Sensitive and rapid detection of 2,4-dicholorophenoxyacetic acid in water samples by using evanescent wave all-fiber immunosensor

Sensitive and rapid detection of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was achieved with a newly developed evanescent wave all-fiber immunosensor (EWAI). A reusable functional sensing surface of the immunosensor is prepared by covalent binding of 2,4-D-bovine serum albumin (2,4-D-BSA) conjugate to a self-assembled alkanethiol monolayer formed onto the fiber optic probe through heterobifunctional reagent. The quantification of free 2,4-D in samples was based on indirect competitive immunoreaction principle. Under optimum conditions, calibration curve obtained for 2,4-D had detection limits of 0.07 microg L(-1), the 50% inhibition concentration (IC(50)) was 3.93+/-0.03 microg L(-1) and the quantitative detection range was 0.22-69.5 microg L(-1). The antibodies binding on the sensor surface could be removed simply by the flow of a pepsin solution (pH 1.9), facilitating reuse of the same probe. The regeneration of the sensor surface allowed the performance of more than 100 assay cycles without significant loss of reactivity. The antibody showed negligible cross-reactivity against a few compounds structurally similar to 2,4-D. The immunosensor developed was successfully applied to the monitoring of 2,4-D in spiked water samples without significant effect of the matrix. The proposed portable immunosensor is promising for real-time on-site analysis of small molecules of environmental interest.     

EFFECT OF ETHYLENE ON CELLULAR DIFFERENTIATION IN ETIOLATED PEA SEEDLINGS

Ethylene (50 ppm) and 2,4-D (1 mm ) inhibit xylogenesis and completely prevent fiber lignification in the third internode of etiolated pea seedlings (Pisum sativum var. Alaska) during 96–240 hr of treatment. When ethylene is removed after 72 hr, normal differentiation resumes; thus the gas causes no irreversible damage to the cells. A combined treatment with 50 ppm ethylene and 50 μm 2,4-D or treatment with 1 mm 2,4-D alone causes inhibition similar to that observed with 50 ppm ethylene, and 50 μm 2,4-D is partially effective. The inhibition of xylogenesis and fiber lignification caused by 2,4-D is partly or wholly due to auxin-induced ethylene production.     

Genotoxic effect of 2,4-dichlorophenoxy acetic acid and its metabolite 2,4-dichlorophenol in mouse.

The cytogenetic effect of 2,4-dichlorophenoxy acetic acid (2,4-D) and its metabolite 2,4-dichlorophenol (2,4-DCP) was studied in bone-marrow, germ cells and sperm head abnormalities in the treated mice. Swiss mice were treated orally by gavage with 2,4-D at 1.7, 3.3 and 33 mg kg(-1)BW (1/200, 1/100 and 1/10 of LD(50)). 2,4-DCP was intraperitoneally (i.p.) injected at 36, 72 and 180 mg kg(-1)BW (1/10, 1/5, 1/2 of LD(50)). A significant increase in the percentage of chromosome aberrations in bone-marrow and spermatocyte cells was observed after oral administration of 2,4-D at 3.3 mg kg(-1)BW for three and five consecutive days. This percentage increased and reached 10.8+/-0.87 (P<0.01) in bone-marrow and 9.8+/-0.45 (P<0.01) in spermatocyte cells after oral administration of 2,4-D at 33 mg kg(-1)BW for 24 h. This percentage was, however, lower than that induced in bone-marrow and spermatocyte cells by mitomycin C (positive control). 2,4-D induced a dose-dependent increase in the percentage of sperm head abnormalities. The genotoxic effect of 2,4-DCP is weaker than that of 2,4-D, as indicated by the lower percentage of the induced chromosome aberrations (in bone-marrow and spermatocyte cells) and sperm head abnormalities. Only the highest tested concentration of 2,4-DCP (180 mg kg(-1)BW, 1/2 LD(50)) induced a significant percentage of chromosome aberrations and sperm head abnormalities after i.p. injection. The obtained results indicate that 2,4-D is genotoxic in mice in vivo under the conditions tested. Hence, more care should be given to the application of 2,4-D on edible crops since repeated uses may underlie a health hazard.     

Determination of SRS-A release from guinea-pig lungs by a radioimmunoassay

A sensitive radioimmunoassay for leukotrienes (LTs) has been developed. Rabbits were immunized with a conjugate of LTD4 and bovine serum albumin, prepared by using 1,5-difluoro-2,4-dinitrobenzene as the coupling agent. The assay can detect 0.045 pmol LTD4 at a final plasma dilution of 1:72. 50% displacement of bound 3H-LTD4 was obtained with 0.43 +/- 0.03 pmol LTD4. LTC4, LTE4 and LTF4 cross-react 159%, 57% and 85%, respectively, whereas LTB4, 5-HETE and prostaglandins did not. The assay was validated by measuring the antigen-induced release of LTs from sensitized guinea-pig chopped lungs. High correlation (0.9434, p less than 0.05) was found when LTs were simultaneously determined by this assay and a bioassay on guinea pig ileum.     

Biodegradation kinetics of 2,4-D by bacterial strains isolated from soil

The aim of the study was to characterize the 2,4-dichlorophenoxyacetic acid (2,4-D) degradative potential of three bacterial strains identified by MIDI-FAME profiling as Burkholderia cepacia (DS-1), Pseudomonas sp. (DS-2) and Sphingomonas paucimobilis (DS-3) isolated from soil with herbicide treatment history. All strains were capable of using herbicide as the only source of carbon and energy when grown in mineral salt medium (MSM) containing 2,4-D (50 mg/l). Over a 10 day incubation period, 69%, 73% and 54% of the initial dose of 2,4-D were degraded by strains DS-1, DS-2 and DS-3, respectively. Analysis of 2,4-dichlorophenol (2,4-DCP) concentration, the main metabolite of 2,4-D degradation, revealed that strains DS-1 and DS-2 may also have the potential to metabolize this compound. The percentage of 2,4-DCP removal was 67% and 77% in relation to maximum values of 9.5 and 9.2 mg/l determined after 4 and 2 days for MSM+DS-1 and MSM+DS-2, respectively. The degradation kinetics of 2,4-D (50 mg/kg) in sterile soil (SS) showed different potential of tested strains to degrade 2,4-D. The times within which the initial 2,4-D concentration was reduced by 50% (DT₅₀) were 6.3, 5.0 and 9.4 days for SS+DS-1, SS+DS-2 and SS+DS-3, respectively.     

Detection of Clostridium botulinum toxin A using a fiber optic-based biosensor.

A rapid, sensitive, analytical method for the detection of Clostridium botulinum toxin has been developed. The fiber optic-based biosensor utilizes the evanescent wave of a tapered optical fiber for signal discrimination. A 50 mW argon-ion laser, which generates laser light at 514 nm, is used in conjunction with an optical fiber probe that is tapered at the distal end. Antibodies specific for C. botulinum are covalently attached to the surface of the tapered fiber. The principle of the system is a sandwich immunoassay using rhodamine-labeled polyclonal anti-toxin A immunoglobin G (IgG) antibodies for generation of the specific fluorescent signal. Various anti-toxin antibodies were immobilized to the fibers. Affinity-purified polyclonal horse anti-toxin A antibodies performed better than the IgG fraction from the same horse serum or than the monoclonal anti-toxin A antibody BA11-3. Botulinum toxin could be detected within a minute, at concentrations as low as 5 ng/ml. The reaction was highly specific and no response was observed against tetanus toxin.     

Highly sensitive amperometric immunosensors for microcystin detection in algae

The presence of cyanobacterial toxins in water and algae pose a health hazard for animals and humans, due to their tumour-promoting activity and carcinogen effects. The use of simple, rapid and reliable tools for routine analysis is becoming a necessity. With this purpose, our group has developed two electrochemical immunosensors for the detection of microcystin-LR (MC-LR) based on the affinity between this cyanotoxin and the corresponding monoclonal and polyclonal antibodies. A competitive direct enzyme-linked immunosorbent assays (ELISAs) was designed and, after validation of the approach on microtiter wells, screen-printed graphite electrodes were used as supports. Colorimetry was used to optimise the experimental parameters and to compare the performance of monoclonal and polyclonal antibodies. Afterwards, electrochemical measurements were performed at -200 mV (versus Ag/AgCl) using 5-methyl-phenazinium methyl sulfate (MPMS) as mediator for horseradish peroxidase (HRP), the enzymatic label of the competitor. The IC(50) values were 0.10 and 1.73 microgL(-1) for MAb and PAb, respectively. Whereas Mab provided higher sensitivities, the reproducibility was better when using PAb. The developed amperometric immunosensors were applied to the analysis of cyanobacterial samples from the Tarn River (Midi-Pyrénées, France) and the presence of MC was confirmed by the colorimetric protein phosphatase inhibition (PPI) assay and high performance liquid chromatography (HPLC). The limits of detection attained from the calibration curves and the results obtained for the real samples demonstrate the potential use of the immunosensors as screening tools for routine use in the assessment of water quality and the control of toxins in algae.     

Intrinsic tryptophan fluorescence as a probe of metal and alpha-ketoglutarate binding to TfdA,a mononuclear non-heme iron dioxygenase

2,4-Dichlorophenoxyacetic acid (2,4-D)/alpha-ketoglutarate (alphaKG) dioxygenase, TfdA, couples the oxidative decarboxylation of alphaKG to the oxidation of the herbicide 2,4-D using a mononuclear non-heme Fe(II) active site. The intrinsic tryptophan fluorescence associated with the four Trp residues in TfdA allows for the use of fluorescence spectroscopy to monitor the binding of iron and alphaKG to the enzyme. The fluorescence spectrum of TfdA is quenched by 50-85% upon addition of Fe(II) or alphaKG, allowing determination of their binding affinities (K(d)=7.45+/-0.61 and 3.35+/-0.35 microM, respectively). Cu, Zn, Mn, Co, Mg, and Ca dictations also quench the TfdA fluorescence with affinities similar to that of Fe(II), whereas monovalent cations such as Na, K, and Li do not. H114A and D116A mutant forms of TfdA, lacking either a histidine or aspartate metallocenter ligand, exhibit weaker affinity for both Fe(II) and alphaKG based on the fluorescence changes. Trp256 is predicted to lie within 5 A of the metal and alphaKG binding sites; however, its substitution by Phe or Leu has negligible effects on the Fe(II)- and alphaKG-dependent fluorescence quenching. Because Trp195 is predicted to be quite distant ( approximately 15 A) from the active site, we conclude that some combination of Trp113 and Trp248 serves as the reporter that senses metal and cofactor binding to TfdA.     

Clastogenicity of pentachlorophenol, 2,4-D and butachlor evaluated by Allium root tip test

The meristematic mitotic cells of Allium cepa is an efficient cytogenetic material for chromosome aberration assay on environmental pollutants. For assessing genotoxicity of pentachlorophenol (PCP), 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-chloro-2,6-diethyl-N-(butoxymethyl) acetanilide (butachlor), 50% effective concentration (EC(50)), c-mitosis, stickiness, chromosome breaks and mitotic index (MI) were used as endpoints of genotoxicity. EC(50) values for PCP and butachlor are 0.73 and 5.13 ppm, respectively. 2,4-D evidently induced morphological changes at higher concentrations. Some changes like crochet hooks, c-tumours and broken roots were unique to 2,4-D at 5-20 ppm. No such abnormalities were found in PCP and butachlor treated groups, however, root deteriorated and degenerated at higher concentrations (<3 ppm) in PCP. MI in 2,4-D showed a low average of 14.32% followed by PCP (19.53%), while in butachlor it was recorded 71.6%, which is near to the control value. All chemicals induced chromosome aberrations at statistically significant level. The highest chromosome aberration frequency (11.90%) was recorded in PCP at 3 ppm. Large number of c-mitotic anaphases indicated that butachlor acts as potent spindle inhibitor, whereas, breaks, bridges, stickiness and laggards were most frequently found in PCP showing that it is a potent clastogen.     

Gene probe analysis of soil microbial populations selected by amendment with 2,4-dichlorophenoxyacetic acid.

Soils with a history of 2,4-dichlorophenoxyacetic acid (2,4-D) treatment at field application rates and control soils with no prior exposure to 2,4-D were amended with 2,4-D in the laboratory. Before and during these treatments, the populations of 2,4-D-degrading bacteria were monitored by most-probable-number (MPN) enumeration and hybridization analyses, using probes for the tfd genes of plasmid pJP4, which encode enzymes for 2,4-D degradation. Data obtained by these alternate methods were compared. Several months after the most recent field application of 2,4-D (approximately 1 ppm), soils with a 42-year history of 2,4-D treatment did not have significantly higher numbers of 2,4-D-degrading organisms than did control soils with no prior history of treatment. In response to laboratory amendments with 2,4-D, both the previously treated soils and those with no prior history of exposure exhibited a dramatic increase in the number of 2,4-D-metabolizing organisms. The MPN data indicate a 4- to 5-log population increase after one amendment with 250 ppm of 2,4-D and ultimately a 6- to 7-log increase after four additional amendments, each with 400 ppm of 2,4-D. Similarly, when total bacterial DNA from the soil microbial community of these samples was analyzed by using a probe for the tfdA gene (2,4-D monoxygenase) or the tfdB gene (2,4-dichlorophenol hydroxylase) a dramatic increase in the level of hybridization was observed in both soils.(ABSTRACT TRUNCATED AT 250 WORDS)     

Herbicide 2,4-dichlorophenoxyacetic acid (2,4-D)-induced cytogenetic damage in human lymphocytes in vitro in presence of erythrocytes

The genotoxic effects of 2,4-D and its commercial derivative 2,4-D DMA were studied by measuring sister chromatid exchange (SCE), cell-cycle progression and mitotic index in human whole blood (WBC) and plasma leukocyte cultures (PLC). Concentrations of 10, 25, 50 and 100 microg herbicide/ml were used during 72 h. In WBC, a significant increase in SCE frequency was observed within the 10-50 microg 2,4-D/ml and 25-100 microg 2,4-D DMA/ml dose range. Contrarily, in PLC, none of the concentrations employed affected the SCEs frequency. A significant delay in cell proliferation was observed in WBC after treatments with 25 and 50 microg 2,4-D/ml and 50 and 100 microg 2,4-D DMA/ml. In PLC, only 100.0 microg 2,4-D/ml altered cell-cycle progression. For both chemicals, a progressive dose-related inhibition of mitotic activity was observed. The results demonstrated that the presence of erythrocytes in the culture system modulated the DNA and cellular damage inflicted by 2,4-D and 2,4-D DMA into human lymphocytes in vitro as well as both 2,4-D and 2,4-D DMA were more potent genotoxic agents in the presence of human red cells.     

Gene probe analysis of soil microbial populations selected by amendment with 2,4-dichlorophenoxyacetic acid.

Soils with a history of 2,4-dichlorophenoxyacetic acid (2,4-D) treatment at field application rates and control soils with no prior exposure to 2,4-D were amended with 2,4-D in the laboratory. Before and during these treatments, the populations of 2,4-D-degrading bacteria were monitored by most-probable-number (MPN) enumeration and hybridization analyses, using probes for the tfd genes of plasmid pJP4, which encode enzymes for 2,4-D degradation. Data obtained by these alternate methods were compared. Several months after the most recent field application of 2,4-D (approximately 1 ppm), soils with a 42-year history of 2,4-D treatment did not have significantly higher numbers of 2,4-D-degrading organisms than did control soils with no prior history of treatment. In response to laboratory amendments with 2,4-D, both the previously treated soils and those with no prior history of exposure exhibited a dramatic increase in the number of 2,4-D-metabolizing organisms. The MPN data indicate a 4- to 5-log population increase after one amendment with 250 ppm of 2,4-D and ultimately a 6- to 7-log increase after four additional amendments, each with 400 ppm of 2,4-D. Similarly, when total bacterial DNA from the soil microbial community of these samples was analyzed by using a probe for the tfdA gene (2,4-D monoxygenase) or the tfdB gene (2,4-dichlorophenol hydroxylase) a dramatic increase in the level of hybridization was observed in both soils.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molybdenum in human whole blood of adult residents of the Merida State (Venezuela)

The concentration of molybdenum was measured in whole blood samples of 418 (244 males and 174 females) apparently normal donors ranging in age from 18 to 27-years old and living in nine different locations in the Mérida State (Venezuela). The geometric mean concentration of molybdenum of 418 subjects was of 2.66+/-0.66 microgL(-1) (range: 1.20-4.80 microgL(-1)). The levels of molybdenum in whole blood samples found in this work were of 2.57+/-0.52 and 2.54+/-0.51 (range: 1.20-4.80 and 1.40-4.20) microgL(-1) for males and females, respectively. The data of the content molybdenum in whole blood had no statistical correlation with age, sex or height above the sea level of the sampling sites. However, there was a tendency to decrease the levels of the element in those sampling sites located in highlands (> or = 1900 m above the sea level). This variability may be due to the source of molybdenum from the soil to the food chain that has affected its levels in donors from these areas under study. The results of this study are compared with values previously reported for subjects studied in other populations.     

Preparation of anti-2,4-dichlorophenol and 2,4-dichlorophenoxyacetic acid monoclonal antibodies

The ratios of hapten and bovine serum albumin (BSA) in an antigen conjugate were determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Hybridomas secreting monoclonal antibodies against 2,4-dichlorophenoxyacetic acid (2,4-D) were produced by fusing 2,4-D-BSA conjugate-immunized splenocytes with a HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. A substantial cross-reaction was observed for 2,4-dichlorophenol (2,4-DP) when compared with that observed for 2,4-D. The full measurement range for this assay is 0.2–3 μg ml⁻¹ for 2,4-DP. On the other hand, the range for 2,4-D is between 1 and 20 μg ml⁻¹. This revised version was published online in July 2006 with corrections to the Cover Date.     

Phenanthrenes and a dihydrophenanthrene from Tamus communis and their cytotoxic activity

From the petroleum ether extract of the rhizomes of Tamus communis, the 7-hydroxy-2,3,4,8-tetramethoxyphenanthrene (1) was isolated, together with the known 2,3,4-trimethoxy-7,8-methylenedioxyphenanthrene (2), 3-hydroxy-2,4,-dimethoxy-7,8-methylenedioxyphenanthrene (3), 2-hydroxy-3,5,7-trimethoxyphenanthrene (4) and 2-hydroxy-3,5,7-trimethoxy-9,10-dihydrophenanthrene (5), through cytotoxic assay guidance. The structures were determined by means of HREIMS, (1)H NMR, JMOD and NOESY experiments. The cytotoxic effects of the isolated compounds were tested on cervix adenocarcinoma (HeLa) cells, with the MTT assay. The results demonstrated that, with the exception of 2, all these compounds displayed pronounced cytotoxic activity; especially 1 and 3 exhibited significant cell growth inhibitory effects, with IC(50)=8.52+/-0.70 and 3.64+/-0.12 microM, respectively.     

Enzymatic recycling-based amperometric immunosensor for the ultrasensitive detection of okadaic acid in shellfish

Electrochemical immunosensors based on a competitive indirect enzyme-linked immunosorbent assay (ciELISA) and an enzymatic recycling system were developed for the detection of okadaic acid (OA). OA-ovalbumin (OA-OVA) conjugate was immobilised on screen-printed electrodes (SPEs) and competition of a newly generated monoclonal antibody (MAb) for free and immobilised OA was subsequently performed. Secondary antibodies labelled with alkaline phosphatase (ALP) or horseradish peroxidase (HRP) were used for signal generation. Experimental parameters were firstly optimised by colorimetric ELISA on microtiter wells and on SPEs. The ELISA system was then tested by amperometry at +300 mV vs. Ag/AgCl (detection of p-aminophenol produced by the reaction of p-aminophenyl phosphate with ALP) or -200 mV vs. Ag/AgCl (detection of 5-methyl-phenazinium methyl sulfate, redox mediator in the HRP bioelectrocatalysis). The limits of detection (LODs) with standard solutions were 1.07 and 1.98 microgL(-1) when using ALP and HRP labels, respectively. An electrochemical signal amplification system based on diaphorase (DI) recycling was integrated into the ALP-based immunosensor, decreasing the LOD to 0.03 microgL(-1) and enlarging the working range by two orders of magnitude. Preliminary results with mussel and oyster extracts were obtained and compared with the colorimetric immunoassay, the colorimetric protein phosphatase inhibition assay (PPIA) and LC-MS/MS.     

A packed Cytodex microbead array for three-dimensional cell-based biosensing

A packed Cytodex 3 microbead array was fabricated as a simple three-dimensional (3-D) cell-based biosensing format. Resting membrane potentials and voltage-gated calcium channel (VGCC) function of SH-SY5Y human neuroblastoma cells cultured on the microbead array versus collagen-coated flat (2-D) substrates were evaluated by confocal microscopy with a potentiometric dye, tetramethylrhodamine methyl ester, and a calcium fluorescent indicator, Calcium Green-1. SH-SY5Y cells, differentiated with 1mM dibutyryl cAMP and 2.5 microM 5-bromodeoxyuridine, showed significant resting membrane potential establishment on the topographical scaffolds in a period of 13 days into differentiation, in contrast to the previously reported insignificant resting membrane potential establishment of the same cells within collagen hydrogels. On days 2, 8 and 13 into differentiation, cells on collagen-coated flat substrates developed resting membrane potentials of -6.0+/-19.5 mV (n=198), -30.5+/-19.9 mV (n=191) and -21.7+/-18.9 mV (n=308), in contrast to values for cells on 3-D scaffolds of -25.8+/-14.7 mV (n=112), -37.6+/-13.1 mV (n=120) and -28.7+/-12.2 mV (n=158), respectively. The development of VGCC function, as measured by percentage of cells responsive to 50 mM high K(+) depolarization, was significantly slower for cells on 3-D scaffolds (20.0% on day 13 into differentiation) than for cells on 2-D substrates (30.7% on day 8 into differentiation). The exaggerated 2-D cell calcium dynamics, in comparison with those of 3-D cells, is consistent with previous 2-D/3-D comparative studies. This study established the rationale and feasibility of the microbead array format for 3-D cell-based biosensing.     

Transformation of mono- and dichlorinated phenoxybenzoates by phenoxybenzoate-dioxygenase in pseudomonas pseudoalcaligenes POB310 and a modified diarylether-metabolizing bacterium

Pseudomonas pseudoalcaligenes POB310 contains genes that encode phenoxybenzoate dioxygenase. The enzyme transforms mono- and dichlorinated phenoxybenzoates to yield protocatechuate that is used as a growth substrate and chlorophenols that are nonmetabolizable. Mass spectral analysis of (18)O metabolites obtained from the protocatechuate 3,4-dioxygenase-deficient mutant, POB310-B1, suggested that the reaction mechanism is a regioselective angular dioxygenation. A cloning vector containing reaction relevant genes (pD30.9) was transferred into Pseudomonas sp. strain B13 containing a modified ortho-cleavage pathway for aromatic compounds. The resultant Pseudomonas sp. strain B13-D5 (pD30.9) completely metabolized 3-(4-chlorophenoxy)benzoate. During growth on 3-phenoxybenzoate, strain B13-D5 (pD30.9) (K(s) = 0.70+/-0.04 mM, mu(max) = 0.45+/-0.03 h(-1), t(d) = 1.5 h, Y = 0.45+/-0.03 g bio- mass x g substrate(-1)) was better adapted to low substrate concentrations, had a faster rate of growth, and a greater yield than POB310 (K(s) = 1.13+/-0.06 mM, mu(max) = 0.31+/-0.02 h(-1), t(d) = 2.2 h, Y = 0.39+/-0.02 g biomass. g substrate(-1)).     

Selected contribution: skeletal muscle capillarity and enzyme activity in rats selectively bred for running endurance.

To attempt to explain the difference in intrinsic (untrained) endurance running capacity in rats selectively bred over seven generations for either low (LCR) or high running capacity (HCR), the relationship among skeletal muscle capillarity, fiber composition, enzyme activity, and O(2) transport was studied. Ten females from each group [body wt: 228 g (HCR), 247 g (LCR); P = 0.03] were studied at 25 wk of age. Peak normoxic maximum O(2) consumption and muscle O(2) conductance were previously reported to be 12 and 33% higher, respectively, in HCR, despite similar ventilation, arterial O(2) saturation, and a cardiac output that was <10% greater in HCR compared with LCR. Total capillary and fiber number in the medial gastrocnemius were similar in HCR and LCR, but, because fiber area was 37% lower in HCR, the number of capillaries per unit area (or mass) of muscle was higher in HCR by 32% (P < 0.001). A positive correlation (r = 0.92) was seen between capillary density and muscle O(2) conductance. Skeletal muscle enzymes citrate synthase and beta-hydroxyacyl-CoA dehydrogenase were both approximately 40% higher (P < 0.001) in HCR (12.4 +/- 0.7 vs. 8.7 +/- 0.4 and 3.4 +/- 0.2 vs. 2.4 +/- 0.2 mmol. kg(-1). min(-1), respectively), whereas phosphofructokinase was significantly (P = 0.02) lower in HCR (27.8 +/- 1.2 vs. 35.2 +/- 2.5 mmol. kg(-1). min(-1)) and hexokinase was the same (0.65 +/- 0.04 vs. 0.65 +/- 0.03 mmol. kg(-1). min(-1)). Resting muscle ATP, phosphocreatine, and glycogen contents were not different between groups. Taken together, these data suggest that, in rats selectively bred for high-endurance exercise capacity, most of the adaptations for improved O(2) utilization occur peripherally in the skeletal muscles and not in differences at the level of the heart or lung.     

Probing the 2,4-dichlorophenoxyacetate/alpha-ketoglutarate dioxygenase substrate-binding site by site-directed mutagenesis and mechanism-based inactivation

TfdA is an Fe(II)- and alpha-ketoglutarate- (alphaKG-) dependent dioxygenase that hydroxylates the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) producing a hemiacetal that spontaneously decomposes to 2,4-dichlorophenol and glyoxylate. On the basis of a recently published TfdA structural model [Elkins et al. (2002) Biochemistry 41, 5185-5192], His214, Lys71, Arg278, and the backbone amide of Ser117 are suggested to bind the 2,4-D carboxylate; Lys95 and possibly Lys71 are hypothesized to interact with the 2,4-D ether atom; and Arg274 and Thr141 are suspected to bind alphaKG. TfdA variants with substitutions at these and other positions were purified and characterized in order to explore the roles of these residues in catalysis. The K71L, K71Q, K95L, K95Q, R274Q, R274L, and R278Q variants exhibited significantly increased 2,4-D K(m), alphaKG K(m), and alphaKG K(d) values, consistent with their proposed roles in substrate binding. A protease-sensitive site was successfully eliminated in the R78Q variant, which also exhibited decreased affinity for 2,4-D. In contrast, the Y81F, Y126F, T141V, Y169F, and Y244F variants showed only modest changes in their kinetics. An observed 4-fold lower K(m) of the K95L variant compared to wild-type protein with the alternative substrate 2,4-dichlorocinnamic acid provided additional evidence for an interaction between Lys95 and the 2,4-D ether atom. Phenylpropiolic acid was identified as a mechanism-based inactivator of the enzyme [K(i) = 38.1 +/- 6.0 microM and k(inact)(max) = 2.3 +/- 0.1 min(-1)]. This acetylenic compound covalently modifies a peptide (166-AEHYALNSR-174) that is predicted to form one side of the substrate-binding pocket. The K95L variant of TfdA was not inactivated by phenylpropiolic acid, providing added support that Lys95 is present at the active site. These results support the identity of suspected substrate-binding residues derived from structural modeling studies and extend our understanding of the oxidative chemistry carried out by TfdA.