CO(2) Fixation in Opuntia Roots

Nonautotrophic CO₂ metabolism in Opuntia echinocarpa roots was studied with techniques of manometry and radiometry. The roots were grown in a one-quarter strength nutrient solution for several days; the distal 2 cm was used for physiological studies. The roots assimilated significant quantities of ¹⁴CO₂ and appeared to show a crassulacean-type acid metabolism with respect to quality and quantity. Most of the ¹⁴C activity was associated with the distal portion of the elongating root indicating correlation with metabolic activity. The ¹⁴CO₂ assimilation was comparable to a crassulacean leaf succulent, but 3 times greater than that found for stem tissue of the same Opuntia species.

The rates of O₂ and CO₂ exchange and estimated CO₂ fixation were 180, 123, and 57 μl/g per hour. A respiratory quotient of 0.66 was found.

The products of ¹⁴CO₂ fixation were similar in most respects to reported experiments with leaf succulents. Equilibration of the predominant malic acid with isocitric, succinic, and fumaric acids was not evident. The latter observation was interpreted as metabolic isolation of the fixation products rather than poor citric acid cycle activity.

A rapid turnover of the fixed ¹⁴CO₂ was measured by following decarboxlyation kinetics and by product analysis after a postincubation period. The first order rate constant for the steady state release was 4.4 × 10⁻³ min⁻¹ with a half-time of 157.5 minutes. Amino acids decayed at a more rapid rate than organic acids.

     

Continuous monitoring of rhizosphere respiration after labelling of plant shoots with 14CO2

The present work describes an original method to follow rate of ¹⁴CO₂ and total CO₂ production from rhizosphere respiration after plant shoots had been pulse-labelled with ¹⁴CO₂. We used a radioactivity detector equipped with a plastic cell for flow detection of beta radiation by solid scintillation counting. The radioactivity detector was coupled with an infrared gas analyser. The flow detection of ¹⁴CO₂ was compared to trapping of ¹⁴CO₂ in NaOH and counting by liquid scintillation. First, we demonstrated that NaOH (1 M) trapped 95% of the CO₂ of a gaseous sample. Then, we determined that the counting efficiency of the radioactivity flow cell was 41% of the activity of gaseous samples as determined by trapping in NaOH (1 M) and by counting by static liquid scintillation. The sensitivity of the ¹⁴CO₂- flow detection was 0.08 Bq mL⁻¹ air and the precision was 2.9% of the activity measured compared to 0.9% for NaOH trapping method. We presented two applications which illustrate the relevance of ¹⁴CO₂-flow detection to investigations using 14C to trace photoassimilates within the plant-soil system. First, we examined the kinetics of 14CO₂ production when concentrated acid is added to NaH¹⁴CO₃. This method is the most commonly used to label photoassimilates with ¹⁴C. Then, we monitored ¹⁴CO₂ activity in rhizosphere respiration of 5-week old maize cultivated in soil and whose shoots had been pulse-labelled with ¹⁴CO₂. We conclude that alkali traps should be used for a cumulative determination of ¹⁴CO₂ because they are cheap and accurate. On the other hand, we demonstrated that the flow detection of ¹⁴CO₂ had a finer temporal resolution and was consequently a relevant tool to study C dynamics in the rhizosphere at a short time scale. This revised version was published online in June 2006 with corrections to the Cover Date.     

Intermediary metabolism of carbon compounds by nitrifying bacteria

Summary The assimilation of¹⁴CO₂ and [2-¹⁴C] acetate, [3-¹⁴C] pyruvate, [5-¹⁴C] -ketoglutarate, [2,3-¹⁴C] succinate, [U-¹⁴C] glutamate and [U-¹⁴C] aspartate was followed in cell suspensions ofNitrosomonas europaea andNitrobacter agilis respectively. There was appreciable incorporation of these substrates even without adding the inorganic nitrogen compounds that are oxidized by these bacteria yielding ATP. In the soluble amino acid fraction most of¹⁴C label was recovered in glutamate while in the protein amino acids a more uniform distribution was found. Acetate was rapidly incorporated to a high level in both nitrifying bacteria while inNitrobacter there was a relatively lower uptake of the other substrates especially succinate. High levels of the NAD malate dehydrogenase and NADP isocitrate dehydrogenase were measured but no significant amounts of the other tricarboxylic acid cycle enzymes or NADH oxidase were found. Glutamate decarboxylase was detected in both organisms and the transferase assay for glutamine synthetase indicated a 30-fold higher activity for this enzyme inNitrobacter. The amino acid composition of the water soluble fraction was determined in both bacteria.     

Radiorespirometer for continuous monitoring of chick embryo development.

A radiorespirometer is described that is capable of continuous monitoring of O₂ utilization, CO₂ and/or ¹⁴CO₂ production per minute, heart rate, and body activity of an embryonated egg while it develops for several days in a closed chamber with near normal pO₂ or other desired pO₂. Oxygen is supplied to the embryo by an electrolytic cell, and CO₂ is removed by a KOH solution flowing through a diffusion cell separated from the embryo chamber by a CO₂ permeable rubber membrane. The instrument permits selecting embryos for viability and developmental stage, according to their O₂ utilization per minute and respiratory quotient, without breaking the eggshell. Inoculation of the embryonated egg with ¹⁴C-labeled substrates, drugs, or toxins can occur without interfering with continuous recording of metabolic activity.     

Isotope Labeling and Microautoradiography of Active Heterotrophic Bacteria on the Basis of Assimilation of 14CO2

Most heterotrophic bacteria assimilate CO₂ in various carboxylation reactions during biosynthesis. In this study, assimilation of ¹⁴CO₂ by heterotrophic bacteria was used for isotope labeling of active microorganisms in pure cultures and environmental samples. Labeled cells were visualized by microautoradiography (MAR) combined with fluorescence in situ hybridization (FISH) to obtain simultaneous information about activity and identity. Cultures of Escherichia coli and Pseudomonas putida assimilated sufficient ¹⁴CO₂ during growth on various organic substrates to obtain positive MAR signals. The MAR signals were comparable with the traditional MAR approach based on uptake of ¹⁴C-labeled organic substrates. Experiments with E. coli showed that ¹⁴CO₂ was assimilated during both fermentation and aerobic and anaerobic respiration. The new MAR approach, HetCO₂-MAR, was evaluated by targeting metabolic active filamentous bacteria, including “Candidatus Microthrix parvicella” in activated sludge. “Ca. Microthrix parvicella” was able to take up oleic acid under anaerobic conditions, as shown by the traditional MAR approach with [¹⁴C]oleic acid. However, the new HetCO₂-MAR approach indicated that “Ca. Microthrix parvicella,” did not significantly grow on oleic acid under anaerobic conditions with or without addition of NO₂⁻, whereas the addition of O₂ or NO₃⁻ initiated growth, as indicated by detectable ¹⁴CO₂ assimilation. This is a metabolic feature that has not been described previously for filamentous bacteria. Such information could not have been derived by using the traditional MAR procedure, whereas the new HetCO₂-MAR approach differentiates better between substrate uptake and substrate metabolism that result in growth. The HetCO₂-MAR results were supported by stable isotope analysis of ¹³C-labeled phospholipid fatty acids from activated sludge incubated under aerobic and anaerobic conditions in the presence of ¹³CO₂. In conclusion, the novel HetCO₂-MAR approach expands the possibility for studies of the ecophysiology of uncultivated microorganisms.     

Carbon dioxide fixation by detached cereal caryopses

Immature detached cereal caryopses from barley (Hordeum vulgare L. var distichum cv Midas) and wheat (Triticum aestivum L. cv Sicco) were shown to be capable of fixing externally supplied ¹⁴CO₂ in the light or dark. Green cross cells and the testa contained the majority of the ¹⁴C-labeled material. Some ¹⁴C-labeled material was also found in the outer, or transparent, layer and in the endosperm/embryo fraction. More ¹⁴C was recovered from caryopses when they were incubated in ¹⁴CO₂ without the transparent layer, thus suggesting that this layer is a barrier to the uptake of CO₂. In all cases, significant amounts of ¹⁴C-labeled material were found in caryopses after dark incubation with ¹⁴CO₂. Interestingly, CO₂ fixation in the chlorophyll-less mutant Albino lemma was significantly greater in the light than in the dark. The results indicate that intact caryopses have the ability to translocate ¹⁴C-labeled assimilate derived from external CO₂ to the endosperm/embryo. Carboxylating activity in the transparent layer appears to be confined to phosphoenolpyruvate carboxylase activity but that in the chloroplast-containing cross-cells may be accounted for by both ribulose-1,5-bisphosphate carboxylase-oxygenase and phosphoenolpyruvate carboxylase activity. Depending on a number of assumptions, the amount of CO₂ fixed is sufficient to account for about 2% of the weight of starch found in the mature caryopsis.     

Comparison of unitrophic and mixotrophic substrate metabolism by acetate-adapted strain of Methanosarcina barkeri

We examined the unitrophic metabolism of acetate and methanol individually and the mixotrophic utilization of these compounds by using detailed ¹⁴C-labeled tracer studies in a strain of Methanosarcina barkeri adapted to grow on acetate as the sole carbon and energy source. The substrate consumption rate and methane production rate were significantly lower on acetate alone than during the unitrophic or mixotrophic metabolism of methanol. Cell yields (in grams per mole of substrate) were identical during exponential growth on acetate and exponential growth on methanol. During unitrophic metabolism of acetate, the methyl moiety accounted for the majority of the CH₄ produced, but 14% of the CO₂ generated originated from the methyl moiety. This correlated with the concurrent reduction of equivalent amounts of the C-1 of acetate to CH₄. ¹⁴CH₄ was also produced from added ¹⁴CO₂, although to a lesser extent than from reduction of the C-1 of acetate. During mixotrophic metabolism, methanol and acetate were catabolized simultaneously. The rates of ¹⁴CH₄ and ¹⁴CO₂ generation from [2-¹⁴C]acetate were logarithmic and higher in mixotrophic than in unitrophic cultures at substrate concentrations of 50 mM. A comparison of the oxidoreductase activities in cell extracts of the acetate-adapted strain grown on acetate and of strain MS grown on methanol or on H₂ plus CO₂ indicated that the pyruvate, α-ketoglutarate, and isocitrate dehydrogenase activities remained constant, whereas the CO dehydrogenase activity was significantly higher (5,000 nmol/min per mg of protein) in the acetate-adapted strain. These results suggested that a significant intramolecular redox pathway is possible for the generation of CH₄ from acetate, that energy metabolism from acetate by M. barkeri is not catabolite repressed by methanol, and that the acetate-adapted strain is a metabolic mutant with derepressed CO dehydrogenase activity.     

Compartmentation of malate in relation to ion absorption in beet

Malate in beet discs treated in different salt solutions was labeled by a 30 min pulse of ¹⁴CO₂, and subsequent changes in specific activity were followed for several hr. In treatments which resulted in net acid synthesis in response to excess cation absorption, malate specific activity fell slowly after removal of ¹⁴CO₂. In solutions where no net acid synthesis occurred, and from which cation and anion were absorbed equally, malate specific activity fell rapidly when ¹⁴CO₂ was removed. The foregoing suggests that the net synthesis of organic acids in response to excess cation absorption leads to the removal of organic anions from cytoplasmic metabolic pools as counter-ions in salt transport to the vacuole.     

Oriented translocation of energy in grafted reef corals

Colonies of the Red Sea reef coral Stylophora pistillata were grafted with alien branches (alografts), which had been labelled by NaH¹⁴CO₃ in the light. The “cold” host-colonies translocated the ¹⁴C-containing photosynthetic metabolites in an oriented pathway from the grafted branches into their own tissues. The highest accumulations of ¹⁴C products were detected in specific branch-tips of the host, away from the contact zones. The “recipient” colonies utilize these energy-rich materials for their metabolic requirements. The ¹⁴CO₂ produced through respiration is consequently detected in the skeletal-carbonate of the tips as Ca¹⁴CO₃. The purple morph of S. pistillata is found to be superior to the yellow morph.     

Biochemical components of the photosynthetic CO2 compensation point of higher plants.

Barley, Panicum milioides and Panicum maximum were exposed to ¹⁴CO₂ near their photosynthetic CO₂ compensation points and their respective ¹⁴C-products were determined. In short exposure times Panicum maximum had 100% of its ¹⁴C in malate and aspartate whereas Panicum milioides and barley had 16 and 3% of their respective ¹⁴C in C₄ organic acids. Near the respective CO₂ compensation points a linear relationship occurs in plotting the ratio of glycine, serine, and glycerate to C₄ organic acids. The ratio of ribulose 1,5-bisphosphate oxygenase to phosphoenolpyruvate carboxylase is linear with their CO₂ compensation points. The photosynthetic CO₂ compensation point apparently is controlled by the activity of enzymes producing photorespiration metabolites and the activity of phospheonolpyruvate carboxylase.     

Morphine metabolism and normorphine in Papaver somniferum

Normorphine has been established as an active metabolite of morphine in P. somniferum. This was done by, (a) demonstrating the presence of normorphine throughout the life cycle of the plant, (b) finding normorphine-¹⁴C after feeding morphine-¹⁴C via the roots, and (c) exposing opium poppies to ¹⁴CO₂ under steady state conditions which led to morphine and normorphine of the same specific activity. Feeding normorphine-¹⁴C showed that the N-demethylation step is irreversible. A sensitive procedure was developed for the detection of normorphine in the presence of large amounts of morphine; using this procedure, normorphine was found in raw opium. These results indicate that the major, if not the sole, morphine degradative pathway involves an initial demethylation to normorphine, which is subsequently degraded to non-alkaloidal metabolites. The high rates of turnover observed led to the conclusion that the morphine alkaloids do play an active metabolic role, perhaps as specific methylating agents.     

In Vivo Measurement of Indole-3-acetic Acid Decarboxylation in Aging Coleus Petiole Sections

The concentration of indoleacetic acid (IAA) in plant tissues is regulated, in part, by its rate of decarboxylation. However, the commonly used in vitro assays for IAA oxidase may not accurately reflect total in vivo decarboxylation rates. A method for measuring in vivo decarboxylation was utilized in which ¹⁴CO₂ is collected following uptake of [1-¹⁴C]IAA by excised tissue sections. After a 30-minute equilibration period, the evolution of ¹⁴CO₂ was found to follow an approximately linear course with respect to both time and tissue weight.

Decarboxylation rates were measured by this method in petiole sections of the Princeton clone of Coleus blumei Benth. Both the ¹⁴CO₂ evolved per milligram tissue and the percent of [1-¹⁴C]IAA uptake decarboxylated were highest in sections from the youngest petioles tested, and declined in the older tissue. Thin layer chromatography of acetonitrile extracts from the [1-¹⁴C]IAA-treated petioles showed a decreasing amount of free IAA and an increase at the retardation factor of indoleacetylaspartate in the older sections. The decreased decarboxylation rates in the older petioles may be attributable to a generally lower metabolic rate and increased protection of the IAA by conjugation.

     

Quantification de bactéries nitrifiantes du genre Nitrobacter en milieux aquatiques (l'estuaire de la Seine,France)

In the Seine estuary, nitrification is a major process involved in the oxygen budget, together with photosynthesis, heterotrophic bacterial degradation and reaeration. Within the framework of the Seine-Aval programme, we attempted to further understand the process using a molecular approach that was expected to detect nitrifying bacteria, and also to quantify them. On the basis of the PCR approach using specific primers of the genus Nitrobacter, we coupled a competitive PCR method in order to quantify these Nitrobacter, a genus widely distributed in freshwater aquatic ecosystems. This molecular approach was conducted in parallel with a measurement of potential nitrifying with a ¹⁴C radiotracing method. We showed that Nitrobacter represent less than 0.1% of the total bacterial community, as determined by epifluorescence microscopy, and that potential nitrifying activity increased with Nitrobacter numbers. Following that methodological step demonstrating a real feasibility, a possible relationship between these two measurements is expected on the basis of five longitudinal field studies already realised.     

Biosynthesis and metabolism of steryl glycosides in Sinapis alba seedlings

With ¹⁴CO₂, d-glucose-[U-¹⁴C] and dl-mevalonate-[4R-4-³H₁] used as precursors, a study was made of the labelling dynamics of the steryl glucosides (SG) and steryl acylglucosides (ASG) in Sinapis alba seedlings. The radioactivity of the sterol and sugar moieties, as well as of the fatty acid moieties in the case of ASG, was analysed separately. The course of incorporation of ¹⁴C from ¹⁴ CO₂ and glucose-[U-¹⁴C] into the sugar part of SG and ASG indicated that about $\text{2}\text{3}$ of the whole pool of the newly synthesized sterol glycosides of both types underwent rapid deglucosylation. Likewise, fatty acids in the ASG pool were rapidly exchanged. The present results point to a high metabolic activity of the sterol glycoside derivatives in plant cells.     

13[C]-Urea Breath Test as a Novel Point-of-Care Biomarker for Tuberculosis Treatment and Diagnosis

Background

Pathogen-specific metabolic pathways may be detected by breath tests based on introduction of stable isotopically-labeled substrates and detection of labeled products in exhaled breath using portable infrared spectrometers.

Methodology/Principal Findings

We tested whether mycobacterial urease activity could be utilized in such a breath test format as the basis of a novel biomarker and diagnostic for pulmonary TB. Sensitized New-Zealand White Rabbits underwent bronchoscopic infection with either Mycobacterium bovis or Mycobacterium tuberculosis. Rabbits were treated with 25 mg/kg of isoniazid (INH) approximately 2 months after infection when significant cavitary lung pathology was present. [¹³C] urea was instilled directly into the lungs of intubated rabbits at selected time points, exhaled air samples analyzed, and the kinetics of δ¹³CO₂ formation were determined. Samples obtained prior to inoculation served as control samples for background ¹³CO₂ conversion in the rabbit model. ¹³CO₂, from metabolic conversion of [¹³C]-urea by mycobacterial urease activity, was readily detectable in the exhaled breath of infected rabbits within 15 minutes of administration. Analyses showed a rapid increase in the rate of ¹³CO₂ formation both early in disease and prior to treatment with INH. Following INH treatment, all evaluable rabbits showed a decrease in the rate of ¹³CO₂ formation.

Conclusions/Significance

Urea breath testing may provide a useful diagnostic and biomarker assay for tuberculosis and for treatment response. Future work will test specificity for M. tuberculosis using lung-targeted dry powder inhalation formulations, combined with co-administering oral urease inhibitors together with a saturating oral dose of unlabeled urea, which would prevent the δ¹³CO₂ signal from urease-positive gastrointestinal organisms.     

Dark metabolism of carbon monoxide in lettuce leaf discs

In the dark, leaf tissue of crisphead lettuce (Lactuca sativa L.) metabolized ¹⁴CO to ¹⁴CO₂ and acid-stable products. Tissue incubated at 2.5°C for 3.5 hours and 48 hours converted about 1% and 17%, respectively, of the applied ¹⁴CO to ¹⁴CO₂, and incorporated about 0.04% and 0.6% of the ¹⁴C in acid-stable products. Examination of soluble acid-stable products from ¹⁴CO and ¹⁴CO₂-treated leaf tissue revealed that the labeling patterns of both treatments were identical during the 3.5-hour and the 48-hour incubation periods. Malate, citrate, and aspartate together comprised 70% or more of the soluble radioactivity from both treatments. Incorporation of radioactivity from CO into soluble acid-stable products during a 3-hour incubation period at 2.5°C was inhibited 90% by adding 3% nonradioactive CO₂. These results indicate that in head lettuce in the dark ¹⁴CO is metabolized primarily to ¹⁴CO₂ which is the precursor of acid-stable products. In leaf discs at 2.5°C, the apparent K_m for CO oxidation to CO₂ was 5.3 microliters per liter and the V_max was 9.7 nanoliters per gram per hour. The mitochondrial fraction of the leaf homogenate was the most active fraction to oxidize CO to CO₂, and this activity was heat-labile and cyanide-sensitive.     

Effect of starvation and refeeding on amino acid metabolism in muscle of crab Neohelice granulata previously fed protein- or carbohydrate-rich diets

The present study assesses the effects of starvation and refeeding on 1-[¹⁴C]-methyl aminoisobutyric acid (¹⁴C-MeAIB) uptake, ¹⁴C-total lipids, ¹⁴CO₂ production from ¹⁴C-glycine, ¹⁴C-protein synthesis from ¹⁴C-leucine and Na⁺–K⁺-ATPase activity in jaw muscle of Neohelice granulata previously maintained on a carbohydrate-rich (HC) or high-protein (HP) diet. In N. granulata the metabolic adjustments during starvation and refeeding use different pathways according to the composition of the diet previously offered to the crabs. During starvation, ¹⁴CO₂ production from ¹⁴C-glycine, and ¹⁴C-protein synthesis from ¹⁴C-leucine were reduced in HC-fed crabs. In crabs maintained on the HP or HC diet, ¹⁴C-total lipid synthesis increased after 15 days of starvation. In crabs fed HP diet, ¹⁴C-MeAIB uptake and Na⁺–K⁺-ATPase activity decreased in refeeding state. In crabs refeeding HC diet, ¹⁴C-MeAIB uptake and ¹⁴CO₂ production decreased during the refeeding. In contrast, the ¹⁴C-protein synthesis increased after 120 h of refeeding. In both dietary groups, ¹⁴C-total lipid synthesis increased during refeeding. Changes in the carbon amino acid flux between different metabolic pathways in muscle are among the strategies used by this crab to face starvation and refeeding. Protein or carbohydrate levels in the diet administered to this crab modulate the carbon flux between the different metabolic pathways.     

Randomization of carbon atoms in the glucose molecule and changes of specific radioactivity of14CO2 liberated by the callus tissue ofDaucus carota L. from glucose-6- and -1-14C

On incubation of the callus tissue ofDaucus carota L. in solutions of glucose-6-¹⁴C and -1-¹⁴C the distribution of radioactivity in the molecule of endogenous glucose will change and the ratio of activities of liberated¹⁴CO₂ (C₆/C₁) will rise The limits of possible changes of specific activity of¹⁴CO₂ and of the C₆/C₁ ratio were calculated with respect to the observed randomization and it was shown that the mutual exchange of carbon atoms in the molecules is not the decisive cause of the rise of the ratio. The specific radioactivity of¹⁴C in CO₂ is as much as 12 times higher than that of endogenous glucose and fructose and about twice as high as the theoretical maximum. This might indicate that in addition to the cytoplasmic fraction of glucose the callus cells contain a fraction of low metabolic activity, most likely in the vacuoles, that could account for some of the increase of the C₆/C₁ value. The main reason for the changes in the C₆/C₁ ratio is envisaged in the establishment of isotopic equilibrium between the pentose cycle and glycolysis and other metabolic systems, in particular via triose phosphates, the radioactivity of which can greatly affect the C₆/C₁ ratio, as was shown in a model experiment.     

Exchange Properties of the Activator CO(2) of Spinach Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

The exchange properties of the activator CO₂ of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase were characterized both in vitro with the purified enzyme, and in situ within isolated chloroplasts. Carboxyarabinitol-1,5-bisphosphate, a proposed reaction intermediate analog for the carboxylase activity of the enzyme, was used to trap the activator CO₂ on the enzyme both in vitro and in situ. Modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in intact chloroplasts during a light/dark cycle was associated with a similar modulation in carboxyarabinitol-1,5-bisphosphate-trapped CO₂. The exchange kinetics of the activator CO₂ were monitored by activation of the enzyme to steady state in the presence of ¹²CO₂, followed by addition of ¹⁴CO₂ and determination of the amount of labeled CO₂ trapped on the enzyme by carboxyarabinitol-1,5-bisphosphate. Rate constants (K_obs) for exchange with both the purified enzyme (0.45 min⁻¹) and in illuminated chloroplasts (0.18 min⁻¹) were comparable to the observed rate constants for enzyme activation under the two conditions. A similar exchange of the activator CO₂ was not observed in chloroplasts in the dark. Kinetic analysis of the exchange properties of the purified enzyme were consistent with an equilibrium between active and inactive forms of the enzyme during steady state activation.