Oxidation of Methyl Halides by the Facultative Methylotroph Strain IMB-1

Washed cell suspensions of the facultative methylotroph strain IMB-1 grown on methyl bromide (MeBr) were able to consume methyl chloride (MeCl) and methyl iodide (MeI) as well as MeBr. Consumption of >100 μM MeBr by cells grown on glucose, acetate, or monomethylamine required induction. Induction was inhibited by chloramphenicol. However, cells had a constitutive ability to consume low concentrations (<20 nM) of MeBr. Glucose-grown cells were able to readily oxidize [¹⁴C]formaldehyde to ¹⁴CO₂ but had only a small capacity for oxidation of [¹⁴C]methanol. Preincubation of cells with MeBr did not affect either activity, but MeBr-induced cells had a greater capacity for [¹⁴C]MeBr oxidation than did cells without preincubation. Consumption of MeBr was inhibited by MeI, and MeCl consumption was inhibited by MeBr. No inhibition of MeBr consumption occurred with methyl fluoride, propyl iodide, dibromomethane, dichloromethane, or difluoromethane, and in addition cells did not oxidize any of these compounds. Cells displayed Michaelis-Menten kinetics for the various methyl halides, with apparent K_s values of 190, 280, and 6,100 nM for MeBr, MeI, and MeCl, respectively. These results suggest the presence of a single oxidation enzyme system specific for methyl halides (other than methyl fluoride) which runs through formaldehyde to CO₂. The ease of induction of methyl halide oxidation in strain IMB-1 should facilitate its mass culture for the purpose of reducing MeBr emissions to the atmosphere from fumigated soils.     

Consumption of tropospheric levels of methyl bromide by C(1) compound-utilizing bacteria and comparison to saturation kinetics.

Pure cultures of methylotrophs and methanotrophs are known to oxidize methyl bromide (MeBr); however, their ability to oxidize tropospheric concentrations (parts per trillion by volume [pptv]) has not been tested. Methylotrophs and methanotrophs were able to consume MeBr provided at levels that mimicked the tropospheric mixing ratio of MeBr (12 pptv) at equilibrium with surface waters ( approximately 2 pM). Kinetic investigations using picomolar concentrations of MeBr in a continuously stirred tank reactor (CSTR) were performed using strain IMB-1 and Leisingeria methylohalidivorans strain MB2(T) - terrestrial and marine methylotrophs capable of halorespiration. First-order uptake of MeBr with no indication of threshold was observed for both strains. Strain MB2(T) displayed saturation kinetics in batch experiments using micromolar MeBr concentrations, with an apparent K(s) of 2.4 microM MeBr and a V(max) of 1.6 nmol h(-1) (10(6) cells)(-1). Apparent first-order degradation rate constants measured with the CSTR were consistent with kinetic parameters determined in batch experiments, which used 35- to 1 x 10(7)-fold-higher MeBr concentrations. Ruegeria algicola (a phylogenetic relative of strain MB2(T)), the common heterotrophs Escherichia coli and Bacillus pumilus, and a toluene oxidizer, Pseudomonas mendocina KR1, were also tested. These bacteria showed no significant consumption of 12 pptv MeBr; thus, the ability to consume ambient mixing ratios of MeBr was limited to C(1) compound-oxidizing bacteria in this study. Aerobic C(1) bacteria may provide model organisms for the biological oxidation of tropospheric MeBr in soils and waters.     

Degradation of methyl bromide by methanotrophic bacteria in cell suspensions and soils.

Cell suspensions of Methylococcus capsulatus mineralized methyl bromide (MeBr), as evidence by its removal from the gas phase, the quantitative recovery of Br- in the spent medium, and the production of 14CO2 from [14C]MeBr. Methyl fluoride fluoride (MeF) inhibited oxidation of methane as well as that of [14C]MeBr. The rate of MeBr consumption by cells varied inversely with the supply of methane, which suggested a competitive relationship between these two substrates. However, MeBr did not support growth of the methanotroph. In soils exposed to high levels (10,000 ppm) of MeBr, methane oxidation was completely inhibited. At this concentration, MeBr removal rates were equivalent in killed and live controls, which indicated a chemical rather than biological removal reaction. At lower concentration (1,000 ppm) of MeBr, methanotrophs were active and MeBr consumption rates were 10-fold higher in live controls than in killed controls. Soils exposed to trace levels (10 ppm) of MeBr demonstrated complete consumption within 5 h of incubation, while controls inhibited with MeF or incubated without O2 had 50% lower removal rates. Aerobic soils oxidized [14C]MeBr to 14CO2, and MeF inhibited oxidation by 72%. Field experiments demonstrated slightly lower MeBr removal rates in chambers containing MeF than in chambers lacking MeF. Collectively, these results show that soil methanotrophic bacteria, as well as other microbes, can degrade MeBr present in the environment.     

Bacterial Oxidation of Methyl Bromide in Fumigated Agricultural Soils

The oxidation of [(sup14)C]methyl bromide ([(sup14)C]MeBr) to (sup14)CO(inf2) was measured in field experiments with soils collected from two strawberry plots fumigated with mixtures of MeBr and chloropicrin (CCl(inf3)NO(inf2)). Although these fumigants are considered potent biocides, we found that the highest rates of MeBr oxidation occurred 1 to 2 days after injection when the fields were tarped, rather than before or several days after injection. No oxidation of MeBr occurred in heat-killed soils, indicating that microbes were the causative agents of the oxidation. Degradation of MeBr by chemical and/or biological processes accounted for 20 to 50% of the loss of MeBr during fumigation, with evasion to the atmosphere inferred to comprise the remainder. In laboratory incubations, complete removal of [(sup14)C]MeBr occurred within a few days, with 47 to 67% of the added MeBr oxidized to (sup14)CO(inf2) and the remainder of counts associated with the solid phase. Chloropicrin inhibited the oxidation of MeBr, implying that use of this substance constrains the extent of microbial degradation of MeBr during fumigation. Oxidation was by direct bacterial attack of MeBr and not of methanol, a product of the chemical hydrolysis of MeBr. Neither nitrifying nor methane-oxidizing bacteria were sufficiently active in these soils to account for the observed oxidation of MeBr, nor could the microbial degradation of MeBr be linked to cooxidation with exogenously supplied electron donors. However, repeated addition of MeBr to live soils resulted in higher rates of its removal, suggesting that soil bacteria used MeBr as an electron donor for growth. To support this interpretation, we isolated a gram-negative, aerobic bacterium from these soils which grew with MeBr as a sole source of carbon and energy.     

Strain IMB-1, a Novel Bacterium for the Removal of Methyl Bromide in Fumigated Agricultural Soils

A facultatively methylotrophic bacterium, strain IMB-1, that has been isolated from agricultural soil grows on methyl bromide (MeBr), methyl iodide, methyl chloride, and methylated amines, as well as on glucose, pyruvate, or acetate. Phylogenetic analysis of its 16S rRNA gene sequence indicates that strain IMB-1 classes in the alpha subgroup of the class Proteobacteria and is closely related to members of the genus Rhizobium. The ability of strain IMB-1 to oxidize MeBr to CO₂ is constitutive in cells regardless of the growth substrate. Addition of cell suspensions of strain IMB-1 to soils greatly accelerates the oxidation of MeBr, as does pretreatment of soils with low concentrations of methyl iodide. These results suggest that soil treatment strategies can be devised whereby bacteria can effectively consume MeBr during field fumigations, which would diminish or eliminate the outward flux of MeBr to the atmosphere.Methyl bromide (MeBr) is a fumigant used in the cultivation of selected fruits, vegetables, and flowers and in the preservation of stored grains and structures. Use of MeBr as a pesticide increases the yield and quality of crops without leaving behind toxic residues characteristic of more complex organopesticides. However, because bromine released from MeBr destroys stratospheric ozone (18, 22, 29, 33), its use will be eliminated in the United States and elsewhere under the auspices of the Clean Air Act and the Montreal Protocol unless effective mechanisms which prevent its escape to the atmosphere can be found (36). Currently, much uncertainty exists with regard to the tropospheric residence time (τ) of MeBr, a factor which is used to calculate its ozone degradation potential (2). Estimates of τ range from ∼1.7 years when only oxidation by tropospheric OH radicals is considered (22) to less than 1.2 years when oceanic sinks are factored in (20). The discovery that soil bacteria oxidize MeBr from the atmosphere, when quantified and combined with the two preceding sinks, lowers τ to ∼0.8 years (32). Chemical destruction of MeBr occurs by hydrolysis, exchange with other halides, and reaction with organic matter (8, 9, 12), but its destruction by microorganisms has been noted in soils and aquatic environments (3, 16, 17a, 19, 23, 27, 28, 32). In aerobic environments, MeBr is oxidized to CO₂ and Br⁻ (3, 16, 23, 27).Bacterial oxidation of MeBr in soils has been reported both at very low (∼5 to 15 parts per trillion) ambient atmospheric mixing ratios (17a) and at the very high concentrations employed for field fumigation (23). The relative contributions that chemical reactions and bacterial oxidation make to the destruction of MeBr during agricultural fumigation are not yet known, but their combined effect will constrain the emissions of MeBr from soils. Reported destruction of MeBr within the soil matrix, as evidenced by the accumulation of Br⁻, can be substantial and account for as much as 39 to 70% of the applied MeBr in some cases (39, 40). Physical manipulations (e.g., soil compaction and deeper injection of MeBr) have been proposed to increase the retention time of MeBr within the soil matrix, thereby allowing for its more extensive degradation and subsequent decrease in its outward flux to the atmosphere (13). In addition, use of thicker, impermeable covering tarps has been proposed to reduce losses (14, 37), as has the substitution of methyl iodide for MeBr (11, 25). However, enhancement of microbial degradation of MeBr while it is present in the soil matrix may also be a means to eliminate emissions. This could be achieved by exploiting the ability of certain soil bacteria that use MeBr as a carbon and energy source (23). Here, we report further details on the characteristics of such an isolate (23), which we designate strain IMB-1. We demonstrate how the properties of IMB-1 can be used to greatly accelerate the oxidation of MeBr in fumigated soils. Because agricultural field fumigation represents the largest source of anthropogenic emissions of MeBr to the atmosphere, it is at least possible in theory that the overall goal of eliminating most human-derived emission of MeBr could be achieved by in situ biodegradation of this substance.     

Consumption of Tropospheric Levels of Methyl Bromide by C1 Compound-Utilizing Bacteria and Comparison to Saturation Kinetics

Pure cultures of methylotrophs and methanotrophs are known to oxidize methyl bromide (MeBr); however, their ability to oxidize tropospheric concentrations (parts per trillion by volume [pptv]) has not been tested. Methylotrophs and methanotrophs were able to consume MeBr provided at levels that mimicked the tropospheric mixing ratio of MeBr (12 pptv) at equilibrium with surface waters (≈2 pM). Kinetic investigations using picomolar concentrations of MeBr in a continuously stirred tank reactor (CSTR) were performed using strain IMB-1 and Leisingeria methylohalidivorans strain MB2^T — terrestrial and marine methylotrophs capable of halorespiration. First-order uptake of MeBr with no indication of threshold was observed for both strains. Strain MB2^T displayed saturation kinetics in batch experiments using micromolar MeBr concentrations, with an apparent K_s of 2.4 μM MeBr and a V_max of 1.6 nmol h⁻¹ (10⁶ cells)⁻¹. Apparent first-order degradation rate constants measured with the CSTR were consistent with kinetic parameters determined in batch experiments, which used 35- to 1 × 10⁷-fold-higher MeBr concentrations. Ruegeria algicola (a phylogenetic relative of strain MB2^T), the common heterotrophs Escherichia coli and Bacillus pumilus, and a toluene oxidizer, Pseudomonas mendocina KR1, were also tested. These bacteria showed no significant consumption of 12 pptv MeBr; thus, the ability to consume ambient mixing ratios of MeBr was limited to C₁ compound-oxidizing bacteria in this study. Aerobic C₁ bacteria may provide model organisms for the biological oxidation of tropospheric MeBr in soils and waters.     

The bacterial oxidation of N-methylisonicotinate, a photolytic product of Paraquat

Two bacteria have been isolated that are capable of oxidizing N-methylisonicotinate, a photodegradation product of Paraquat (1.1'-dimethyl-4,4'-bipyridylium ion). N-Methylisonicotinate-grown cells of strain 4C1, a Gram-positive rod, oxidized 2-hydroxy-N-methylisonicotinate without lag. Cell-free extracts of these cells converted 2-hydroxyisonicotinate into 2,6-dihydroxyisonicotinate; the reaction did not require molecular oxygen. Maleamate was deamidated and maleate isomerized to fumarate by soluble enzyme systems. [(14)C]Formaldehyde was isolated as the dimedone derivative from the supernatant of a cell suspension oxidizing N-[(14)C]methylisonicotinate, and no [(14)C]-methylamine was detected. Whole cells incubated with N-methyl[carboxy-(14)C]isonicotinate released 95% of the radioactivity as (14)CO(2). The second bacterium, strain 4C2, a Gram-negative rod, did not oxidize any of the mono- or di-hydroxypyridines or their N-methyl derivatives that were available or could be synthesized; nor did cell-free extracts oxidize any of these compounds. Methylamine was oxidized by whole cells without lag; cell-free extracts converted methylamine into formaldehyde when a soluble enzyme system requiring an electron acceptor was used; formaldehyde was oxidized to formate and formate to CO(2) by enzyme systems requiring NAD(+).     

Methyl iodide reactions on the surface of mechanically pre-activated platinum(II) salt in the heterogeneous system: K2PtCl4 powder–MeI vapor

Mechanically pre-activated K₂PtCl₄ salt consumes methyl iodide producing methyl chloride at room temperature. The reaction mechanism includes the following steps sequence: oxidative addition of methyl iodide to platinum(II) complexes with intermediate formation of methyl platinum(IV) complexes and further decomposition of the latter in the course of innersphere reductive elimination yielding methyl chloride. The first step of the reaction proceeds owing to the assistance of active centers regenerated in the course of each event of MeI into MeCl transformation taking part in the chain halogen substitution process. It could be assumed that the role of active centers is played by coordinatively unsaturated platinum(II) complexes located on the surface. These species bearing a positive efficient charge can render electrophilic assistance for the nucleophilic substitution. The chain termination can be caused by recombination of coordinatively unsaturated platinum(II) complexes and interstitial chloride ions forming an inactive K₂PtCl₄ complex.     

New insights into methyl bromide cooxidation by Nitrosomonas europaea obtained by experimenting with moderately low density cell suspensions

We examined the rates and sustainability of methyl bromide (MeBr) oxidation in moderately low density cell suspensions ( approximately 6 x 10(7) cells ml(-1)) of the NH(3)-oxidizing bacterium Nitrosomonas europaea. In the presence of 10 mM NH(4)(+) and 0.44, 0. 22, and 0.11 mM MeBr, the initial rates of MeBr oxidation were sustained for 12, 12, and 24 h, respectively, despite the fact that only 10% of the NH(4)(+), 18% of the NH(4)(+), and 35% of the NH(4)(+), respectively, were consumed. Although the duration of active MeBr oxidation generally decreased as the MeBr concentration increased, similar amounts of MeBr were oxidized with a large number of the NH(4)(+)-MeBr combinations examined (10 to 20 micromol mg [dry weight] of cells(-1)). Approximately 90% of the NH(3)-dependent O(2) uptake activity and the NO(2)(-)-producing activity were lost after N. europaea was exposed to 0.44 mM MeBr for 24 h. After MeBr was removed and the cells were resuspended in fresh growth medium, NO(2)(-) production increased exponentially, and 48 to 60 h was required to reach the level of activity observed initially in control cells that were not exposed to MeBr. It is not clear what percentage of the cells were capable of cell division after MeBr oxidation because NO(2)(-) accumulated more slowly in the exposed cells than in the unexposed cells despite the fact that the latter were diluted 10-fold to create inocula which exhibited equal initial activities. The decreases in NO(2)(-)-producing and MeBr-oxidizing activities could not be attributed directly to NH(4)(+) or NH(3) limitation, to a decrease in the pH, to the composition of the incubation medium, or to toxic effects caused by accumulation of the end products of oxidation (NO(2)(-) and formaldehyde) in the medium. Additional cooxidation-related studies of N. europaea are needed to identify the mechanism(s) responsible for the MeBr-induced loss of cell activity and/or viability, to determine what percentages of cells damaged by cooxidative activities are culturable, and to determine if cooxidative activity interferes with the regulation of NH(3)-oxidizing activity.     

Peroxidative oxidation of halides catalysed by myeloperoxidase. Effect of fluoride on halide oxidation

It was found that all halides can compete with cyanide for binding with myeloperoxidase. The lower is the pH, the higher is the affinity of halides. The apparent dissociation constants (Kd) of myeloperoxidase-cyanide complex were determined in the presence of F-, Cl-, Br- and I- in the pH range of 4 to 7. In slightly acidic pH (4 - 6) fluoride and chloride exhibit a higher affinity towards the enzyme than bromide and iodide. Taking into account competition between cyanide and halides for binding with myeloperoxidase the dissociation constants of halide-myeloperoxidase complexes were calculated. All halides except fluoride can be oxidized by H2O2 in the presence of myeloperoxidase. However, since fluoride can bind with myeloperoxidase, it can competitively inhibit the oxidation of other halides. Fluoride was a competitive inhibitor with respect to other halides as well as to H2O2. Inhibition constants (Ki) for fluoride as a competitive inhibitor with respect to H2O2 increased from iodide oxidation through bromide to chloride oxidation.     

(14)C radiolabeling of proteins to monitor biodistribution of ingested proteins

The economical preparation of microgram quantities of (14)C-labeled proteins by in vacuo methylation with methyl iodide is described. The (14)C radiolabeling was achieved by the covalent attachment of [(14)C]methyl groups onto amino and imidazole groups by reaction in vacuo with [(14)C]methyl iodide. The method was tested by investigating the biodistribution of (14)C in rats that were fed (14)C-labeled human soluble cluster of differentiation 14 (CD14) protein, a receptor for bacterial lipopolysaccharide. Two other control proteins, bovine serum albumin (BSA) and casein, were also labeled with (14)C and used for comparative analysis to determine the following: (i) the efficacy and cost efficiency of the in vacuo radiolabeling procedure and (ii) the extent of incorporation of the (14)C label into the organs of orogastrically fed 10-day-old Sprague-Dawley rats. [(14)C]BSA, [(14)C]casein, and [(14)C]CD14 were individually prepared with specific radioactivities of 34,400, 18,800, and 163,000 disintegrations per minute (dpm)/microg, respectively. It was found that the accumulation of (14)C label in the organs of [(14)C]CD14-fed rats, most notably the persistence of (14)C in the stomach 480 min postgavage, was temporally and spatially distinct from [(14)C]BSA and [(14)C]casein-fed rats.     

Autotrophic synthesis of activated acetic acid from two CO2 in Methanobacterium thermoautotrophicum

In a previous study with Methanobacterium thermoautotrophicum evidence was presented that methanogenesis and autotrophic synthesis of activated acetic acid from CO₂ are linked processes. In this study one-carbon metabolism was investigated with growing cultures and in vitro.Serine was shown to be converted into glycine and activated formaldehyde, but only traces of label from [¹⁴C-3] of serine appeared in biosynthetic one-carbon positions. This seeming discrepancy could be explained if the same activated formaldehyde is an intermediate in biosynthesis and in methanogenesis from CO₂. This hypothesis was supported by demonstrating that [¹⁴C-3] of serine and [¹⁴C] formaldehyde were rapidly converted into methane, but a small portion of the label was also specifically incorporated into the methyl group of acetate. Methane and acetate synthesis in vitro were similarly stimulated by various compounds. These experiments indicate that the methyl of acetate and methane share common one-carbon precursor(s), i.e. methylene tetrahydromethanopterin, which can also be formed enzymatically from C-3 of serine or chemically from formaldehyde.Propyl iodide 20–40 M) and methyl iodide (1–3 M) completely inhibited growth in the dark. This effect was abolished by light. Methane formation was hardly affected. When ¹⁴CH₃I was applied at an only slightly inhibitory concentration, ¹⁴C was incorporated into the methyl of acetate. In vitro, similar effects on [¹⁴C] acetate formation from ¹⁴CO₂ or from [¹⁴C-3] of serine were observed, except that methyl iodide did not inhibit, but even stimulated acetate synthesis. These experiments indicate that a corrinoid is involved in acetate synthesis and probably not in methanogenesis from CO₂; the metal is light-reversibly alkylated and functions in methyl transfer to the acetate methyl.     

Inhibition of the low-Km mitochondrial aldehyde dehydrogenase by diethyl maleate and phorone in vivo and in vitro. Implications for formaldehyde metabolism.

Formaldehyde can be oxidized primarily by two different enzymes, the low-Km mitochondrial aldehyde dehydrogenase and the cytosolic GSH-dependent formaldehyde dehydrogenase. Experiments were carried out to evaluate the effects of diethyl maleate or phorone, agents that deplete GSH from the liver, on the oxidation of formaldehyde. The addition of diethyl maleate or phorone to intact mitochondria or to disrupted mitochondrial fractions produced inhibition of formaldehyde oxidation. The kinetics of inhibition of the low-Km mitochondrial aldehyde dehydrogenase were mixed. Mitochondria isolated from rats treated in vivo with diethyl maleate or phorone had a decreased capacity to oxidize either formaldehyde or acetaldehyde. The activity of the low-Km, but not the high-Km, mitochondrial aldehyde dehydrogenase was also inhibited. The production of CO2 plus formate from 0.2 mM-[14C]formaldehyde by isolated hepatocytes was only slightly inhibited (15-30%) by incubation with diethyl maleate or addition of cyanamide, suggesting oxidation primarily via formaldehyde dehydrogenase. However, the production of CO2 plus formate was increased 2.5-fold when the concentration of [14C]formaldehyde was raised to 1 mM. This increase in product formation at higher formaldehyde concentrations was much more sensitive to inhibition by diethyl maleate or cyanamide, suggesting an important contribution by mitochondrial aldehyde dehydrogenase. Thus diethyl maleate and phorone, besides depleting GSH, can also serve as effective inhibitors in vivo or in vitro of the low-Km mitochondrial aldehyde dehydrogenase. Inhibition of formaldehyde oxidation by these agents could be due to impairment of both enzyme systems known to be capable of oxidizing formaldehyde. It would appear that a critical amount of GSH, e.g. 90%, must be depleted before the activity of formaldehyde dehydrogenase becomes impaired.     

Synthesis, radiosynthesis and in vivo preliminary evaluation of [11C]LBT-999, a selective radioligand for the visualisation of the dopamine transporter with PET

LBT-999 (8-((E)-4-fluoro-but-2-enyl)-3beta-p-tolyl-8-aza-bicyclo[3.2.1]octane-2beta-carboxylic acid methyl ester), a cocaine derivative belonging to a new generation of highly selective dopamine transporter (DAT) ligands, and its corresponding carboxylic acid derivative, the latter used as precursor for labelling both with tritium and the positron-emitter carbon-11 (half-life: 20.38 min), were synthesized from (R)-cocaine. [(3)H]LBT-999 (>99% radiochemically pure, specific radioactivity of 3.1 TBq/mmol) was prepared from [(3)H]methyl iodide, allowing its in vitro pharmacological evaluation (K(D): 9 nM for DAT and IC(50) > 1000 nM for SERT and NET). Routine production batches of 4.5-9.0 GBq of iv injectable solutions of [(11)C]LBT-999 (with specific radioactivities ranging from 30 to 45 GBq/mumol) were prepared in 25-30 min (HPLC purification and formulation included) using the efficient methylation reagent [(11)C]methyl triflate. The preliminary in vivo pharmacological evaluation of [(11)C]LBT-999, using both biodistributions in rats and brain imaging in monkeys with positron emission tomography (PET), clearly illustrates that this ligand is an excellent candidate for quantification with PET of DAT in humans.     

Identification of intermediates of in vivo trichloroethylene oxidation by the membrane-associated methane monooxygenase

The rate and products of trichloroethylene (TCE) oxidation by Methylomicrobium album BG8 expressing membrane-associated methane monooxygenase (pMMO) were determined using 14C radiotracer techniques. [(14)C]TCE was degraded at a rate of 1.24 nmol (min mg protein)(-1) with the initial production of glyoxylate and then formate. Radiolabeled CO(2) was also found after incubating M. album BG8 for 5 h with [(14)C]TCE. Experiments with purified pMMO from Methylococcus capsulatus Bath showed that TCE could be mineralized to CO(2) by pMMO. Oxygen uptake studies verified that M. album BG8 could oxidize glyoxylate and that pMMO was responsible for the oxidation based on acetylene inactivation studies. Here we propose a pathway of TCE oxidation by pMMO-expressing cells in which TCE is first converted to TCE-epoxide. The epoxide then spontaneously undergoes HCl elimination to form glyoxylate which can be further oxidized by pMMO to formate and CO(2).     

Metabolism of methylamine in the tea plant (Thea sinensis L.)

1. The metabolism of methylamine in excised shoot tips of tea was studied with micromolar amounts of [(14)C]methylamine. Of the [(14)C]methylamine supplied 57% was utilized by tea shoots during the 10h experimental period. 2. The main products of [(14)C]methylamine metabolism in tea shoots were serine, gamma-glutamylmethylamide, theobromine, caffeine and CO(2). There was also incorporation of the label into glutamate, aspartate, RNA purine nucleotides and S-adenosylmethionine. 3. The formation of methylamine from gamma-glutamylmethylamide was confirmed by feeding tea shoots with gamma-glutamyl[(14)C]methylamide. The products of gamma-glutamyl[(14)C]methylamide metabolism in tea plants were serine, theobromine, caffeine, glutamate and aspartate. 4. The results indicate that the oxidation of methylamine to formaldehyde is the first step of methylamine utilization. Labelled formaldehyde released by the metabolism of methylamine leads to the incorporation of the label into metabolites on the C(1) pathways of this compound. It is also suggested that formaldehyde is further oxidized via formate to CO(2). 5. The role of gamma-glutamylmethylamide in methylamine metabolism in tea plants is discussed. 6. Results support the view that theobromine is the immediate precursor of caffeine.     

INTERACTION OF FATTY ACID AND GLUCOSE OXIDATION BY CULTURED HEART CELLS

Chick embryo heart cells in tissue culture actively oxidize [1-¹⁴C]palmitate to ¹⁴CO₂. Fatty acid oxidation by cell monolayers was linear with time and increasing protein concentration. The addition of carnitine to the assay medium resulted in a 30–70% increase in the rate of fatty acid oxidation. The specific activity of palmitic acid oxidation did not change significantly with time in culture and was also the same in rapidly proliferating and density-inhibited cell cultures. Addition of unlabeled glucose to the assay medium resulted in a 50% decrease in ¹⁴CO₂ production from [1-¹⁴C]palmitate. Conversely, palmitate had a similar sparing effect on [¹⁴C]glucose oxidation to ¹⁴CO₂. Lactate production accounted for most of the glucose depleted from the medium and was not inhibited by the presence of palmitate in the assay. Thus, the sparing action of the fatty acids on glucose oxidation appears to be at the mitochondrial level. The results indicate that although chick heart cells in culture are primarily anaerobic, they can oxidize fatty acid actively.     

Induction of chromatid aberrations by TEM and maleic hydrazide is differently affected by pretreatment of Vicia faba root-tip meristems with methyl iodide

Treatment of Vicia faba main root meristems with methyl iodide (MeI) 2 h before challenge treatment with triethylene melamine (TEM) significantly reduced the yield of metaphases with chromatid aberrations, i.e., resulted in clastogenic adaptation. Combined treatment with MeI and TEM increased the aberration yield; MeI treatment alone (10(-3) M, 0.5 h) was without clastogenic effect. No protective effects were observed after MeI pretreatment and challenge treatment by maleic hydrazide (MH). The data obtained in V. faba are compared to those previously reported for E. coli.     

Synthesis and characterization of [¹²⁵I]2-iodo N-[(S)-{(S)-1-methylpiperidin-2-yl}(phenyl)methyl]3-trifluoromethyl-benzamide as novel imaging probe for glycine transporter 1

In this study, 2-iodo substituted 1-methylpiperidin-2-yl benzamide derivatives were synthesized and evaluated as candidate SPECT imaging agents for glycine transporter 1 (GlyT1). In JAR cells, which predominantly express GlyT1, 2-iodo N-[(S)-{(S)-1-methylpiperidin-2-yl}(phenyl)methyl]3-trifluoromethyl-benzamide (5) showed excellent inhibitory activity of [(3)H]glycine uptake (IC(50)=2.4 nM). Saturation assay in rat cortical membranes revealed that [(125)I]5 had a single high affinity binding site with a K(d) of 1.54 nM and a B(max) of 3.40 pmol/mg protein. In vitro autoradiography demonstrated that [(125)I]5 showed consistent accumulation with GlyT1 expression. The in vitro binding was greatly inhibited by GlyT1 inhibitors but not by other site ligands, which suggested the high specific binding of [(125)I]5 with GlyT1. In the biodistribution and ex vivo autoradiography studies using mice, [(125)I]5 showed high blood-brain barrier permeability (1.68-2.17% dose/g at 15-60 min) and similar regional brain distribution pattern with in vitro results. In addition, pre-treatment of GlyT1 ligands resulted in significant decrease of [(125)I]5 binding in the GlyT1-rich regions. This preliminary study demonstrated that radio-iodinated 5 is a promising SPECT imaging probe for GlyT1.