Selective Inhibition of Ammonium Oxidation and Nitrification-Linked N2O Formation by Methyl Fluoride and Dimethyl Ether

Methyl fluoride (CH₃F) and dimethyl ether (DME) inhibited nitrification in washed-cell suspensions of Nitrosomonas europaea and in a variety of oxygenated soils and sediments. Headspace additions of CH₃F (10% [vol/vol]) and DME (25% [vol/vol]) fully inhibited NO₂^- and N₂O production from NH₄⁺ in incubations of N. europaea, while lower concentrations of these gases resulted in partial inhibition. Oxidation of hydroxylamine (NH₂OH) by N. europaea and oxidation of NO₂^- by a Nitrobacter sp. were unaffected by CH₃F or DME. In nitrifying soils, CH₃F and DME inhibited N₂O production. In field experiments with surface flux chambers and intact cores, CH₃F reduced the release of N₂O from soils to the atmosphere by 20- to 30-fold. Inhibition by CH₃F also resulted in decreased NO₃^- + NO₂^- levels and increased NH₄⁺ levels in soils. CH₃F did not affect patterns of dissimilatory nitrate reduction to ammonia in cell suspensions of a nitrate-respiring bacterium, nor did it affect N₂O metabolism in denitrifying soils. CH₃F and DME will be useful in discriminating N₂O production via nitrification and denitrification when both processes occur and in decoupling these processes by blocking NO₂^- and NO₃^- production.     

Difluoromethane, a New and Improved Inhibitor of Methanotrophy

Difluoromethane (HFC-32; DFM) is compared to acetylene and methyl fluoride as an inhibitor of methanotrophy in cultures and soils. DFM was found to be a reversible inhibitor of CH₄ oxidation by Methylococcus capsulatus (Bath). Consumption of CH₄ in soil was blocked by additions of low levels of DFM (0.03 kPa), and this inhibition was reversed by DFM removal. Although a small quantity of DFM was consumed during these incubations, its remaining concentration was sufficiently elevated to sustain inhibition. Methanogenesis in anaerobic soil slurries, including acetoclastic methanogenesis, was unaffected by levels of DFM which inhibit methanotrophy. Low levels of DFM (0.03 kPa) also inhibited nitrification and N₂O production by soils. DFM is proposed as an improved inhibitor of CH₄ oxidation over acetylene and/or methyl fluoride on the basis of its reversibility, its efficacy at low concentrations, its lack of inhibition of methanogenesis, and its low cost.     

Tracer Analysis of Methanogenesis in Salt Marsh Soils

Differences in paths of carbon flow have been found in soils of the tall (TS) and short (SS) Spartina alterniflora marshes of Sapelo Island, Ga. Gaseous end products of [U-¹⁴C]glucose metabolism were ¹⁴CO₂ and ¹⁴CH₄ in the SS region and primarily ¹⁴CO₂ in the TS region. Sulfate concentration did not demonstrably affect glucose catabolism or the distribution of end products in either zone. [U-¹⁴C]acetate was converted to ¹⁴CO₂ and ¹⁴CH₄ in the SS soils and almost exclusively to ¹⁴CO₂ in the TS soils. Sulfate concentration did not affect acetate metabolism in the SS soils; however, a noticeable effect of sulfate dilution was seen in TS soils. Sulfate dilution in TS samples resulted in increased methane formation. Total glucose and acetate metabolism were similar in TS and SS soils despite differences in end products. A microbial community characterized by fermentative/sulfate-reducing processes has developed in TS soils as opposed to the fermentative/methanogenic/sulfate-reducing community found in SS soils.     

Evidence for the enzymatic transfer of N-acetylglucosamine from UDP-N-acetylglucosamine into dolichol derivatives and glycoproteins by calf brain membranes

Incubating white matter membranes with UDP-N-acetyl-[¹⁴C]glucosamine in the presence of Mg²⁺ and AMP resulted in the labeling of two major glycolipids, a minor glycolipid and several membrane-associated glycoproteins. The addition of AMP protected the labeled sugar nucleotide from degradation by a membrane-bound sugar nucleotide pyrophosphatase activity. While no labeled oligosaccharide lipid was recovered in a CHCl₃CH₃OHH₂O (10:10:3) extract after incubating with only UDP-N-acetyl-[¹⁴C] glucosamine, Mg²⁺, and AMP, the inclusion of unlabeled GDP-mannose led to the formation of an N-acetyl-[¹⁴C]glucosamine-labeled oligosaccharide lipid that was soluble in CHCl₃CH₃OHH₂O (10:10:3). The [GlcNAc-¹⁴C]oligosaccharide unit was released by treatment with 0.1 N HCl in 80% tetrahydrofuran at 50 °C for 30 min and appears to have the same molecular size as the lipid-linked [mannose-¹⁴C] oligosaccharide, formed enzymatically by white matter membranes as judged by their elution behavior on Bio-Gel P-6. The incorporation of N-acetyl-[¹⁴C]glucosamine into glycolipid was stimulated by exogenous dolichol monophosphate, but inhibited by UMP or tunicamycin, a glucosamine-containing antibiotic. Although UMP and tunicamycin drastically inhibited the labeling of glycolipid, these compounds had very little effect on the labeling of glycoproteins. The major glycolipids have the chemical and Chromatographic characteristics of N-acetylglucosaminylpyrophosphoryldolichol and N,N′-diacetylchitobiosylpyrophosphoryldolichol. When the labeled glycoproteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, four labeled polypeptides were observed, having apparent molecular weights of 145,000, 105,000, 54,000, and 35,000. Virtually all of the N-acetyl-[¹⁴C]glucosamine was released when the labeled glycopeptides, produced by pronase digestion, were incubated with an exo-β-N-acetylglucosaminidase, indicating that all of the N-acetyl-[¹⁴C]glucosamine incorporated under these conditions is attached to white matter membrane glycoproteins at nonreducing termini.     

Consumption of Methane and CO2 by Methanotrophic Microbial Mats from Gas Seeps of the Anoxic Black Sea

The deep anoxic shelf of the northwestern Black Sea has numerous gas seeps, which are populated by methanotrophic microbial mats in and above the seafloor. Above the seafloor, the mats can form tall reef-like structures composed of porous carbonate and microbial biomass. Here, we investigated the spatial patterns of CH₄ and CO₂ assimilation in relation to the distribution of ANME groups and their associated bacteria in mat samples obtained from the surface of a large reef structure. A combination of different methods, including radiotracer incubation, beta microimaging, secondary ion mass spectrometry, and catalyzed reporter deposition fluorescence in situ hybridization, was applied to sections of mat obtained from the large reef structure to locate hot spots of methanotrophy and to identify the responsible microbial consortia. In addition, CO₂ reduction to methane was investigated in the presence or absence of methane, sulfate, and hydrogen. The mat had an average δ¹³C carbon isotopic signature of −67.1‰, indicating that methane was the main carbon source. Regions dominated by ANME-1 had isotope signatures that were significantly heavier (−66.4‰ ± 3.9 ‰ [mean ± standard deviation; n = 7]) than those of the more central regions dominated by ANME-2 (−72.9‰ ± 2.2 ‰; n = 7). Incorporation of ¹⁴C from radiolabeled CH₄ or CO₂ revealed one hot spot for methanotrophy and CO₂ fixation close to the surface of the mat and a low assimilation efficiency (1 to 2% of methane oxidized). Replicate incubations of the mat with ¹⁴CH₄ or ¹⁴CO₂ revealed that there was interconversion of CH₄ and CO_2. The level of CO₂ reduction was about 10% of the level of anaerobic oxidation of methane. However, since considerable methane formation was observed only in the presence of methane and sulfate, the process appeared to be a rereaction of anaerobic oxidation of methane rather than net methanogenesis.     

Substrates for Sulfate Reduction and Methane Production in Intertidal Sediments

The activity of and potential substrates for methane-producing bacteria and sulfate-reducing bacteria were examined in marsh, estuary, and beach intertidal sediments. Slow rates of methane production were detected in all sediments, although rates of sulfate reduction were 100- to 1,000-fold higher. After sulfate was depleted in sediments, the rates of methane production sharply increased. The addition of methylamine stimulated methanogenesis in the presence of sulfate, and [¹⁴C]methylamine was rapidly converted to ¹⁴CH₄ and ¹⁴CO₂ in freshly collected marsh sediment. Acetate, hydrogen, or methionine additions did not stimulate methanogenesis. [methyl-¹⁴C]methionine and [2-¹⁴C]acetate were converted to ¹⁴CO₂ and not to ¹⁴CH₄ in fresh sediment. No reduction of ¹⁴CO₂ to ¹⁴CH₄ occurred in fresh sediment. Molybdate, an inhibitor of sulfate reduction, inhibited [2-¹⁴C]acetate metabolism by 98.5%. Fluoracetate, an inhibitor of acetate metabolism, inhibited sulfate reduction by 61%. These results suggest that acetate is a major electron donor for sulfate reduction in marine sediments. In the presence of high concentrations of sulfate, methane may be derived from novel substrates such as methylamine.     

Magnitude and biophysical regulators of methane emission and consumption in the Australian agricultural, forest, and submerged landscapes: a review

Increases in the concentrations of atmospheric greenhouse gases, carbon dioxide (CO₂), methane (CH₄), nitrous oxide (N₂O) due to human activities are associated with global climate change. CO₂ concentration in the atmosphere has increased by 33% (to 380 ppm) since 1750 ad, whilst CH₄ concentration has increased by 75% (to 1,750 ppb), and as the global warming potential (GWP) of CH₄ is 25 fold greater than CO₂ it represents about 20% of the global warming effect. The purpose of this review is to: (a) address recent findings regarding biophysical factors governing production and consumption of CH₄, (b) identify the current level of knowledge regarding the main sources and sinks of CH₄ in Australia, and (c) identify CH₄ mitigation options and their potential application in Australian ecosystems. Almost one-third of CH₄ emissions are from natural sources such as wetlands and lake sediments, which is poorly documented in Australia. For Australia, the major anthropogenic sources of CH₄ emissions include energy production from fossil fuels (~24%), enteric fermentation in the guts of ruminant animals (~59%), landfills, animal wastes and domestic sewage (~15%), and biomass burning (~5%), with minor contributions from manure management (1.7%), land use, land-use change and forestry (1.6%), and rice cultivation (0.2%). A significant sink exists for CH₄ (~6%) in aerobic soils, including agricultural and forestry soils, and potentially large areas of arid soils, however, due to limited information available in Australia, it is not accounted for in the Australian National Greenhouse Gas Inventory. CH₄ emission rates from submerged soils vary greatly, but mean values ≤10 mg m^?2 h^?1 are common. Landfill sites may emit CH₄ at one to three orders of magnitude greater than submerged soils. CH₄ consumption rates in non-flooded, aerobic agricultural, pastoral and forest soils also vary greatly, but mean values are restricted to ≤100 μg m^?2 h^?1, and generally greatest in forest soils and least in agricultural soils, and decrease from temperate to tropical regions. Mitigation options for soil CH₄ production primarily relate to enhancing soil oxygen diffusion through water management, land use change, minimised compaction and soil fertility management. Improved management of animal manure could include biogas capture for energy production or arable composting as opposed to open stockpiling or pond storage. Balanced fertiliser use may increase soil CH₄ uptake, reduce soil N₂O emissions whilst improving nutrient and water use efficiency, with a positive net greenhouse gas (CO₂-e) effect. Similarly, the conversion of agricultural land to pasture, and pastoral land to forestry should increase soil CH₄ sink. Conservation of native forests and afforestation of degraded agricultural land would effectively mitigate CH₄ emissions by maintaining and enhancing CH₄ consumption in these soils, but also by reducing N₂O emissions and increasing C sequestration. The overall impact of climate change on methanogenesis and methanotrophy is poorly understood in Australia, with a lack of data highlighting the need for long-term research and process understanding in this area. For policy addressing land-based greenhouse gas mitigation, all three major greenhouse gases (CO₂, CH₄ and N₂O) should be monitored simultaneously, combined with improved understanding at process-level.     

Catabolism of porphobilinogen by etiolated barley leaves

When [2,4-¹⁴C]porphobilinogen (PBG) or [2 (aminomethyl),5-¹⁴C]PBG is administered to etiolated barley (Hordeum vulgare L. var. Larker) leaves in darkness, label becomes incorporated into CO₂, organic and amino acids, sugars, lipids, and proteins during a 4-hour incubation. Less than 1% of the label, however, is incorporated into porphyrins. The rate of ¹⁴CO₂ evolution from leaves fed [2,4-¹⁴C]PBG is strongly inhibited by anaerobiosis but is unaffected by aminooxyacetic acid, while the rate of ¹⁴CO₂ evolution from [2(aminomethyl),5-¹⁴C]PBG is strongly inhibited by aminooxyacetic acid but is not affected by anaerobiosis.     

Autotrophic synthesis of activated acetic acid from two CO2 inMethanobacterium thermoautotrophicum

An in vitro system of autotropic synthesis of activated acetic acid from¹⁴CO₂ inMethanobacterium thermoautotrophicum was developed.

1. A recognized¹⁴CO₂-fixation product in vitro was activated [¹⁴C] acetic acid. It could be trapped enzymatically into citrate and released again as [¹⁴C] acetate by citrate synthase and citrate lyase, respectively.
2. The synthesis of both activated acetic acid and methane from CO₂ proceeded in parallel under a variety of conditions. Both of these processes were stimulated greatly and to the same extent by the addition of methyl coenzyme M to the assay.
3. Various inhibitors of methanogenesis tested also inhibited acetate synthesis, e.g. CH₂Cl₂, CHCl₃, CCl₄, N₂O, and bromoethane sulfonic acid. Cyanide specifically inhibited the synthesis of activated acetic acid, whereas methane formation was unaffected. Cyanide inhibition was relieved by adding CO, whereas the inhibition by the other compounds was not.

The data suggest: The product studied in vitro was acetyl CoA. Its synthesis involves intermediates of CO₂ reduction to methane. In addition, a cyanide-sensitive reaction is required which does not participate in CO₂ reduction to methane.     

温带针阔混交林土壤碳氮气体通量的主控因子与耦合关系

中高纬度森林地区由于气候条件变化剧烈,土壤温室气体排放量的估算存在很大的不确定性,并且不同碳氮气体通量的主控因子与耦合关系尚不明确。以长白山温带针阔混交林为研究对象,采用静态箱-气相色谱法连续4a(2005—2009年)测定土壤二氧化碳(CO2)、甲烷(CH4)和氧化亚氮(N2O)净交换通量以及温度、水分等相关环境因子。研究结果表明:温带针阔混交林土壤整体上表现为CO2和N2O的排放源和CH4的吸收汇。土壤CH4、CO2和N2O通量的年均值分别为-1.3 kg CH4hm-2a-1、15102.2 kg CO2hm-2a-1和6.13 kg N2O hm-2a-1。土壤CO2通量呈现明显的季节性规律,主要受土壤温度的影响,水分次之;土壤CH4通量的季节变化不明显,与土壤水分显著正相关;土壤N2O通量季节变化与土壤CO2通量相似,与土壤水分、温度显著正相关。土壤CO2通量和CH4通量不存在任何类型的耦合关系,与N2O通量也不存在耦合关系;土壤CH4和N2O通量之间表现为消长型耦合关系。这项研究显示温带针阔混交林土壤碳氮气体通量主要受环境因子驱动,不同气体通量产生与消耗之间存在复杂的耦合关系,下一步研究需要深入探讨环境变化对其耦合关系的影响以及内在的生物驱动机制。     

Effect of tolbutamide on the metabolism of Tetrahymena

Tolbutamide partially inhibited the growth but increased the glycogen content of Tetrahymena pyriformis in logarithmically growing cultures. Tolbutamide slightly increased ¹⁴CO² production from [1-¹⁴C] and [6-¹⁴HC] glucose and [2-¹⁴C] pyruvate, but had little effect on the oxidation of [1-¹⁴C] acetate when any of these substrates were added to the proteose-peptone medium in which the cells had been grown. Measurement of ¹⁴CO₂ production from [1-¹⁴C] and [2-^I4C]-glyoxylate showed that this substrate was primarily oxidized via the glyoxylate cycle, with little if any oxidation occurring via the peroxisomal glyoxylate oxidase. Addition of tolbutamide inhibited the glyoxylate cycle as indicated by a marked reduction in label appearing in CO₂ and in glycogen from labeled acetate. In control cells, addition of acetate strongly inhibited the oxidation of [2-¹⁴C]-pyruvate whereas addition of pyruvate had little effect on the oxidation of [1-¹⁴C]-acetate. Acetate was more effective than pyruvate in preventing the growth inhibitory and glycogen-increasing effects of tolbutamide. The data suggest that one effect of tolbutamide may be to interfere with the transfer of isocitrate and acetyl CoA across mitochondrial membranes.     

Palmitate oxidation by rat skeletal muscle mitochondria: Comparison of polarographic and radiochemical experiments

Oxidation of palmitate by rat skeletal muscle mitochondria was determined polarographically and radiochemically under state 3 conditions. Maximal oxidation rate is reached at 4 μm palmitate, palmitoyl-CoA, or palmitoyl-l-carnitine. At palmitoyl-CoA concentrations higher than 30 μm oxidation is inhibited. At limiting substrate concentrations as used in polarographic experiments palmitate is totally degraded to CO₂. At higher concentrations the palmitate molecule is only partially degraded, due to the accumulation of intermediates. Citric acid cycle intermediates, especially 2-oxoglutarate, accumulate during oxidation of palmitate in the presence of malate. It is suggested that this accumulation is stimulated by dicarboxylate exchange. The rate of formation of ¹⁴CO₂ and ¹⁴C-labeled perchloric acid-soluble products is higher from [1-¹⁴C]palmitate than that from [U-¹⁴C]palmitate. This difference, which is enhanced by higher carnitine concentrations indicates incomplete oxidation during the β-oxidation in state 3. The simultaneous determination of ¹⁴CO₂ production and ¹⁴C-labeled perchloric acid-soluble products appears to be a more accurate and sensitive method for measuring ¹⁴C-fatty acid oxidation than that of ¹⁴CO₂ production alone.     

The effect of methionine on the metabolism of serine and glycine in vitamin B-12-deficient rats

The effect of methionine supplementation on glycine and serine metabolism was studied in vitamin B-12-deficient rats which received only 0.2% methionine in the diet. In the perfused liver, incorporation of the C-2 of glycine to the C-3 of serine was increased by addition of methionine to the perfusate. The oxidation of [1-¹⁴C]glycine to ¹⁴CO₂ was however depressed. Unlike methionine, glycine did not have any significant effect on the liver folate coenzyme distribution. Oxidation of [3-¹⁴C]serine to ¹⁴CO₂ both in vivo and in perfused liver was increased by methionine. A major portion of the C-3 radioactivity however was recovered in glucose. Data presented indicate that the rate of oxidation of [2-¹⁴C]histidine to ¹⁴CO₂ is more sensitive indicator of folate deficiency than the rate of oxidation of [3-¹⁴C] serine to ¹⁴CO₂ although both are presumably tetrahydrofolate dependent.     

Catabolism of 5-Aminolevulinic Acid to CO(2) by Etiolated Barley Leaves

The in vivo oxidation of the C₄ and C₅ of 5-aminolevulinic acid (ALA) to CO₂ has been studied in etiolated barley (Hordeum vulgare L. var. Larker) leaves in darkness. The rate of ¹⁴CO₂ evolution from leaves fed [4-¹⁴C]ALA is strongly inhibited by aminooxyacetate, anaerobiosis, and malonate. The rate of ¹⁴CO₂ evolution from leaves fed [5-¹⁴C]ALA is also inhibited by these treatments but to a lesser extent. These results suggest that (a) one step in ALA catabolism is a transamination reaction and (b) the C₄ is oxidized to CO₂ via the tricarboxylic acid cycle to a greater extent than is the C₅.     

Responses of CO2, N2O and CH4 fluxes between atmosphere and forest soil to changes in multiple environmental conditions

To investigate the effects of multiple environmental conditions on greenhouse gas (CO₂, N₂O, CH₄) fluxes, we transferred three soil monoliths from Masson pine forest (PF) or coniferous and broadleaved mixed forest (MF) at Jigongshan to corresponding forest type at Dinghushan. Greenhouse gas fluxes at the in situ (Jigongshan), transported and ambient (Dinghushan) soil monoliths were measured using static chambers. When the transported soil monoliths experienced the external environmental factors (temperature, precipitation and nitrogen deposition) at Dinghushan, its annual soil CO₂ emissions were 54% in PF and 60% in MF higher than those from the respective in situ treatment. Annual soil N₂O emissions were 45% in PF and 44% in MF higher than those from the respective in situ treatment. There were no significant differences in annual soil CO₂ or N₂O emissions between the transported and ambient treatments. However, annual CH₄ uptake by the transported soil monoliths in PF or MF was not significantly different from that at the respective in situ treatment, and was significantly lower than that at the respective ambient treatment. Therefore, external environmental factors were the major drivers of soil CO₂ and N₂O emissions, while soil was the dominant controller of soil CH₄ uptake. We further tested the results by developing simple empirical models using the observed fluxes of CO₂ and N₂O from the in situ treatment and found that the empirical models can explain about 90% for CO₂ and 40% for N₂O of the observed variations at the transported treatment. Results from this study suggest that the different responses of soil CO₂, N₂O, CH₄ fluxes to changes in multiple environmental conditions need to be considered in global change study.     

Soil-atmosphere exchange of nitrous oxide and methane in New Zealand terrestrial ecosystems and their mitigation options: a review

The two non-CO₂ greenhouse gases (GHGs) nitrous oxide (N₂O) and methane (CH₄) comprise 54.8% of total New Zealand emissions. Nitrous oxide is mainly generated from mineral N originating from animal dung and urine, applied fertiliser N, biologically fixed N₂, and mineralisation of soil organic N. Even though about 96% of the anthropogenic CH₄ emitted in New Zealand is from ruminant animals (methanogenesis), methane uptake by aerobic soils (methanotrophy) can significantly contribute to the removal of CH₄ from the atmpsphere, as the global estimates confirm. Both the net uptake of CH₄ by soils and N₂O emissions from soils are strongly influenced by changes in land use and land management. Quantitative information on the fluxes of these two non-CO₂ GHGs is required for a range of land-use and land-management ecosystems to determine their contribution to the national emissions inventory, and for assessing the potential of mitigation options. Here we report soil N₂O fluxes and CH₄ uptake for a range of land-use and land-management systems collated from published and unpublished New Zealand studies. Nitrous oxide emissions are highest in dairy-grazed pastures (10–12 kg N₂O–N ha^?1 year^{? 1}), intermediate in sheep-grazed pastures, (4–6 kg N₂O–N ha^?1 year^?1), and lowest in forest, shrubland and ungrazed pasture soils (1–2 kg N₂O–N ha^?1 year^?1). N deposited in the form of animal urine and dung, and N applied as fertiliser, are the principal sources of N₂O production. Generally, N₂O emissions from grazed pasture soils are high when the soil water-filled pore-space is above field capacity, and net CH₄ uptake is low or absent. Although nitrification inhibitors have shown some promise in reducing N₂O emissions from grazed pasture systems, their efficacy as an integral part of farm management has yet to be tested. Methane uptake was highest for a New Zealand Beech forest soil (10–11 kg CH₄ ha^?1 year^?1), intermediate in some pine forest soils (4–6 kg CH₄ ha^?1 year^?1), and lowest in most pasture (<1 kg CH₄ ha^?1 year^?1) and cropped soils (1.5 kg CH₄ ha^?1 year^?1). Afforestation /reforestation of pastures results in increases in soil CH₄ uptake, largely as a result of increases in soil aeration status and changes in the population and activities of methanotrophs. Soil CH₄ uptake is also seasonally dependent, being about two to three times higher in a dry summer and autumn than in a wet winter. There are no practical ways yet available to reduce CH₄ emissions from agricultural systems. The mitigation options to reduce gaseous emissions are discussed and future research needs identified.     

Anaerobic C1 Metabolism of the O-Methyl-14C-Labeled Substituent of Vanillate

The O-methyl substituents of aromatic compounds constitute a C₁ growth substrate for a number of taxonomically diverse anaerobic acetogens. In this study, strain TH-001, an O-demethylating obligate anaerobe, was chosen to represent this physiological group, and the carbon flow when cells were grown on O-methyl substituents as a C₁ substrate was determined by ¹⁴C radiotracer techniques. O-[methyl-¹⁴C]vanillate (4-hydroxy-3-methoxy-benzoate) was used as the labeled C₁ substrate. The data showed that for every O-methyl carbon converted to [¹⁴C]acetate, two were oxidized to ¹⁴CO₂. Quantitation of the carbon recovered in the two products, acetate and CO₂, indicated that acetate was formed in part by the fixation of unlabeled CO₂. The specific activity of ¹⁴C in acetate was 70% of that in the O-methyl substrate, suggesting that only one carbon of acetate was derived from the O-methyl group. Thus, it is postulated that the carboxyl carbon of the product acetate is derived from CO₂ and the methyl carbon is derived from the O-methyl substituent of vanillate. The metabolism of O-[methyl-¹⁴C]vanillate by strain TH-001 can be described as follows: 3¹⁴CH₃OC₇H₅O₃ + CO₂ + 4H₂O → ¹⁴CH₃COOH + 2¹⁴CO₂ + 10H⁺ + 10e^- + 3HOC₇H₅O₃.     

THE METABOLISM OF LEUCINE BY DEVELOPING RAT BRAIN: EFFECT OF LEUCINE AND 2-OXO-4-METHYLVALERATE ON LIPID SYNTHESIS FROM GLUCOSE AND KETONE BODIES

Abstract— The oxidation of l -[U-¹⁴C]leucine and l -[l-¹⁴C]leucine at varying concentrations from 0.1 to 5mM to CO₂ and the incorporation into cerebral lipids and proteins by brain slices from 1-week old rats were markedly stimulated by glucose. Although the addition of S mM-dl -3-hydroxybutyrate had no effect on the metabolism of [U-¹⁴C]leucine by brain slices from suckling rats, the stimulatory effects of glucose on the metabolism of l -[U-¹⁴C]leucine were markedly reduced in the presence of dl -3-hydroxybutyrate. The stimulatory effect of glucose on leucine oxidation was, however, not observed in adult rat brain. Furthermore, the incorporation of leucine-carbon into cerebral lipids and proteins was also very low in the adult brain. The incorporation of l -[U-¹⁴C]leucine into cerebral lipids by cortex slices was higher during the first 2 postnatal weeks, which then declined to the adult level. During this time span, the oxidation of l -[U-¹⁴C]leucine to CO₂ remained relatively unchanged. The incorporation in vivo of D-3-hydroxy[3-¹⁴C]butyrate into cerebral lipids was markedly decreased by acute hyperleucinemia induced by injecting leucine into 9-day old rats. In in vitro experiments, 5 mM-leucine had no effect on the oxidation of [U-¹⁴C]glucose to CO₂ or its incorporation into lipids by brain slices from 1-week old rats. However, 5 mM-leucine inhibited the oxidation of d -3-hydroxy-[3-¹⁴C]butyrate, [3-¹⁴C]acetoacetate and [1-¹⁴C]acetate to CO₂ by brain slices, but their incorporation into cerebral lipids was not affected by leucine. In contrast 2-oxo-4-methylvalerate, a deaminated metabolite of leucine, markedly inhibited both the oxidation to CO₂ and the incorporation into lipids of labelled glucose, ketone bodies and acetate by cortex slices from 1-week old rats. These findings suggest that the reduction in the incorporation in vivo of d -3-hydroxy[3-¹⁴C]butyrate into cerebral lipids in rats injected with leucine is most likely caused by 2-oxo-4-methylvalerate formed from leucine. Since the concentrations of leucine and 2-oxo-4-methylvalerate in plasma of untreated patients with maple-syrup urine disease are markedly elevated, our findings are compatible with the possibility that an alteration in the metabolism of glucose and ketone bodies in the brain may contribute to the pathophysiology of this disease.     

Validation of the design of feeding experiments involving [(14)C]substrates used to monitor metabolic flux in higher plants

The aim of this study was to examine whether flux through the pathways of carbohydrate oxidation is accurately reflected in the pattern of ¹⁴CO₂ release from positionally labelled [¹⁴C]substrates in conventional radiolabel feeding studies. Heterotrophic cell suspension cultures of Arabidopsis thaliana were used for this work. The presence of an alkaline trap to capture metabolically generated ¹⁴CO₂ had no significant effect on the ratio of ¹⁴CO₂ release from specifically labelled [¹⁴C]substrates, or on the metabolism of [U-¹⁴C]glucose by the cells. Although the amount of ¹⁴CO₂ captured in a conventional time-course study was only about half of that released from a sample acidified at an equivalent time point, the ratios of ¹⁴CO₂ released from different positionally labelled [¹⁴C]glucose and [1-¹⁴C]gluconate were the same in untreated and acidified samples. Less than 5% of radioactivity supplied to the growth medium as [¹⁴C]bicarbonate was incorporated into acid-stable compounds, and there was no evidence for appreciable reassimilation of ¹⁴CO₂ generated intracellularly during oxidation of [1-¹⁴C]gluconate by the cells. It is concluded that the ratio of label captured from specifically labelled [¹⁴C]glucose is a valid and convenient measure of the relative rates of oxidation of the different positional carbon atoms within the supplied respiratory substrate. However, it is argued that failure to compensate for the incomplete absorption of ¹⁴CO₂ by an alkaline trap may distort estimates of respiration that rely on an absolute measure of the amount of ¹⁴CO₂ generated by metabolism.     

Effects of nitrogen deposition on the greenhouse gas fluxes from forest soils

Nitrogen (N) deposition can alter the rates of microbial N- and C- turnover, and thus can affect the fluxes of greenhouse gases (GHG, e.g., CO₂, CH₄, and N₂O) from forest soils. The effects of N deposition on the GHG fluxes from forest soils were reviewed in this paper. N deposition to forest soils have shown variable effects on the soil GHG fluxes from forest, including increases, decreases or unchanged rates depending on forest type, N status of the soil, and the rate and type of atmospheric N deposition. In forest ecosystems where biological processes are limited by N supply, N additions either stimulate soil respiration or have no significant effect, whereas in “N saturated” forest ecosystems, N additions decrease CO₂ emission, reduce CH₄ oxidation and elevate N₂O flux from the soil. The mechanisms and research methods about the effects of N deposition on GHG fluxes from forest soils were also reviewed in this paper. Finally, the present and future research needs about the effects of N deposition on the GHG fluxes from forest soils were discussed.