Electrokinetic movement and biodegradation of 2,4-dichlorophenoxyacetic acid in silt soil.

The coupling of electrokinetic movement of an organic contaminant, 2,4-dichlorophenoxyacetic acid (2,4-D), through soil and its biodegradation in situ has been demonstrated. In a first experiment, the direction and rate of movement of 2,4-D were determined using homogeneously contaminated soil (864 mg 2,4-D/kg dry weight soil) compacted into six individual compartments, 6 cm long, 3 cm wide, and 4 cm deep. Each compartment was bordered by a carbon felt anode and a stainless steel cathode. The application of a current density of 3.72 A/m(2) led to migration of 2,4-D towards the anode at a rate of approximately 4 cm/day. In a second experiment, electrokinetic movement and biodegradation were combined in situ. Sterilized silt soil contaminated with ring-labeled 14C-2,4-D (811 mg 2,4-D/kg dry weight soil) was compacted into a single soil compartment, 22 cm long, 7 cm wide, and 4 cm deep, in a 4.5 cm region adjacent to the cathode. The remainder of the compartment was filled with sterilized soil (to a total weight of 1,015 g). Burkholderia spp. RASC c2 (1.88 x 10(11) cells), a tetracycline-resistant bacterium with chromosomally encoded degradative genes for 2,4-D, was inoculated into the soil at a position 14-16 cm from the cathode. The reactor was placed within a sealed perspex box, with a constant air flow connected to sodium hydroxide traps. Under an applied current density of 0.89 A/m(2), the pollutant moved towards the bacteria. As it reached the inoculated region, its concentration decreased in the soil and 14CO2 was recovered in the traps. At the end of the experiment, 87.1% of radiolabel had been removed from the soil, 5.8% of which was recovered as 14CO2. A third, control, experiment showed a significant contrast in the absence of an electric current, where a slow rate of diffusion controlled the movement of both 2,4-D and bacteria in the soil and biodegradation occurred at the interface between the diffusing fronts.

Dihydrolipoamide dehydrogenase in the trypanosoma subgenus, trypanozoon.

The enzyme dihydrolipoamide dehydrogenase has been discovered and characterised in four salivarian trypanosomes of the subgenus trypanozoon: Trypanosoma brucei brucei, T. b. gambiense, T. b. rhodesiense, and Trypanosoma evansi. The three T. brucei species, which have insect procyclic forms biochemically distinct from their mammalian bloodstream forms, express dihydrolipoamide dehydrogenase in both cell types, but have higher levels in the procyclic forms. Determination of Michaelis constants for the enzyme from each of the three T. brucei species did not reveal any significant kinetic differences between the bloodstream and procyclic enzymes. On Western blots, antibodies raised against dihydrolipoamide dehydrogenase from the stereorarian trypanosome, Trypanosoma cruzi, cross-react strongly with the dihydrolipoamide dehydrogenase from all three T. brucei species; by this method, the relative molecular masses of their dihydrolipoamide dehydrogenases are indistinguishable. Dihydrolipoamide dehydrogenase was purified from both the bloodstream and the procyclic forms of T. b. brucei, and the N-terminal have been sequenced. These sequences are identical to the derived protein sequence of the cloned gene (Else et al., Eur. J. Biochem. 212 (1993) 423-429), but have a nine amino acid N-terminal truncation, giving an N-terminus equivalent to that of T. cruzi dihydrolipoamide dehydrogenase. The T. b. brucei dihydrolipoamide dehydrogenase gene has been expressed in Escherichia coli and the resultant protein purified; its N-terminus is processed in a similar fashion to that in the trypanosome, but with reduced specificity.

Subcellular localisation of dihydrolipoamide dehydrogenase and detection of lipoic acid in bloodstream forms of Trypanosoma brucei.

In the long-slender bloodstream form of Trypanosoma brucei, the enzyme dihydrolipoamide dehydrogenase exists in the absence of the 2-oxo-acid dehydrogenase complexes of which it is normally a component, and appears to be associated with the plasma membrane of the organism [Danson, M. J., Conroy, K., McQuattie, A. & Stevenson, K. J. (1987) Biochem. J. 243, 661-665]. In the present paper, a complete subcellular fractionation of T. brucei has been carried out and, by comparison with marker enzymes, it is confirmed that the dihydrolipoamide dehydrogenase is indeed associated with the plasma membrane. In addition, we now provide evidence that the distribution of the enzyme is over the whole surface of the membrane, including the flagellar pocket region, and that the enzyme is not found in any other cellular fraction. A study of the latency of the enzyme suggests that it is located on the cytoplasmic surface of the plasma membrane. The discovery of the presumed substrate of dihydrolipoamide dehydrogenase, lipoic acid, is reported for T. brucei. Using a biological assay involving a strain of Escherichia coli that requires lipoic acid for growth, we have found that acid-hydrolysed extracts of T. brucei contain 1.7 (+/- 0.2) ng of the cofactor/mg protein. The chemical nature of the lipoic acid was confirmed by gas chromatography/mass spectrometry.

The ability of denbufylline to inhibit cyclic nucleotide phosphodiesterase and its affinity for adenosine receptors and the adenosine re-uptake site.

1. Denbufylline has been examined for its ability to inhibit cyclic nucleotide phosphodiesterase isoenzymes from rat cardiac ventricle and cerebrum, as well as for its affinity for adenosine A1 and A2 receptors and the re-uptake site. For comparison, SK&F 94120, theophylline and 3-isobutyl-1-methyl-xanthine (IBMX) were examined as phosphodiesterase inhibitors whilst N6-cyclohexyladenosine, R(-)-N6-(2-phenylisopropyl)-adenosine, 5'-N-ethylcarboxamido-adenosine, 2-nitrobenzylthioinosine, theophylline and IBMX were examined for their affinity for adenosine binding sites. 2. This investigation confirmed the presence of four phosphodiesterase activities in rat cardiac ventricle; in rat cerebrum only three were present. 3. Denbufylline selective inhibited one form of Ca2+-independent, low Km cyclic AMP phosphodiesterase. The form inhibited was one of two present in cardiac ventricle and the sole one in cerebrum. This form was not inhibited by cyclic GMP. The inotropic agent SK&F 94120 selectively inhibited the form of cyclic AMP phosphodiesterase which was inhibited by cyclic GMP present in cardiac ventricle. Theophylline and IBMX were relatively non-selective phosphodiesterase inhibitors. 4. Denbufylline was a less potent inhibitor of ligand binding to adenosine receptors than of cyclic AMP phosphodiesterase. This contrasted with theophylline, which had a higher affinity for adenosine receptors, and IBMX which showed no marked selectivity. Denbufylline, theophylline and IBMX all had a low affinity for the adenosine re-uptake site. 5. Denbufylline is being developed as an agent for the therapy of multi-infarct dementia. The selective inhibition of a particular low Km cyclic AMP phosphodiesterase may account for the activity of this compound.