The Movement of 2,4-dichlorophenyl methanesulfonate 35S in Plants

Movement of 2,4-dichlorophenyl methanesulfonate ³⁵S in plant roots was investigated using plastic containers which physically isolated portions of the plant root system. Results with bean and cotton plants showed that this compound can be absorbed in one part of the root system and distributed to other parts of the root system and also to the top of the plant. These tests confirm field observations of nematode control on roots outside a treated zone which indicated lateral movement of this compound in the roots. There was no evidence of its downward movement from treated leaves to stems or roots of cotton or beans.     

脊椎动物线粒体DNA的基因重排

将GenBank上已公布的321种脊椎动物mtDNA全序列,按纲整理归类,绘制基因排布图并进行比对。比对结果表明：81个物种的mtDNA中观察到基因重排现象,涉及脊椎动物各纲,其中9个物种同时存在基因顺序变化和基因倒置现象,所有的基因重排都涉及tRNA的变化。脊椎动物mtDNA基因顺序变化可分为3类：1)邻接的基因或片段的位置交换;2)接近于控制序列或轻链起始位点的基因或片段的位置变化,有时还伴随着控制序列的倍增;3)I-Q-M区域的变化。所有鸟类、蛇类、鳄类和有袋类的mtDNA具有各自独特的基因排列顺序。基因倒置现象常见于鱼类和哺乳类,且多表现为tRNA从轻链往重链上迁移。本文就这些基因重排现象、发生重排的机制和mtDNA基因重排在系统发生研究中的应用做一简要概述。     

2,4-Dichlorophenoxyacetic Acid (2,4-D) Utilization by Delftia acidovorans MC1 at Alkaline pH and in the Presence of Dichlorprop is Improved by Introduction of the tfdK Gene

Growth of Delftia acidovorans MC1 on 2,4-dichlorophenoxyacetic acid (2,4-D) and on racemic 2-(2,4-dichlorophenoxy)propanoic acid ((RS)-2,4-DP) was studied in the perspective of an extension of the strain’s degradation capacity at alkaline pH. At pH 6.8 the strain grew on 2,4-D at a maximum rate (μ_max) of 0.158 h⁻¹. The half-maximum rate-associated substrate concentration (K_s) was 45 μM. At pH 8.5 μ_max was only 0.05 h⁻¹ and the substrate affinity was mucher lower than at pH 6.8. The initial attack of 2,4-D was not the limiting step at pH 8.5 as was seen from high dioxygenase activity in cells grown at this pH. High stationary 2,4-D concentrations and the fact that μ_max with dichlorprop was around 0.2 h⁻¹ at both pHs rather pointed at limited 2,4-D uptake at pH 8.5. Introduction of tfdK from D. acidovorans P4a by conjugation, coding for a 2,4-D-specific transporter resulted in improved growth on 2,4-D at pH 8.5 with μ_max of 0.147 h⁻¹ and K_s of 267 μM. Experiments with labeled substrates showed significantly enhanced 2,4-D uptake by the transconjugant TK62. This is taken as an indication of expression of the tfdK gene and proper function of the transporter. The uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) reduced the influx of 2,4-D. At a concentration of 195 μM 2,4-D, the effect amounted to 90% and 50%, respectively, with TK62 and MC1. Cloning of tfdK also improved the utilization of 2,4-D in the presence of (RS)−2,4-DP. Simultaneous and almost complete degradation of both compounds occurred in TK62 up to D = 0.23 h⁻¹ at pH 6.8 and up to D = 0.2 h⁻¹ at pH 8.5. In contrast, MC1 left 2,4-D largely unutilized even at low dilution rates when growing on herbicide mixtures at pH 8.5.     

Bioremediation of 2,4-dinitrotoluene (2,4-DNT) in immobilized micro-organism biological filter

Aims: To evaluate the biodegradability of 2,4‐DNT using an anaerobic filter (AF) combined with a biological aerated filter (BAF), and elucidate the degradation mechanism of 2,4‐DNT and analyze the bacterial community of the reactors over a long period of operation. Methods and Results: The pilot test experienced wide fluctuations influent concentrations and there was lower than 0.50 mg l^?1 of 2,4‐DNT in the effluent of the system. The removal efficiency was above 99%. GC‐MS analysis demonstrated that 2,4‐DNT was mainly reduced to 2‐amino‐4‐nitrotoluene (2‐A‐4‐NT), 4‐amino‐2‐nitrotoluene (4‐A‐2‐NT), and 2,4‐diaminotoluene (2,4‐DAT) during the anaerobic reaction. In addition, ethanol was added into the influent as the electron donor. Because of the use of part ethanol as an auxiliary carbon source, more than twice the theoretical requirement of ethanol was needed to achieve a high 2,4‐DNT removal efficiency (>93%). ESEM observations showed that the carrier could immobilize micro‐organisms, which flourished more in reactors operating over longer periods. Further research by PCR‐DGGE revealed that new 2,4‐DNT‐resistant bacterial had been generated during the stress of 2,4‐DNT for 150 days. The dominant species for 2,4‐DNT degradation were identified by a comparison with gene sequences in GenBank. Conclusions: 2,4‐DNT could be effectively degraded by the combined process and ethanol played an important role in the biotransformation. The proposed transformation pathway of 2,4‐DNT was concluded. During the 150‐day operation, some microbial taxa unaccustomed to 2,4‐DNT died out and some new 2,4‐DNT‐resistant microbial taxa appeared. Significance and Impact of the Study: The study provides a novel method for the bioremediation of 2,4‐DNT, which is difficult to degrade by traditional biological methods. The most 2,4‐DNT‐resistant microbial taxa have not been reported elsewhere and they may be helpful to the treatment of actual 2,4‐DNT wastewater.     

Enhancement of 2,4-dichlorophenoxyacetic acid (2,4-D) degradation in soil by dissemination of catabolic plasmids

Few studies have been done to evaluate the transfer of catabolic plasmids from an introduced donor strain to indigenous microbial populations as a means to remediate contaminated soils. In this work we determined the effect of the conjugative transfer of two 2,4-D degradative plasmids to indigenous soil bacterial populations on the rate of 2,4-D degradation in soil. We also assessed the influence of the presence of 2,4-D on the number of transconjugants formed. The two plasmids used, pEMT1k and pEMT3k, encode 2,4-D degradative genes (tfd) that differ in DNA sequence as well as gene organisation, and confer different growth rates to Ralstonia eutropha JMP228 when grown with 2,4-D as a sole carbon source. In an agricultural soil (Ardoyen) treated with 2,4-D (100 ppm) there were ca. 10⁷CFU of transconjugants per gram bearing pEMT1k as well as a high number of pEMT3k bearing transconjugants (ca. 10⁶ CFU/g). In this soil the formation of a high number of 2,4-D degrading transconjugants resulted in faster degradation of 2,4-D as compared to the uninoculated control soil. In contrast, only transconjugants with pEMT1k were detected (at a level of ca. 10³ CFU/g soil) in the untreated Ardoyen soil. High numbers of transconjugants that carried pEMT1k were also found in a second experiment done using forest soil (Lembeke) treated with 100 ppm 2,4-D. However, unlike in the Ardoyen soil, no transconjugants with pEMT3k were detected and the transfer of plasmid pEMT1k to indigenous bacteria did not result in a higher rate of decrease of 2,4-D. This may be because 2,4-D was readily metabolised by indigenous bacteria in this soil. The results indicate that bioaugmentation with catabolic plasmids may be a viable means to enhance the bioremediation of soils which lack an adequate intrinsic ability to degrade a given xenobiotic.     

Measurement of free and bound malondialdehyde in plasma by high-performance liquid chromatography as the 2,4-dinitrophenylhydrazine derivative

We established a method for the detection of free and total (free and bound) malondialdehyde (MDA) in human plasma samples after derivatisation with 2,4-dinitrophenylhydrazine (DNPH). Free MDA was prepared by perchloric acid deproteinisation whereas an alkaline hydrolysation step for 30 min at 60°C was introduced prior to protein precipitation for the determination of total MDA. Derivatisation was accomplished in 10 min at room temperature subsequently chromatographed by HPLC on a reversed-phase 3 μm C₁₈ column with UV detection (310 nm). The detection limit was 25 pmol/ml for free and 0.3 nmol/ml for total MDA. The recovery of MDA added to different human plasma samples was 93.6% (n=11; RSD 7.1%) for the hydrolysation procedure. In samples from 12 healthy volunteers who underwent a hypoxic treatment (13% O₂ for 6 h) we estimated a baseline value of total MDA of 2.16 nmol/ml (SD 0.29) (ambient air) with a significant increase to 2.92 (nmol/ml, SD 0.57; P=0.01) after the end of this physiological oxidative stress challenge. Plasma values of free MDA in these samples were close to our detection limit. The presented technique can easily performed with an isocratic HPLC apparatus and provides highly specific results for MDA as do sophisticated GC–MS methods.     

Domain-Specific Rearrangement between the Two Arg-Gingipain-Encoding Genes in Porphyromonas gingivalis: Possible Involvement of Nonreciprocal Recombination

Porphyromonas gingivalis has two functional genes (rgpA and rgpB) encoding an arginine-specific cysteine proteinase (Arg-gingipain, RGP). Several RGP-encoding genes have been cloned and sequenced from various P. gingivalis strains, but all of the genes seem to be essentially equivalent to rgpA. In this study, we cloned and sequenced the second rgp gene (rgpB). A comparison of the rgpB gene and the rgp1 gene, one of the rgpA-equivalent genes, revealed that their gene structures were very similar to each other, except that the rgpB gene did not possess most of the hemagglutinin domain present in the C-terminal region of the rgpl gene, and provided strong evidence for homologous recombination between the proteinase domain regions of the two rgp genes. The presence of nonreciprocal recombination in P. gingivalis was experimentally proven using suicide/integration plasmid systems. The results provide one of the hypothetical scenarios of the generation of the two rgp genes; that is, they have been generated through the duplication of an ancestor rgp gene, insertion of the hemagglutinin domain region into one copy of the two resulting rgp genes (or deletion of the region from one rgp) and homologous recombination between the proteinase domain regions of the two rgp genes.     

Determination of paromomycin in human plasma and urine by reversed-phase high-performance liquid chromatography using 2,4-dinitrofluorobenzene derivatization

A sensitive high-performance liquid chromatographic method for the determination of paromomycin in human plasma and urine was developed. Paromomycin was quantitated following pre-column derivatization with 2,4-dinitrofluorobenzene (DNFB). The chromatographic separation was carried out on a C₁₈ column at 50°C using a mobile phase consisting of 64% methanol in water adjusted to pH 3.0 with phosphoric acid. The eluents were monitored by UV detection at 350 nm. The linearity of response for paromomycin was demonstrated at concentrations from 0.5 to 50 μg/ml in plasma and 1 to 50 μg/ml in urine. The relative standard deviation of the assay procedure is less than 5%.     

Peroxidase-catalyzed Oxidation of 3,3",5,5"-Tetramethylbenzidine in the Presence of 2,4-Dinitrosoresorcinol and Polydisulfide Derivatives of Resorcinol and 2,4-Dinitrosoresorcinol

A comparative study of the kinetics of peroxidase-catalyzed oxidation of 3,3",5,5"-tetramethylbenzidine (TMB) in the presence of 2,4-dinitrosoresorcinol (DNR), its polydisulfide derivative [poly(DNRDS)], and resorcinol polydisulfide [poly(RDS)], substances that competitively inhibit the formation of TMB conversion product, was carried out. The inhibition constants K _i for DNR, poly(DNRDS), and poly(RSD) were determined at 20°C and pH 6.4 to be 110, 13.5, and 0.78 M, respectively. The stoichiometric coefficients of inhibition were calculated to be 0.38 and 76 for poly(DNRDS) and poly(RDS), respectively. In the pH range 6.4–7.0, the initial rates of the peroxidative oxidation of TMB, and its mixtures with DNR and poly(DNRDS) and the K _i value for poly(RDS) substantially decreased with increasing pH. The kinetic parameters of poly(RDS) (K _i 0.22–0.78 M and f 76) suggest that it is the most efficient inhibitor of peroxidase oxidation of TMB: in micromolar concentrations, it completely stops this process and can be used in EIA.     

Biological monitoring of dinitrotoluene by gas chromatographic–mass spectrometric analysis of 2,4-dinitrobenzoic acid in human urine

The method of analysis described permits the determination of 2,4-dinitrobenzoic acid down to the lower μg l⁻¹ range in the urine of persons exposed to dinitrotoluene. 2,4-Dinitrobenzoic acid is the main metabolite of 2,4-dinitrotoluene and technical dinitrotoluene. After acidic hydrolysis, which served to release the conjugated part of the 2,4-dinitrobenzoic acid, the analyte was selectively separated from the urine matrix via various extraction steps and then derivatised to the methyl ester. Quantitative analysis was carried out using capillary gas chromatography and mass selective detection. 3,5-Dinitrobenzoic acid was used as an internal standard. The detection limit was 1 μg l⁻¹ urine. The relative standard deviations of within-series imprecision were between 5 and 6%. The relative recoveries were between 91 and 110% depending on the concentration. The analytical method developed as part of this study was used to investigate a collective consisting of 82 urine samples from persons working in the area of explosives disposal. The concentrations of 2,4-dinitrobenzoic acid determined ranged from the detection limit to 95 μg l⁻¹ urine. The method allowed the quantification of low-level internal exposure to dinitrotoluene.     

The use of biomarkers in the antioxidant responses of Daphnia magna to the acute and chronic exposure to no. 20 diesel oil and 2,4-dichlorophenol

Abstract

The effects of no. 20 diesel oil exposure, 2,4-dichlorophenol (2,4-DCP) exposure and combined exposure on the antioxidant defences of Daphnia magna have been studied systematically for the first time. Daphnia magna was exposed for 1 day or 10 days to several concentrations of 0, 0.005, 0.01, 0.05, 0.1, 0.5 or 1.0 mg L^?1 solutions. Antioxidant defences consisting of the levels of reduced glutathione (GSH) and glutathione disulfide (GSSG) and activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), selenium dependent glutathione peroxidase (Se-GPx) and glutathione S-transferase (GST) of daphnids were determined to evaluate their protective roles and to analyse the occurrence of oxidative stress. The possible antioxidant defence mechanisms are discussed. Furthermore, GST can be a potential biomarker and an early-warning index for the pollutants in waters in that GST responded sensitively to 1 day and 10 days of exposure to diesel oil and 2,4-DCP and 10 days of combined exposure. Crossover comparisons showed an antagonistic action about the no-observed-effect concentration (NOEC) against Daphnia magna, which needs further studies.     

Modification of replicon operation in HeLa cells by 2,4-dinitrophenol

Cycloheximide causes inhibition of semiconservative DNA replication in HeLa cells by reducing the average rate of DNA chain elongation. 2,4-Dinitrophenol inhibits semiconservative DNA replication (50 to 80% inhibitions at 10^?3 to 5 × 10^?3 M-2,4-dinitrophenol) without affecting the average rate of DNA chain elongation. Therefore, at any given time the number of replicating sections of DNA per DNA-synthesizing (S-phase) cell appears to be reduced in the presence of 2,4-dinitrophenol.Radioactivity profiles of pulse-labeled DNA in alkaline sucrose gradients suggest that 2,4-dinitrophenol modifies initiation and termination patterns of replicating sections, most of which are found to be 10 to 80 μm (mode: 15 to 30 μm) under control conditions. DNA synthesized in the presence of 2,4-dinitrophenol has the density of control DNA, is metabolically stable, and after mitosis, functions normally as a template in the next round of replication.     

The toxicity of 2,4-D to Cyprinus carpio var. Communis in relation to the seasonal variation in the temperature

The paper deals with the variations in the toxicity of 2,4-D to Cyprinus carpio at different temperatures during different seasons of the year. The higher temperature has higher toxicity and vice versa at the same concentration. The TLm values indicate that there is a 7–7.5 fold increase in toxicity with the rise in temperature from 17°C in February (winter) to 39°C in May (summer). These results have significance in manipulating the 2,4-D doses for the eradication of aquatic weeds in different seasons.     

Rearrangement and Reactivation of the Myelin Basic Protein Locus in Myelin-Deficient (mld) Mouse Brain

Abstract: Myelin-deficient ( mld ) is a complex mutation affecting the myelin basic protein (MBP) locus of the mouse. It consists of duplication and partial inversion of the MBP gene and results in a dysfunctional MBP locus. The mutant phenotype is reversed, both in vivo and in vitro, in ∼5% of mld oligodendrocytes. One possible mechanism for the somatic reversion is recombination between homologous sequences of the duplicated gene copies to reconstitute a functional MBP locus. There are several possible recombination events that could reconstitute a functional MBP locus by DNA rearrangement. Two of these would result in reinversion and circularization of specific MBP gene sequences, respectively. In this work polymerase chain reaction analysis was used to detect both reinverted and circularized MBP gene sequences in mld mouse tissues, indicating that DNA rearrangement at the MBP locus does occur. Analysis of individually harvested cells showed that in revertant MBP-positive mld oligodendrocytes DNA rearrangement at the MBP locus was correlated with reactivation of the MBP gene. Fluctuation analysis showed that reactivation of the MBP locus is a stochastic event occurring with a frequency of ∼1.4 × 10⁻⁶ per cell per cell cycle during oligodendrocyte development. The frequency of rearrangement and reactivation of the MBP locus was comparable in double mutant ( mld/mld , scid/scid ) and single mutant ( mld/mld , + ^scid /+ ^scid ) mice, indicating that the scid factor is not required for MBP gene reactivation in mld . The significance of DNA rearrangement in mammalian development is discussed.     

Induction of apoptosis in cerebellar granule cells by 2,4-dichlorophenoxyacetic acid

2,4-Dichlorophenoxyacetic acid (2,4-D) and derivatives are herbicides widely used in Argentina and other parts of the world. Exposure to 2,4-D, its ester and salt formulations, have been associated with a range of adverse health effects in humans and different animal species, from embryotoxicity and teratogenicity to neurotoxicity. In this work, we demonstrate that after 24 hs of treatment with 1 and 2 mM 2,4-D there is an induction of apoptosis in cerebellar granule cells (CGC) in culture. However, with 2 mM 2,4-D one population of CGC developed features of apoptosis while another appeared to die by necrosis. This process is associated with an increase in caspase-3 activity after 12 hs of treatment with the herbicide, which is preceded by cytochrome c release from the mitochondria. Treatment of CGC with 2,4-D appears to induce apoptosis by a direct effect on mitochondria producing cytochrome c release and consequently activation of caspase-3, being mitochondrial damage sufficient for triggering the events that may cause apoptosis.     

The Relationship Between the Rate of Molecular Evolution and the Rate of Genome Rearrangement in Animal Mitochondrial Genomes

Evolution of mitochondrial genes is far from clock-like. The substitution rate varies considerably between species, and there are many species that have a significantly increased rate with respect to their close relatives. There is also considerable variation among species in the rate of gene order rearrangement. Using a set of 55 complete arthropod mitochondrial genomes, we estimate the evolutionary distance from the common ancestor to each species using protein sequences, tRNA sequences, and breakpoint distances (a measure of the degree of genome rearrangement). All these distance measures are correlated. We use relative rate tests to compare pairs of related species in several animal phyla. In the majority of cases, the species with the more highly rearranged genome also has a significantly higher rate of sequence evolution. Species with higher amino acid substitution rates in mitochondria also have more variable amino acid composition in response to mutation pressure. We discuss the possible causes of variation in rates of sequence evolution and gene rearrangement among species and the possible reasons for the observed correlation between the two rates. [Reviewing Editor: Dr. David Pollock]     

Effect of 2,4-dichlorophenoxyacetic acid on invertases in chicory root

The invertase present in roots of chicory (Cichoriun intybus) has a pH optimum of 7.5 and a MW of ca 260 000. It requires relatively high ionic strength to remove it from DEAE cellulose. Treatment of chicory root tissue with 2,4-dichlorophenoxyacetic acid gives rise to a highly active invertase with pH optimum of 5.6 and MW of ca 61 000. It is more easily removed from DEAE cellulose.     

Changes in the nucleotide pools in leaves and buds of tobacco plants upon treatment with 2,4-dichlorophenoxyacetic acid

Tobacco ( Nicotiana tabacum L. cv. Samsun) plants were treated once with 2,4-dichlorophenoxyacetic acid (2,4-D) at the 8-leaf stage. The effect of the herbicide on leaf metabolism was followed over 7 days by determination of the ribonucleotide pools, including NAD⁺, NADP⁺ and UDP-sugars, by high-preformance liquid chromatography. 2,4-D treatment resulted in large changes in the nucleotide concentrations, the magnitude and sign of which were dependent upon the leafage. The nucleotide pools decreased in the apical tissue, but increased strongly in the mature leaves with the highest relative increase in the oldest leaf tested. The time course of the changes revealed a maximum on day 5 after 2,4-D treatment. The increase in the adenine nucleotide pools, energy charge and the NADVNADP⁺ ratio are interpreted to indicate a stress situation. The different responses of young, mature and senescent tissue to the synthetic auxin could reflect their different inherent sensitivity due to the natural auxin gradient.     

Isolation and characterization of 2,4-dichlorophenoxyacetic acid-catabolizing bacteria and their biodegradation efficiency in soil

Bacterial isolates (NJ 10 and NJ 15) capable of degrading the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were isolated from agricultural soil by enrichment culture technique. The isolates exhibited substantial growth in mineral salt medium supplemented with 0.1–0.5% of 2,4-D as a sole source of carbon and energy. Based on their morphological, cultural and biochemical characteristics, the isolates NJ 10 and NJ 15 have been identified as Pseudomonas species and Pseudomonas aeruginosa, respectively. Biodegradation studies in a soil microcosm enriched with pure cultures of the isolates demonstrated a time-dependent disappearance of 2,4-D from the 100 mg/kg herbicide-amended soil. The HPLC data analysis revealed 96.6 and 99.8% degradation in the soil inoculated with the pure cultures of isolates NJ 10 and NJ 15, respectively with in 20 days of incubation at 30 °C. Both the isolates showed significant solubilization of inorganic phosphate [Ca₃(PO₄)₂] on the specific Pikovskaya's medium.     

The regeneration potential of wheat shoot meristems in the presence and absence of 2,4-dichlorophenoxyacetic acid

Summary Tissue cultures capable of plant regeneration were successfully initiated from extremely immature shoot meristems of 21 randomly selected genotypes of wheat on nutrient media containing 2,4-dichlorophenoxyacetic acid (2,4-D). By means of scanning electron microscopy it was demonstrated that cultures consisted of teratomatous primordia, which were kept in a proliferating budding state by the 2,4-D. These are characteristic of cereal tissue cultures. Release of the primordia and outgrowth of normal shoots and roots occurred when the cultures were no longer exposed to 2,4-D. Shoot primordia which were clearly identifiable were always associated with root primordia in a quasi-bipolar fashion. Sometimes regions assumed the shape of zygotic embryos, but the transition from apparently normal embryos with scutellum to abnormal configurations with shoot and root regions was gradual. The differences between genotypes in shoot regeneration potential was minimal compared to cultures derived from explants which were taken from regions temporally and spatially more distant from the shoot apex. It is concluded that the ability to give rise to cultures capable of shoot regeneration was lost within a fraction of a millimeter distance from the apical meristem in many genotypes. The proliferating tissues were subcultured at regular intervals over a period of one year and the regeneration potential was monitored. Areas capable of shoot regeneration tended to deteriorate more or less rapidly and were overgrown by root-type tissue in a number of genotypes. The results are discussed in the context of the frequently observed, but largely unexplained, variability in the regeneration potential of cereal tissue cultures.