately 14-25 mM there was a 90% decline in the naphthalene and a 60% decline in the 2-methylnaphthalene pool within 90 days. These studies demonstrate for the first time that degradation by anaerobic microorganisms can significantly impact the in situ pools of PAHs in petroleum-contaminated, anoxic, sulfate-reducing harbor sediments and suggest that the self-purification capacity of contaminated harbor sediments is greater than previously considered.