Use of tracer tests to evaluate the impact of enhanced-solubilization flushing on in-situ biodegradation.

Tracer tests were conducted to evaluate the effect of a complexing sugar flush (CSF) on in-situ biodegradation potential at a site contaminated by jet fuel, solvents, and other organic compounds. Technical-grade hydroxypropyl-beta-cyclodextrin was used during the CSF study, which was conducted in a hydraulically isolated cell emplaced in a surficial aquifer. In-situ biodegradation potential was assessed with the use of tracer tests, which were conducted prior to and immediately following the CSF study. Ethanol, hexanol, and benzoate were used as the biodegradable tracers, while bromide was used as a nonreactive tracer. The results indicate that the biodegradation of benzoate was similar for both tracer tests. Conversely, the biodegradation of ethanol (23% increase) and hexanol (41% increase) was greater for the post-CSF tracer test. In addition, analysis of core samples collected from within the test cell indicates that the population density of aerobic jet-fuel degraders increased in the vicinity of the injection wells during the CSF. These results indicate that the cyclodextrin flush did not deleteriously affect the indigenous microbial community. This study illustrates that tracer tests can be used to evaluate the impact of remediation activities on in-situ biodegradation potential.

Controlled release of aldicarb from lignin loaded ionotropic hydrogel microspheres.

Aldicarb loaded microspheres of carboxymethyl cellulose, CMC, of various compositions were prepared with the crosslinking action of Al(III). Into these microspheres, lignin was introduced as a filler to further regulate the rate of release. The encapsulation efficiency was low (4.15-13.00%) due to the high water solubility of aldicarb. Release into aqueous media was quite rapid and almost complete within 24h. Initial stages of the release (60% of AS released) was investigated to determine the release mechanism and it was found that release from the microspheres was governed by Fickian diffusion. Among the various parameters, the ratio of lignin to CMC and aldicarb to CMC were found to be influential on the rate of release from the crosslinked CMC-lignin microspheres. The soil behaviour confirmed the delayed release effect of the controlled release formulation compared to Temik (a commercial preparation of aldicarb), especially when lignin was incorporated.

Intensity-modulated radiotherapy: first results with this new technology on neoplasms of the head and neck.

Intensity-modulated beam radiotherapy (IMRT) delivers a highly conformal, three-dimensional (3-D) distribution of radiation doses that is not possible with conventional methods. When administered to patients with head and neck tumors, IMRT allows for the treatment of multiple targets with different doses, while simultaneously minimizing radiation to uninvolved critical structures such as the parotid glands, optic chiasm, and mandible. With 3-D computerized dose optimization, IMRT is a vast improvement over the customary trial-and-error method of treatment planning. We retrospectively reviewed the charts of the first 28 head and neck patients at our institution who were treated with IMRT. All had head and neck neoplasms, including squamous cell carcinoma, adenoid cystic carcinoma, paraganglioma, and angiofibroma. Total radiation doses ranged from 1,400 to 7,100 cGy, and daily doses ranged from 150 to 400 cGy/day. A quality assurance system ensured that computer-generated dosimetry matched film dosimetry in all cases. For midline tumors, this system allowed us to decrease the dose to the parotid glands to less than 3,000 cGy. The incidence of acute toxicity was drastically lower than that seen with conventional radiotherapy delivery to similar sites. This is the first report of the application of IMRT strictly to head and neck neoplasms. We discuss the indications, technique, and initial results of this promising new technology. We also introduce the concept of the Simultaneous Modulated Accelerated Radiation Therapy boost technique, which has several advantages over other altered fractionation schemes.

The modified beta-ketoadipate pathway in Rhodococcus rhodochrous N75: enzymology of 3-methylmuconolactone metabolism.

Rhodococcus rhodochrous N75 is able to metabolize 4-methylcatechol via a modified beta-ketoadipate pathway. This organism has been shown to activate 3-methylmuconolactone by the addition of coenzyme A (CoA) prior to hydrolysis of the butenolide ring. A lactone-CoA synthetase is induced by growth of R. rhodochrous N75 on p-toluate as a sole source of carbon. The enzyme has been purified 221-fold by ammonium sulfate fractionation, hydrophobic chromatography, gel filtration, and anion-exchange chromatography. The enzyme, termed 3-methylmuconolactone-CoA synthetase, has a pH optimum of 8.0, a native Mr of 128,000, and a subunit Mr of 62,000, suggesting that the enzyme is homodimeric. The enzyme is very specific for its 3-methylmuconolactone substrate and displays little or no activity with other monoene and diene lactone analogues. Equimolar amounts of these lactone analogues brought about less than 30% (most brought about less than 15%) inhibition of the CoA synthetase reaction with its natural substrate.

Intensity modulated conformal therapy for intracranial lesions.

The Peacock planning and delivery system was used to create treatment plans and deliver these plans to patients. The system involves an arc therapy delivery of small (2 cm long) slices of radiation combined with indexing of the couch to achieve target coverage. Two clinical examples are shown to demonstrate the system's capability and evaluate the resources required to produce and deliver the plans. One plan is an optic sheath meningioma and the other is a craniopharyngioma that surrounded the optic chiasm. The optic sheath meningioma was treated to 50 Gy in 25 fractions. The treatment involved delivery of two arcs. The total time to set up the patient and deliver the treatment was less than 15 min. Planning and plan validation after computed tomography required approximately 3 days. The patient had 100% restoration of her field of vision and is stable 3 years post therapy. The second patient is a 9-year-old who had a craniopharyngioma which surrounded the optic chiasm. The tumor was treated to 50.4 Gy in 28 fractions and the dose to the optic chiasm was limited to 45 Gy. The treatment required three arcs and total treatment time was less than 20 min. The patient is stable 15 months post therapy. The system is able to create and deliver radiation patterns that are unique. These plans can be created and delivered in times that rival conventional forward planning conformal radiotherapy systems that cannot produce or conveniently deliver such plans.

Biosynthesis of a cyclic tautomer of (3-methylmaleyl)acetone from 4-hydroxy-3,5-dimethylbenzoate by Pseudomonas sp. HH35 but not by Rhodococcus rhodochrous N75.

Here we report that the bacterial catabolism of 4-hydroxy-3,5-dimethylbenzoic acid 1 takes a different course in Rhodococcus rhodochrous N75 and Pseudomonas sp. strain HH35. The former organism accumulates a degradation metabolite of the acid which we isolated and identified as 2,6-dimethylhydroquinone 2. The latter bacterial strain converts the acid and the hydroquinone into a dead-end metabolite. This novel compound was characterised unequivocally by mass spectrometry and 1H and 18C NMR and UV spectroscopy as 4-acetonyl-4-hydroxy-2-methylbut-2-en-1,4-olide 4, a cyclic tautomer of (3-methylmaleyl)acetone, which exists as the enol carboxylate form 8 in aqueous solution.

Biodegradation of phenols by the alga Ochromonas danica.

The eukaryotic alga Ochromonas danica, a nutritionally versatile, mixotrophic chrysophyte, grew on phenol as the sole carbon source in axenic culture and removed the phenol carbon from the growth medium. Respirometric studies confirmed that the enzymes involved in phenol catabolism were inducible and that the alga oxidized phenol; the amount of oxygen consumed per mole of oxidized substrate was approximately 65% of the theoretical value. [U-14C]phenol was completely mineralized, with 65% of the 14C label appearing as 14CO2, approximately 15% remaining in the aqueous medium, and the rest accounted for in the biomass. Analysis of the biomass showed that 14C label had been incorporated into the protein, nucleic acid, and lipid fractions; phenol carbon is thus unequivocally assimilated by the alga. Phenol-grown cultures of O. danica converted phenols to the corresponding catechols, which were further metabolized by the meta-cleavage pathway. This surprising result was rigorously confirmed by taking the working stock culture through a variety of procedures to check that it was axenic and repeating the experiments with algal extracts. This is, as far as is known, the first definitive identification of the meta-cleavage pathway for aromatic ring degradation in a eukaryotic alga, though its incidence in other eukaryotes has been (infrequently) suggested.

Metabolism of phenols by Ochromonas danica.

This study investigated the catabolic potential of a eukaryotic alga to degrade one of the most common organic pollutants, phenol. The alga, Ochromonas danica (993/28), was selected for study after screening for its heterotrophic capabilities. The catabolic versatility of the alga was elucidated by incubating with a variety of phenolic compounds. The alga removed phenol, all the cresol isomers and 3,4-xylenol from its incubation media, with phenol being removed more rapidly than any of its methylated homologues. Consequently, the alga was found to have a greater specificity for phenol than for o- or p-cresols. This study shows that O. danica could catabolize phenol and its methylated homologues.

Characterization of a carbofuran-degrading bacterium and investigation of the role of plasmids in catabolism of the insecticide carbofuran.

A bacterium capable of using the carbamate insecticide carbofuran as a sole source of carbon and energy, was isolated from soil. The ability to catabolise carbofuran phenol, produced by cleavage of the carbamate ester linkage of the insecticide, was lost at very high frequency when the bacterium was grown in the absence of carbofuran. Plasmid analyses together with curing and mating experiments indicated that the presence of a large plasmid (pIH3, greater than 199 kb) was required for the degradation of carbofuran phenol.

Purification and characterization of 4-methylmuconolactone methylisomerase, a novel enzyme of the modified 3-oxoadipate pathway in the gram-negative bacterium Alcaligenes eutrophus JMP 134.

4-Carboxymethyl-4-methylbut-2-en-4-olide (4-methyl-2-enelactone) isomerase, transforming 4-methyl-2-enelactone to 3-methyl-2-enelactone, was purified from a derivative strain of Pseudomonas sp. B13, named B13 FR1, carrying the plasmid pFRC2OP. This plasmid contained the isomerase gene cloned from Alcaligenes eutrophus JMP 134, which uses 4-methyl-2-enelactone as a carbon source. The enzyme consists of a single peptide chain of Mr 40,000 as judged by SDS/PAGE. In addition to 4-methyl-2-enelactone, the putative reaction intermediate, 1-methyl-3,7-dioxo-2,6-dioxy-bicyclo[3.3.0]octane (1-methylbislactone), was a substrate for the enzyme, but kinetic data presented did not favour its role as a reaction intermediate. Isomeric methyl-substituted 4-carboxymethylbut-2-en-4-olides were neither substrates nor inhibitors. Possible reaction mechanisms are discussed.

Purification and characterization of 4-methylmuconolactone methyl-isomerase, a novel enzyme of the modified 3-oxoadipate pathway in nocardioform actinomycetes.

The novel enzyme 4-methyl-2-enelactone methyl-isomerase was detected in, and purified to electrophoretic homogeneity from, p-toluate-grown cells of Rhodococcus rhodocrous N75, a nocardioform actinomycete. The enzyme was very thermostable and had a native Mr of 75,500; as the monomer had an Mr of 17,000, the enzyme is probably tetrameric. The new isomerase is highly specific with respect to its lactone substrate, only accepting (+)-(4S)-4-methylmuconolactone (4-carboxymethyl-4-methylbut-2-en-1,4-olide), and the putative isomerization reaction intermediate 1-methylbislactone ((-)-1-methyl-3,7-dioxo-2,6-dioxabicyclo-[3.3.0]octane) as substrates, and yielding (-)-(4S)-3-methylmuconolactone (4-carboxymethyl-3-methylbut-2-en-1,4-olide) as product. Some other lactone analogues acted as competitive inhibitors. Our data suggest that the isomerization does not involve actual methyl migration, but proceeds via the 1-methybislactone.

Bacterial metabolism of side chain fluorinated aromatics: cometabolism of 3-trifluoromethyl(TFM)-benzoate by Pseudomonas putida (arvilla) mt-2 and Rhodococcus rubropertinctus N657.

The TOL plasmid-encoded enzymes of the methylbenzoate pathway in Pseudomonas putida mt-2 cometabolized 3-trifluoromethyl (TFM)-benzoate. Two products, 3-TFM-1,2-dihydroxy-2-hydrobenzoate (3-TFM-DHB) and 2-hydroxy-6-oxo-7,7,7-trifluoro-hepta-2,4-dienoate (7-TFHOD) were identified chemically and by spectroscopic properties. TFM-substituted analogues of the metabolites of the methylbenzoate pathway were generally converted at drastically reduced rates. The catechol-2,3-dioxygenase from Pseudomonas putida showed moderate turnover rates with 3-TFM-catechol. The catechol-1,2-dioxygenase of Rhodococcus rubropertinctus N657 was totally inhibited by 3-TFM-catechol and did not cleave this substrate. Hammett-type analysis showed the catechol-1,2-dioxygenase reaction to be strongly dependent on the electronic nature of the substituents. Electronegative substituents strongly inhibited catechol cleavage. The catechol-2,3-dioxygenase reaction, however, was only moderately sensitive to electronegative substituents.

Effect of albumin and mannitol on organ blood flow, oxygen delivery, water content, and renal function during hypothermic hemodilution cardiopulmonary bypass.

The present study was designed to determine if the addition of albumin or mannitol to the priming solution of the pump oxygenator would diminish edema in organs, without diminishing some of the beneficial effects of hemodilution on blood flow and renal function. Tissue blood flow (15 mu spheres), water content, and renal clearances were determined in 8 animals during cardiopulmonary bypass. A 2(2) factorial, completely fixed experimental design was used. All animals were placed on cardiopulmonary bypass with hemodilution (hematocrit 25 +/- 2%) and hypothermia (25 degrees +/- 1 degree C). Albumin decreased flow to the midmyocardium of the left ventricle and to the spleen, and increased flow to the inner cortex of the kidney. Albumin caused decreased urine flow and decreased urine sodium, and also diminished renal osmolar, sodium, and free-water clearances. both mannitol and albumin decreased lung water. Mannitol decreased water content of the outer renal cortex, and decreased flow to the inner cortex and medulla of the kidney and to the spleen. Mannitol had no significant effect on urine flow, renal plasma flow, or renal clearances. Neither albumin nor mannitol had any effect on water content of the intestine, stomach, liver, or myocardium where the greatest accumulation of water occurs with hemodilution. The effect of albumin on renal function is potentially deleterious during cardiopulmonary bypass because it decreases urine flow, and osmolar and free-water clearance.

Effects of hypothermia, hemodilution, and pump oxygenation on organ water content, blood flow and oxygen delivery, and renal function.

Hypothermia, hemodilution, and the pump-oxygenator each contribute important effects during cardiopulmonary bypass. We studied their separate effects with a 2(3) factorial, completely fixed experimental design in 16 adult male mongrel dogs. Animals undergoing hypothermia were cooled to 25 degrees +/- 1 degree C. In dogs having hemodilution, hematocrit was adjusted to 25 +/- 2%. An analysis of variance was used to determine the effects of hypothermia, hemodilution, and pump oxygenation. The experiments show that hemodilution produces increased water content in tissue and that edema is greatest in heart and gastrointestinal organs. The pump-oxygenator decreased flow to the subendocardium, whereas hemodilution increased subendocardial flow. Both hypothermia and pump oxygenation diminished flow to the outer kidney cortex, and hemodilution augmented flow to this region. Hypothermia and pump oxygenation decreased and hemodilution raised renal free-water clearance. Although none affected glomerular filtration rate, hypothermia increased filtration fraction while hemodilution diminished it. Hypothermia lessened cerebral cortical flow, an effect opposite that of hemodilution. Thus, hemodilution opposes the adverse effect of hypothermia or pump oxygenation on blood flow, oxygen delivery, or renal function. Increased water content in gastrointestinal organs and myocardium accompanies the beneficial vascular and renal effects of hemodilution.

Similarity of effects of alcohol and sensory stimulation in post-alcoholics and controls.

Derived from the hypothesis that many alcoholics would experience autonomic "arousal" and craving for alcohol under conditions of high sensory stimulation, this study does not support prior findings.

'Loss of control' in alcoholics.

This study evaluates the ability of alcoholics to regulate their blood alcohol levels (BAL) within a designated range by relying primarily on interoceptive cues. Forty male alcoholics and 20 control subjects were exposed to an initial training session in which they received sufficient ethanol to maintain them within a designated BAL range over a 2 1/2-hour period. They were then exposed to two experimental sessions, one providing "overfeedback" and one "underfeedback." During each session, subjects had ten drinking decisions to make with respect to regulation of their BAL. The results indicated that alcoholics displayed greater "loss-of-control" than control subjects. This finding supported the hypothesis that alcoholics may possess a neurophysiologic feedback dysfunction that contributes to their relative inability to regulate ethanol intake.