Carbon isotope fractionation during aerobic biodegradation of trichloroethene by Burkholderia cepacia G4: a tool to map degradation mechanisms

The strain Burkholderia cepacia G4 aerobically mineralized trichloroethene (TCE) to CO(2) over a time period of approximately 20 h. Three biodegradation experiments were conducted with different bacterial optical densities at 540 nm (OD(540)s) in order to test whether isotope fractionation was consistent. The resulting TCE degradation was 93, 83.8, and 57.2% (i.e., 7.0, 16.2, and 42.8% TCE remaining) at OD(540)s of 2.0, 1.1, and 0.6, respectively. ODs also correlated linearly with zero-order degradation rates (1.99, 1.11, and 0.64 micromol h(-1)). While initial nonequilibrium mass losses of TCE produced only minor carbon isotope shifts (expressed in per mille delta(13)C(VPDB)), they were 57.2, 39.6, and 17.0 per thousand between the initial and final TCE levels for the three experiments, in decreasing order of their OD(540)s. Despite these strong isotope shifts, we found a largely uniform isotope fractionation. The latter is expressed with a Rayleigh enrichment factor, epsilon, and was -18.2 when all experiments were grouped to a common point of 42.8% TCE remaining. Although, decreases of epsilon to -20.7 were observed near complete degradation, our enrichment factors were significantly more negative than those reported for anaerobic dehalogenation of TCE. This indicates typical isotope fractionation for specific enzymatic mechanisms that can help to differentiate between degradation pathways.     

A virtual tour of the Guide for zebrafish users

PHS-funded and AAALAC-accredited facilities are required to use the Guide as the basis for setting up a zebrafish care and use program. The authors describe how they accomplished this task at the University of Oregon Zebrafish Facility.     

Identification of metabolites of fluorine-18-labeled M2 muscarinic receptor agonist, 3-(3-[(3-fluoropropyl)thio]-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine, produced by human and rat hepatocytes

An accurate, rapid method for the determination of unmetabolized 3-(3-[(3-[18F]fluoropropyl)thio]-1,2,5-thiadiazol-4-yl)-1.2,5,6-tetrahydro-1-methylpyridine (FP-TZTP), a selective M2 muscarinic agonist, is necessary in order to obtain quantitative information from positron emission tomography (PET) imaging. Using LC-MS-MS to analyze products from cultured human and rat hepatocytes, we identified metabolites resulting from oxidation of the nitrogen in the tetrahydropyridine ring, sulfur-oxidation, demethylation of the tertiary amine, and oxidation of the tetrahydropyridine ring. From the knowledge of the structure of the metabolites, we have developed a two-step extraction sequence that allows rapid determination of the parent fraction in plasma without time-consuming chromatographic analysis.     

A high-throughput Arabidopsis reverse genetics system

A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymmetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from approximately 100000 transformed lines. A total of 85108 TAIL-PCR products from 52964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org.     

Mutants of the CuA site in cytochrome c oxidase of Rhodobacter sphaeroides: II. Rapid kinetic analysis of electron transfer

The function of the binuclear Cu(A) center in cytochrome c oxidase (CcO) was studied using two Rhodobacter sphaeroides CcO mutants involving direct ligands of the Cu(A) center, H260N and M263L. The rapid electron-transfer kinetics of the mutants were studied by flash photolysis of a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine-55. The rate constant for intracomplex electron transfer from heme c to Cu(A) was decreased from 40000 s(-1) for wild-type CcO to 16000 s(-1) and 11000 s(-1) for the M263L and H260N mutants, respectively. The rate constant for electron transfer from Cu(A) to heme a was decreased from 90000 s(-1) for wild-type CcO to 4000 s(-1) for the M263L mutant and only 45 s(-1) for the H260N mutant. The rate constant for the reverse reaction, heme a to Cu(A), was calculated to be 66000 s(-1) for M263L and 180 s(-1) for H260N, compared to 17000 s(-1) for wild-type CcO. It was estimated that the redox potential of Cu(A) was increased by 120 mV for the M263L mutant and 90 mV for the H260N mutant, relative to the potential of heme a. Neither mutation significantly affected the binding interaction with cytochrome c. These results indicate that His-260, but not Met-263, plays a significant role in electron transfer between Cu(A) and heme a.     

A rapid and noninvasive method for detecting tissue-limited mosaicism: detection of i(12)(p10) in buccal smear from a child with Pallister-Killian syndrome

Pallister-Killian syndrome (PKS), a rare disorder, is characterized by tissue-limited or tissue-specific mosaicism. The characteristic chromosome abnormality associated with PKS is i(12p), which is seen predominantly in skin fibroblast cultures. Diagnosis of i(12p) has been carried out on buccal smears before and was shown to be an easy and feasible method. All previously published studies used alpha-satellite probes for the diagnosis and as such have several pitfalls. Our approach, using dual-color, locus-specific probes, has high specificity and sensitivity for the diagnosis of i(12p). Using statistical analysis, we have also confirmed that the signal pattern in interphase nuclei is consistent with isochromosome 12p.     

T-DNA recombination and replication in maize cells

T-DNA recombination and replication was analyzed in 'black mexican sweet' (BMS) cells transformed with T-DNAs containing the replication system from wheat dwarf virus (WDV). Upon recombination between the T-DNA ends, a promoterless marker gene (gusA) was activated. Activation of the recombination marker gene was delayed and increased exponentially over time, suggesting that recombination and amplification of the T-DNA occurred in maize cells. Mutant versions of the viral initiator gene (rep), known to be defective in the replication function, failed to generate recoverable recombinant T-DNA molecules. Circularization of T-DNA by the FLP/FRT site-specific recombination system and/or homologous recombination was not necessary to recover circular T-DNAs. However, replicating T-DNAs appeared to be suitable substrates for site-specific and homologous recombination. Among 33 T-DNA border junctions sequenced, only one pair of identical junction sites was found implying that the population of circular T-DNAs was highly heterogenous. Since no circular T-DNA molecules were detected in treatments without rep, it suggested that T-DNA recombination was linked to replication and might have been stimulated by this process. The border junctions observed in recombinant T-DNA molecules were indicative of illegitimate recombination and were similar to left-border recombination of T-DNA into the genome after Agro-mediated plant transformation. However, recombination between T-DNA molecules differed from T-DNA/genomic DNA junction sites in that few intact right borders were observed. The replicating T-DNA molecules did not enhance genomic random integration of T-DNA in the experimental configuration used for this study.     

The C. elegans apoptotic nuclease NUC-1 is related in sequence and activity to mammalian DNase II

The Caenorhabditis elegans nuc-1 gene has previously been implicated in programmed cell death due to the presence of persistent undegraded apoptotic DNA in nuc-1 mutant animals. In this report, we describe the cloning and characterization of nuc-1, which encodes an acidic nuclease with significant sequence similarity to mammalian DNase II. Database searches performed with human DNase II protein sequence revealed a significant similarity with the predicted C. elegans C07B5.5 ORF. Subsequent analysis of crude C. elegans protein extracts revealed that wild-type animals contained a potent endonuclease activity with a cleavage preference similar to DNase II, while nuc-1 mutant worms demonstrated a marked reduction in this nuclease activity. Sequence analysis of C07B5.5 DNA and mRNA also revealed that nuc-1(e1392), but not wild-type animals contained a nonsense mutation within the CO7B5.5 coding region. Furthermore, nuc-1 transgenic lines carrying the wild-type C07B5.5 locus demonstrated a complete complementation of the nuc-1 mutant phenotype. Our results therefore provide compelling evidence that the C07B5.5 gene encodes the NUC-1 apoptotic nuclease and that this nuclease is related in sequence and activity to DNase II.     

Understanding Disputes: The Politics of Argument

Understanding Disputes: The Politics of Argument. Pat Caplan. ed. Oxford, England: Berg Publishers, 1995. 248 pp.