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PubMed1991

Clinical radiobiology of squamous cell carcinoma of the oropharynx.

Local tumor control is analyzed in a series of 181 patients treated with definitive megavoltage radiotherapy (RT) for histologically proven squamous cell carcinoma of the oropharynx. Considerable variation in treatment time stemmed from the general use of a split-course technique in 49 patients treated from 1978 to 1985. Incomplete follow-up, in those patients alive and well at the termination of the study or who have died from metastases or intercurrent disease before developing a local recurrence, was allowed for by using a multi-variate mixture model. The tumor control probability (TCP) after radiotherapy showed a significant dependence on the following tumor and treatment characteristics: (a) tumor size: the number of tumor target cells increases approximately as the fourth root of estimated tumor volume; (b) sex: the estimated TCP in males is lower than in females with the same characteristics; (c) histopathological differentiation: well-differentiated tumors have a lower TCP than poorly and intermediately differentiated; (d) hemoglobin concentration: patients in the upper normal range have a significantly higher TCP than others; (e) total dose: there is a significant dose-response relationship; and (f) overall treatment time: TCP decreased with increasing overall time, the dose equivalent of proliferation with 2 Gy per fraction was 0.68 Gy/day with 95% confidence limits [0.05, 1.3] Gy/day. The TCP did not depend significantly on subsite within the oropharynx or nodal disease at presentation. The data were consistent with an alpha/beta ratio of the linear-quadratic model of 10 Gy.

Carcinoma, Squamous Cell↗
PubMed1992

Analysis of 3,5,6-trichloropyridinol in human urine using negative-ion chemical ionization gas chromatography-mass spectrometry.

A sensitive gas chromatographic-negative-ion chemical ionization mass spectrometric (GC-NCI-MS) method was developed to measure levels of the chlorpyrifos metabolite 3,5,6-trichloropyridinol (3,5,6-TCP) in human urine. The metabolic 3,5,6-TCP was isolated from urine by acid hydrolysis of urine aliquots, followed by diethyl ether extraction. The residues of ether extraction were taken up in o-xylene and derivatized with N-(tert.-butyldimethylsilyl)-N-methyltrifluoroacetamide (overall five-fold concentration). The structural isomer 3,4,5-trichloropyridinol was used as an internal standard in this analysis. This method was found to be linear for the determination of 3,5,6-TCP over the range 0.8-792 ng/ml. The limit of detection for 3,5,6-TCP in human urine was estimated to be 0.5 ng/ml. Low levels of 3,5,6-TCP (1-18 ng/ml of urine) were identified in control human urine samples. Structural confirmation of the background 3,5,6-TCP was obtained via GC-NCIMS-MS analysis.

Gas Chromatography-Mass Spectrometry↗
PubMedDate not supplied

Threshold, enzymatic, and pathologic changes associated with prolonged transcutaneous pacing in a chronic heart block model.

Previous studies have shown that 30 minutes of transcutaneous cardiac pacing (TCP) can induce mild, clinically insignificant myocardial damage. Longer use of TCP may cause more severe cardiac damage which might result in an increase in the capture threshold for subsequent transvenous cardiac pacing (TVP). To assess this possibility, we examined changes induced by TCP in a canine chronic heart block model. Heart block was induced in conditioned dogs (n = 8) by His bundle ablation. Seven to 10 days after induction of heart block, six animals were paced. Cardiac enzymes were drawn before pacing and at 4, 24, 48, and 72 hours after pacing. Although there was a significant rise in CK at 4 and 24 hours (P less than 0.05), there was no detectable rise in the MB fraction in any of the paced animals. There was no elevation of LDH after pacing, although three animals did develop an LDH1/LDH2 isoenzyme flip indicative of myocardial damage. Animals were sacrificed 72 hours after pacing and their hearts were examined for gross and microscopic changes. The hearts of the paced animals revealed subendocardial, subepicardial, and perivascular areas of basophilic degeneration involving less than 1% of the myocardium in four of six animals. No evidence of such damage was seen in two heart-blocked control animals not undergoing pacing. TVP and TCP capture thresholds assessed before and after a 60-minute TCP pacing period showed no significant change. Hence, use of TCP for a 60-minute period prior to TVP appears to be a safe emergency pacing technique.

Animals↗
PubMed2006

Combining particle size distribution and isothermal calorimetry data to determine the reaction kinetics of alpha-tricalcium phosphate-water mixtures.

Many calcium phosphate bone substitutes are based on the use of alpha-tricalcium phosphate (alpha-TCP) powder. This compound has been intensively studied, but some aspects of alpha-TCP reactivity are still controversial. The goal of this study was to determine the setting kinetics of alpha-TCP based on a new approach that compared particle size distribution data to isothermal calorimetry data. Results indicated that alpha-TCP conversion is mostly controlled by surface reactions, with at later stages a diffusion-controlled mechanism. The presence of an X-ray amorphous alpha-TCP fraction in the crystalline alpha-TCP powder increased the dissolution rate threefold, without modifying the reaction mechanism.

Biocompatible Materials↗
PubMed2006

Graft laceration and pullout strength of soft-tissue anterior cruciate ligament reconstruction: in vitro study comparing titanium, poly-d,l-lactide, and poly-d,l-lactide-tricalcium phosphate screws.

PURPOSE: The aim of part 1 of this study was to compare the structural properties of tibia-graft-interference screw constructs of soft-tissue graft fixation via the anterior cruciate ligament (ACL) by use of different interference screws. In part 2 the influence of graft laceration as a result of 1 single interference screw insertion on the mechanical properties of the graft was evaluated. METHODS: In part 1 of the study, in a bovine knee model, soft-tissue graft fixation with titanium, biodegradable poly-D,L-lactide (PLDLA), and PLDLA-tricalcium phosphate (TCP) interference screws was performed, and the structural properties were determined by use of a materials testing machine at a rate of 12.5 mm/s. In part 2 the bone was cut after 1 single insertion of titanium, PLDLA, or PLDLA-TCP interference screws, and the mechanical properties of the graft were determined. RESULTS: Soft-tissue graft fixation with a biodegradable PLDLA interference screw showed a statistically significantly higher maximum load when compared with PLDLA-TCP and titanium interference screws. PLDLA-TCP screw fixation revealed a significantly higher ultimate load than titanium screw fixation. The mechanical properties of the grafts after 1 single insertion of PLDLA-TCP interference screws showed a statistically significantly higher stiffness, yield load, and maximum load when compared with the PLDLA and titanium interference screw groups. CONCLUSIONS: The results of this study suggest that the use of a titanium screw to fix a soft-tissue graft in cruciate ligament reconstruction surgery may cause damage to the graft. Soft-tissue ACL reconstruction fixed with a PLDLA interference screw resulted in a significantly higher pullout strength than ACL reconstructions fixed with a PLDLA-TCP or titanium interference screw. CLINICAL RELEVANCE: In contrast to fixation with a biodegradable interference screw, fixation with a titanium interference screw may damage the integrity of a soft-tissue graft in cruciate ligament reconstruction and should therefore be avoided.

Absorbable Implants↗
PubMed2005

Novel PCL-based honeycomb scaffolds as drug delivery systems for rhBMP-2.

This study investigated a novel drug delivery system (DDS), consisting of polycaprolactone (PCL) or polycaprolactone 20% tricalcium phosphate (PCL-TCP) biodegradable scaffolds, fibrin Tisseel sealant and recombinant bone morphogenetic protein-2 (rhBMP-2) for bone regeneration. PCL and PCL-TCP-fibrin composites displayed a loading efficiency of 70% and 43%, respectively. Fluorescence and scanning electron microscopy revealed sparse clumps of rhBMP-2 particles, non-uniformly distributed on the rods' surface of PCL-fibrin composites. In contrast, individual rhBMP-2 particles were evident and uniformly distributed on the rods' surface of the PCL-TCP-fibrin composites. PCL-fibrin composites loaded with 10 and 20 microg/ml rhBMP-2 demonstrated a triphasic release profile as quantified by an enzyme-linked immunosorbent assay (ELISA). This consisted of burst releases at 2 h, and days 7 and 16. A biphasic release profile was observed for PCL-TCP-fibrin composites loaded with 10 microg/ml rhBMP-2, consisting of burst releases at 2 h and day 14. PCL-TCP-fibrin composites loaded with 20 microg/ml rhBMP-2 showed a tri-phasic release profile, consisting of burst releases at 2 h, and days 10 and 21. We conclude that the addition of TCP caused a delay in rhBMP-2 release. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and alkaline phosphatase assay verified the stability and bioactivity of eluted rhBMP-2 at all time points.

Body Fluids↗
PubMed2005

The inflammatory potential of biphasic calcium phosphate granules in osteoblast/macrophage co-culture.

Some biological properties of a range of biphasic calcium phosphate (BCP) granules were quantified using a co-culture model of primary unstimulated human osteoblasts and macrophages. The BCP granules were classified in terms of diameter and the ratio of tricalcium phosphate (TCP): hydroxyapatite (HA). All granules were 50% porous and sterilised by gamma-irradiation. Primary unstimulated human osteoblasts and macrophages were cultured in contact with the range of BCP granules for 1, 7 and 14 days. The response of the cells was determined and quantified by the assessment of viable cell adhesion to the substrate, using lactate dehydrogenase assay, and the production and release of the cytokines; interleukin 1beta, (IL-1beta), tumour necrosis factor alpha (TNF-alpha) and prostaglandin E2 (PGE2). Throughout the test period viable cell adhesion on all BCP granules was significantly lower than the tissue culture polystyrene control. Higher content TCP materials, (80% and 100% TCP) did not support viable cell adhesion after 1 day, lower content TCP materials, (20% and 50% TCP) granules did support viable cell adhesion throughout the time period. The percentage content of TCP was a more significant factor than granule size within the test conditions at all time points.

Biocompatible Materials↗
PubMed2006

Osteoinduction with highly purified beta-tricalcium phosphate in dog dorsal muscles and the proliferation of osteoclasts before heterotopic bone formation.

The aim of the study was to examine the chronological histology of osteoinduction of highly purified beta-tricalcium phosphate (beta-TCP) implanted in dog dorsal muscles. Specimens were harvested on days 14, 28, 42, 56, 112 and 168 after implantation, and were analyzed by hematoxylin and eosin (HE) staining, tartrate-resistant acid phosphatase (TRAP) staining, immunohistochemistry, in situ hybridization, and silver impregnation. After day 28, abundant TRAP- and cathepsin K-positive multinucleated cells adhered to beta-TCP, suggesting that these cells are osteoclasts that can resorb beta-TCP. On day 56, new bone was formed and alpha1 chain of type I procollagen mRNA-positive osteoblasts lined the newly formed bone. Silver impregnation showed abundant collagen fibrils within the beta-TCP micropores. These results suggest that micropores function as a storage space for extracellular matrix components, including collagen. Newly formed bone never degenerated in the late stage, suggesting that beta-TCP has good biocompatibility and this material retains the conditions appropriate for osteointegration and bioresorption. In conclusion, beta-TCP has osteoinductivity after implantation in dog dorsal muscles without use of bone marrow cells or osteoinductive cytokines. The appearance of a large number of active osteoclasts precedes new bone formation.

Animals↗
PubMed2004

Univariate analysis of factors correlated with tumor control probability of three-dimensional conformal hypofractionated high-dose radiotherapy for small pulmonary or hepatic tumors.

PURPOSE: To show the factors correlated with tumor control probability (TCP) after three-dimensional conformal hypofractionated high-dose radiotherapy (3D-CHHRT) for small pulmonary or hepatic tumors. METHODS AND MATERIALS: We enrolled 34 patients with 42 lesions (13 lung carcinomas, 6 hepatocellular carcinomas, and 23 lung or liver metastases) treated with 3D-CHHRT alone, with simple immobilization devices, between July 1997 and January 2002. We prescribed 45 Gy in three fractions at the 90-100% isodose line of the planning target volume. The median follow-up period was 18 months (range, 4-46 months). We calculated the TCP using the Kaplan-Meier method and univariate analysis for the following factors: age, gender, primary site, histologic type, tumor size, and previous treatment. RESULTS: Overall, the 2-year TCP of 3D-CHHRT was 83.6%. Local recurrence was observed in 6 lesions within 1 year after treatment. We showed that tumor size was the only significant factor to correlate with the TCP in univariate analysis. The 2-year TCP for tumors <3 cm or > or =3 cm in diameter was 95.0% and 58.3%, respectively (p = 0.0022). No severe adverse effects were observed. CONCLUSION: The TCP for tumors <3 cm in diameter was excellent for 3D-CHHRT.

Adenocarcinoma↗
PubMed2006

Transcutaneous monitoring of partial pressure of carbon dioxide in the elderly patient: a prospective, clinical comparison with end-tidal monitoring.

STUDY OBJECTIVE: To evaluate the accuracy and precision of estimation of partial pressure of carbon dioxide (Pa(CO2)) using end-tidal or transcutaneous CO2 (TcP(CO2)) measurements during mechanical ventilation in the elderly patient. DESIGN: A prospective, observational study was conducted. SETTINGS: The study was done in the anesthesia department of a university hospital. PATIENTS: Seventeen anesthetized, mechanically ventilated patients older than 60 years were studied. INTERVENTIONS AND MEASUREMENTS: During standard sevoflurane anesthesia, and after proper calibration and an equilibration time of 30 minutes with stable hemodynamic and respiratory variables, arterial (Pa(CO2)), end-tidal (Pet(CO2)), and transcutaneous (TcP(CO2)) CO2 partial pressures were determined. In each patient, 1 to 5 sample sets (Pa(CO2), Pet(CO2), and TcP(CO2)) were obtained. MAIN RESULTS: A total of 45 sample sets were obtained from the patients studied. The Pa(CO2) values ranged between 21 and 58 mm Hg. The Pa(CO2) - Pet(CO2) tension gradient was 6 +/- 5 mmHg (95% confidence interval, -3 to 16 mmHg), whereas the Pa(CO2) - TcP(CO2) tension gradient was 2 +/- 4 mmHg (95% confidence interval, -6 to 9 mmHg) (P = 0.0005). The absolute value of the difference between Pa(CO2) and Pet(CO2) was 3 mm Hg or less in 7 of 45 sample sets (15%), whereas the absolute value of the difference between Pa(CO2) and TcP(CO2) was 3 mm Hg or less in 21 of 45 sample sets (46%) (P = 0.003). Linear regression analysis for TcP(CO2) versus Pa(CO2) showed a slope of 0.84 (r(2) = 0.73), whereas the linear regression analysis for Pet(CO2) versus Pa(CO2) showed a slope of 0.54 (r(2) = 0.50). CONCLUSION: Transcutaneous monitoring of CO(2) partial pressure gives a more accurate estimation of arterial CO(2) partial pressure than does Pet(CO2) monitoring.

Aged↗
PubMed2005

Near-infrared spectroscopy and transcutaneous oxygen pressure during exercise to detect arterial ischemia at the buttock level: comparison with arteriography.

BACKGROUND: Noninvasive tests are required to detect (in both male and female subjects and side by side) arteries toward the hypogastric circulation that are likely to present significant lesions as a cause of buttock claudication. METHODS: We compared the accuracy of near-infrared spectroscopy (NIRS) and transcutaneous oxygen pressure (TCP o 2 ) on both buttocks during walking tests to detect lesions on the arteries toward the hypogastric circulation. NIRS was considered abnormal if recovery time to pre-exercise values was greater than 240 seconds for tissue oxygen saturation (absent data being coded 0), and TCP o 2 was coded abnormal if the minimal value of buttock changes minus chest changes was lower than -15 mm Hg. The study was conducted in a university hospital; there were 30 ambulatory patients with stage 2 claudication of the Fontaine classification. RESULTS: Angiography showed 36 abnormal (stenosis > 75%) and 24 normal arterial axes toward the buttocks circulation. NIRS and TCP o 2 provided respectively 55% (range, 41.6% to 67.9%) and 82% (range, 69.6% to 90.5%) accuracy (95% confidence interval) to predict the presence of arteriographically proven lesions; P < .05. CONCLUSIONS: Using available cut-off points proposed in the literature, NIRS showed a lower diagnostic accuracy than TCP o 2 for the prediction of lesions on the arterial tree to the hypogastric circulation. NIRS is a recent technique as compared with TCP o 2 , and its diagnostic accuracy might improve in the future. Currently, one should carefully weigh the advantages and limits of NIRS and TCP o 2 when a choice is to be made between them to monitor exercise-induced changes resulting from lower limb arterial disease at the proximal level.

Aged↗
PubMed2004

Tricalcium phosphate granules or rigid wedge preforms in open wedge high tibial osteotomy: a radiological study with a new evaluation system.

The capacity of two forms of porous beta-tricalcium phosphate bone substitutes (TCP) to promote bone healing in open wedge high tibial osteotomy (OWHTO) was studied. We reviewed the X-rays of 27 osteotomies, with either TCP wedges or TCP granules as filling material, to compare the bone healing rates and bone remodelling, at specific postoperative intervals. A new radiologic rating system for OWHTO was created and tested for clinical applicability. All osteotomies healed uneventfully and complete resorption of TCP was demonstrated at 1 year postoperative in 85% (n = 23) of the procedures. In 44% (n = 10) of these 23 procedures, the osteotomy site was no longer visible. No difference in bone healing rate and bone remodelling was found when comparing the use of granules to a wedge, and no adverse effects of TCP were observed. The good inter- (k = 0.7) and intraobserver (k = 0.6) reliability of the new radiologic rating system enables clinical use. Good bone healing was found in OWHTO with both wedges and granules of TCP.

Adult↗
PubMed1998

Thrombopoietin levels in HIV-associated thrombocytopenia in children.

OBJECTIVES: To determine the mechanism of human immunodeficiency virus (HIV)-associated thrombocytopenia by using thrombopoietin (TPO) levels. STUDY DESIGN: TPO levels were measured in 14 HIV+ children with thrombocytopenia (TCP+), 28 HIV+ children without thrombocytopenia (TCP-), and 15 matched control subjects. RESULTS: For the patients with moderate symptoms, TPO levels were similar for the TCP+ and TCP- groups (251 pg/mL vs 263 pg/mL; P =.98) and similar to those of control subjects. For the patients with severe symptoms, TPO levels were significantly higher for the TCP+ group versus the TCP- group (1172 pg/mL vs 222 pg/mL; P =.03). Patients with severe symptoms and thrombocytopenia had significantly higher TPO levels than those with moderate symptoms and thrombocytopenia (P <.005), were more likely to require growth factors, and did not respond to treatment with intravenous immunoglobulin. CONCLUSIONS: TPO levels can distinguish 2 groups of patients with HIV-associated thrombocytopenia. Patients with severe disease had elevated TPO levels, did not respond to treatment with intravenous immunoglobulin, and were more likely to be growth factor-dependent, suggesting marrow failure.

Child↗
PubMed2003

Bi-phasic change in BDNF gene expression following antidepressant drug treatment.

The gene for brain derived neurotrophic factor (BDNF) has recently received attention in relation to the therapeutic action of antidepressant treatment. This study aimed to clarify the influence of post drug interval on the effect of acute and repeated treatment with antidepressant drugs on BDNF gene expression in the rat brain. It was found that repeated administration of either the monoamine oxidase inhibitor tranylcypromine (TCP) or 5-hydroxytryptamine (5-HT) re-uptake inhibitors (fluoxetine, paroxetine and sertraline), evoke a bi-phasic and time-dependent effect on BDNF gene expression in the rat hippocampus (especially dentate gyrus). A down-regulation of the BDNF gene was detected at 4 h (TCP and fluoxetine) and an up-regulation at 24 h (TCP, paroxetine, fluoxetine, sertraline) after the last of twice daily injections for 14 days. After a single injection the down-regulation was detected at 4 h (TCP, fluoxetine, paroxetine and sertraline) but BDNF mRNA levels were not altered at 24 h post drug (TCP, fluoxetine and paroxetine). Administration of inhibitors of noradrenaline re-uptake (desipramine and maprotiline) or the atypical antidepressant mianserin had no effect on BDNF mRNA levels at either single (4 h post drug, desipramine) or repeated (24 h post drug, desipramine, maprotiline, mianserin) treatment. The gene expression for NT-3, which is distributed in a high density in the dentate gyrus, was not affected by single or repeated injections of antidepressant drugs (TCP, fluoxetine, paroxetine, sertraline, desipramine, maprotiline or mianserin) at 4 or 24 h post drug. In conclusion, these data show that the effect of antidepressant drugs on BDNF gene expression may be more complex and less widespread across treatments than previously thought. Thus, in this study drugs interacting with the central 5-HT system altered BDNF expression but the effect was bi-phasic over the 24 h post drug period.

Adrenergic Uptake Inhibitors↗
PubMed2003

Deriving freshwater quality criteria for 2,4,6-trichlorophenol for protection of aquatic life in China.

Freshwater quality criteria of 2,4,6-trichlorophenol (2,4,6-TCP) were developed with particular reference to the aquatic biota in China, and based on USEPA's guidelines. Acute toxicity tests were performed on nine different domestic species indigenous to China to determine 48 h LC(50) and 96 h LC(50) values for 2,4,6-TCP. In addition, 21 d survival-reproduction test with Daphnia magna, 30 d embryo-larval test with Carassius auratus, 60 d fry-juvenile test with Ctenopharyngodon idellus, 30 d early life stage test with Bufo bufo gargarizans and 96 h growth inhibition test with Scenedesmus obliqaus were also conducted to estimate lower chronic limit and upper chronic limit values. The final acute value (FAV) was 2.01 mg/l 2,4,6-TCP. Acute-to-chronic ratios ranged from 5.01 to 12.2. The final chronic value (FCV) and the final plant value (FPV) of 2,4,6-TCP were 0.226 and 2.24 mg/l respectively. Based on FAV, FCV and FPV for 2,4,6-TCP, a criteria maximum concentration of 1.01 mg/l and a criterion continuous concentration of 0.226 mg/l were derived. The results of this study provide useful data for deriving national or local water quality criteria for 2,4,6-TCP based on aquatic biota in China.

Animals↗
PubMed2002

Impaction grafting with morsellised allograft and tricalcium phosphate-hydroxyapatite: incorporation within ovine metaphyseal bone defects.

An ovine model was used to investigate the in vivo properties of impacted tricalcium phosphate-hydroxyapatite (TCP-HA) aggregates, varying in chemical composition (ratio of TCP to HA) and particle size distribution (8 versus 3 particle size ranges). All pellets were impacted to a standard compactive effort. Eight sheep underwent implantation of pellets in 4 metaphyseal defects in both rear limbs. Treatment groups consisted of: (1) allograft (clinical control). (2) 50/50 allograft/80% HA/20% TCP in 8 particle size ranges, (3) 50/50 allograft/80% TCP/20% HA in 8 sizes and (4) 50/50 allograft/80% HA/20% TCP in only 3 sizes of particles. Healing of defects was evaluated at 14 weeks with computed tomography, histology and histomorphometry. The computer tomography (CT) density measured in all defects containing synthetic agents was higher than in defects filled with allograft alone (p<0.01). Defects containing 8 sizes of 80% HA/ 20% TCP granules (group 2) achieved lower histological scores and contained less bone than the clinical control (p<0.05), whereas groups 3 and 4 did not differ from the control. Although all synthetic agents were osteoconductive, our results suggest that increasing the ratio of TCP over HA and limiting the number of particle size ranges to 3 instead of 8 improve the performance of impacted aggregates as graft expanders. Evaluation under loading conditions of morsellised allograft expanded with 80% TCP/20% HA (BoneSave) in 3 particle size ranges is warranted.

Animals↗
PubMed2004

Magnesia-doped HA/beta-TCP ceramics and evaluation of their biocompatibility.

The sintering behavior, mechanical properties and biocompatibility of magnesia (MgO)-doped HA/TCP biphasic ceramics were studied. Pure HA/TCP ceramics showed poor sinterability due to the phase transformation of beta- to alpha-TCP. MgO-doped HA/TCP ceramics showed high density without any phase transformation of beta-TCP up to 1300 degrees C, for MgO dopants incorporated into the beta-TCP preferably and increased thermal stability of beta-TCP. However, the addition of MgO higher than a critical content, suppressed grain growth of HA/TCP ceramics and lowered sinterability. The optimum amount of MgO doping was 1 wt%, which lead 99% relative density and higher mechanical properties than HA or beta-TCP ceramics. From in vitro test and in vivo test, 1 wt% MgO-doped HA/TCP ceramics showed a good biocompatibility without cytotoxicity. After implantation under the muscle of rabbits, beta-TCP phase was dissolved from the surface and a biological apatite covered the surface. These results proved that MgO addition increased drastically the sintering and mechanical properties of HA/beta-TCP ceramics without altering the biological safety and biocompatibility of the original composite.

Animals↗
PubMed2001

Purification and catalytic properties of the chlorophenol 4-monooxygenase from Burkholderia cepacia strain AC1100.

Burkholderia cepacia strain AC1100 can be induced for the degradation of 2,4,5-trichlorophenol (2,4,5-TCP). We have purified the active enzyme 30-fold to apparent homogeneity with a 44% yield by a two-step chromatographic procedure, and showed that it consists of a single type of subunit of 59 kDa based on SDS-PAGE using Coomassie blue and Sypro staining. This enzyme has no bound prosthetic group but requires exogenous addition of FAD and NADH to perform the dioxygen-dependent hydroxylation in the 4-position of 2,4,6-TCP. Studies of the stoichiometry revealed the consumption of 2 mol of NADH plus 1 mol of dioxygen per mol of 2,4,6-TCP with identification of the reaction product as 2,6-dichlorohydroquinone. Steady state kinetic parameters for cofactors and a variety of substrates were determined. Low K(m) values of 1+/-0.1 microM, 32+/-5 microM and 4+/-2 microM were found for FAD, NADH and 2,6-dichlorophenol (2,6-DCP), respectively, under saturating conditions for the two others. In the presence of 2,6-DCP as a substrate, methimazole (MMI) inhibited the enzyme competitively with a K(i)=27 microM. When other polychlorinated substrates were studied, IC(50) values for MMI were found in a range compatible with their apparent affinity. On the basis of aromatic product formation, NADH and O(2) consumption schemes for 2,4,6-TCP and 2,4,5-TCP degradation are discussed. A Blast search revealed that this enzyme has a high sequence identity (60%) with 2,4,6-TCP-4-monooxygenases from Burkholderia pickettii and from Azotobacter sp. strain GP1 which all of them catalyze para hydroxylative dehalogenation.

Burkholderia cepacia↗