Manometric determination of CO2 combined with scintillation counting of C-14

A procedure is described for rapid transfer to scintillation vials of ¹⁴CO₂ evolved by reactions in the mechanically shaken manometric chamber of Van Slyke and Neill. Results are given with amounts of CO₂ ranging from 2 to 1000 μmoles, and with Hyamine and phenylethylamine to absorb CO₂ in the vials.     

Continuous measurement of 14C-labeled carbon dioxide and lactic acid by cultured cells grown in Leighton tubes.

A procedure for continuous measurement of ¹⁴CO₂ production by cultured cells grown in Leighton tubes has been described. The apparatus developed also permits aliquots of the incubation medium to be taken during the experiments for analysis of labeled metabolites released into the solution. A simple method for determination of [¹⁴C]lactic acid in such aliquots has been described. The reproducibility and usefulness of the apparatus has been demonstrated by incubating fibroblasts with glucose labeled in the C-1 or C-6 position, and examining the effects of selected drugs on CO₂ and lactic acid production.     

Method for Simultaneous Measurement of Radioactive and Inactive CO(2) Evolved from Soil Samples During Incubation with Labeled Substrates

An apparatus is described which allows the simultaneous, continuous, and highly sensitive analysis of inactive and radioactive CO₂ evolved from ¹⁴C-supplemented soils or other materials. The apparatus consists of a control unit, a commercially available conductometric CO₂ analyzer, and fraction collector. A number of model experiments were conducted to demonstrate the potentials of the apparatus. These included analysis of the time course of priming action, when ¹⁴C-glucose was added to soil, separation of CO₂ respiration peaks caused by simultaneous degradation of radioactive and inactive soil supplements, and study of the effects of a fungicide, Benomyl, on degradation of ¹⁴C-labeled glucose. In the last experiment, partial degradation of the fungicide could also be followed.     

Method for Simultaneous Measurement of Radioactive and Inactive CO2 Evolved from Soil Samples During Incubation with Labeled Substrates

An apparatus is described which allows the simultaneous, continuous, and highly sensitive analysis of inactive and radioactive CO₂ evolved from ¹⁴C-supplemented soils or other materials. The apparatus consists of a control unit, a commercially available conductometric CO₂ analyzer, and fraction collector. A number of model experiments were conducted to demonstrate the potentials of the apparatus. These included analysis of the time course of priming action, when ¹⁴C-glucose was added to soil, separation of CO₂ respiration peaks caused by simultaneous degradation of radioactive and inactive soil supplements, and study of the effects of a fungicide, Benomyl, on degradation of ¹⁴C-labeled glucose. In the last experiment, partial degradation of the fungicide could also be followed.     

Radioassay of orotic acid phosphoribosyltransferase and orotidylate decarboxylase utilizing a high-voltage paper electrophoresis technique or an improved 14CO2- release method.

Orotic acid phosphoribosyltransferase (EC 2.4.2.10) and orotidylate decarboxylase (EC 4.1.1.23) can be assayed independently of one another by the high voltage paper electrophoresis method described here, which separates orotic acid, OMP, and UMP, the substrates and/or products of these enzymes, from each other. The relative migration of other compounds, mainly other nucleotides, their bases, or other intermediates of the UMP biosynthetic pathway, has also been recorded. The method has allowed us to observe that OMP is not released to any significant degree from the enzyme complex of these two enzymes that occurs in Ehrlich ascites cells; rather orotic acid is converted stoichiometrically by the enzyme complex to UMP. For purification of the enzyme complex, we have found the release of ¹⁴CO₂ from [¹⁴C]carboxyl-labeled orotic acid (when phosphoribosyl pyrophosphate and Mg²⁺ are present) preferable to the HVPE method as a routine assay procedure. The most economical CO₂-absorbant for the assay of the enzyme complex or for orotidylate decarboxylase (and possibly for other enzymes which release CO₂) is an NaOH-soaked paper strip. As detailed here, its use allows one to repeatedly reuse the scintillation vials and fluid.     

Use of anin situ labeling technique for the determination of seasonal14C distribution in Ponderosa pine

A¹⁴C labeling apparatus was developed to permit the labeling of four-year-old Ponderosa pine with¹⁴CO₂ in the field. The labeling system is a completely closed canopy system with¹⁴CO₂ monitored by a GM tube ratemeter apparatus. The level of¹⁴CO₂ corresponding to ambient levels is monitored by a microloggercomputer which controls a¹⁴CO₂ generating system. The generated¹⁴CO₂ is mixed in the canopy by circulating the atmosphere with 12V diaphram pumps. The portable system requires little operator attention. At approximately monthly intervals over a one-year period two four-year-old Ponderosa pine trees were labeled for three to five days using this labeling apparatus. After an assimilate distribution period, one tree was excavated and analyzed for¹⁴C distribution. During late spring and early summer most of the carbon assimilated (>60%) was found in the active growing tips and new needles, with little being allocated to the roots (<10%) or woody material (<20%). During mid to late fall there was an increase in root labeling along with an increase in carbon going to woody material. Over the winter period, most of the fixed carbon (65%) resided in the older leaves. The early spring labeling period showed another pulse of root labeling along with some labeling of woody tissues.     

Improved technique for accurate and convenient assay of biological reactions liberatingCO2

Use of a convenient and inexpensive apparatus for trapping¹⁴CO₂ from biological reactions in small volumes is described. Alternative scintillants for estimating¹⁴CO₂ trapped in KOH are compared, toluene methoxyethanol (2:1) based scintillants and Instagel (TM-Packard) were efficient and more stable than the dioxane/Cab-O-Sil or toluene/Triton X-100 mixtures usually used. The usefulness of the technique is illustrated with microassays of OMP decarboxylase (EC 4.1.1.23), OMP pyrophosphorylase (EC 2.4.2.10) in the fission yeastSchizosaccharomyces pombe.     

Experiments in whole leaf photosynthesis

The process of photosynthesis is of such importance that most courses of biochemistry and physiology include some reference to it. This paper describes a simple experimental system, which uses radioactive carbon dioxide to study whole leaf photosynthesis under a variety of conditions.

An apparatus of very simple construction, in which the experiments can be safely carried out, is described. The experiments basically consist of the exposure of leaves to ¹⁴CO₂ and the subsequent determination of radioactivity distribution and intensity by counting of leaf discs in a Geiger-Muller system and/or by autoradiography.

Experiments to demonstrate photosynthesis in the presence and absence of light, in variegated leaves of different types and to illustrate the importance of stomata are described.     

Laboratory-Produced CO(2) Calibration Gases

A simple, inexpensive apparatus for making mixtures of accurately known amounts of CO₂ and CO₂-free atmospheric air is described. Calibration gases with CO₂ contents of 200 to 1500 microliters per liter produced with the apparatus had concentrations which were within 10 microliters per liter of the target concentration.     

Analysis of steady state photosynthesis in alfalfa leaves

A method for carrying out kinetic tracer studies of steady state photosynthesis in whole leaves has been developed. An apparatus that exposes whole leaves to ¹⁴CO₂ under steady state conditions, while allowing individual leaf samples to be removed as a function of time, has been constructed. Labeling data on the incorporation of ¹⁴C into Medicago sativa L. metabolite pools are reported. A carbon dioxide uptake rate of 79 micromoles ¹⁴CO₂ per milligram chlorophyll per hour was observed at a CO₂ level slightly below that of air. Several actively turning over pools of early and intermediate metabolites, including 3-phosphoglyceric acid, glycerate, citrate, and uridine diphosphoglucose, showed label saturation after approximately 10 to 20 minutes of photosynthesis with ¹⁴CO₂ under steady state conditions. Alanine labeling increased more rapidly at first, and then at a lower rate as saturation was approached. Sucrose was a major product of photosynthesis and label saturation of the sucrose pool was not observed. Labeled carbon appeared rapidly in secondary metabolites. The steady state apparatus used has numerous advantages, including leaf temperature control, protection against leaf dehydration, high illumination, known ¹⁴CO₂ specific radioactivity, and provision for control and adjustment of ¹⁴CO₂ concentration. The apparatus allows for experiments of long duration and for sufficient sample points to define clearly the metabolic steady state.     

Technique for Measuring 14CO2 Uptake by Soil Microorganisms In Situ

Uptake of ¹⁴CO₂ in soils due to algae or sulfur-oxidizing bacteria was examined by incubation of soil samples with gaseous ¹⁴CO₂ and subsequent chemical oxidation of biologically fixed radioactive isotope to ¹⁴CO₂ for detection with a liquid scintillation counting system. The ¹⁴CO₂ was added to the soil in the gas phase so that no alteration of the moisture or ionic strength of the soil occurred. Wet oxidation of radioactive organic matter was carried out in sealed ampoules, and the ¹⁴CO₂ produced was transferred to a phenethylamine-liquid scintillation counting system with a simply constructed apparatus. The technique is inexpensive and efficient and does not require elaborate traps since several possible interfering factors were found to have no harmful effects. Experiments in coal mine regions and in geothermal habitats have demonstrated the ecological applicability of this technique for measurement of CO₂ fixation by sulfur-oxidizing bacteria and soil algae.     

A simple and sensitive assay for dopamine-beta-hydroxylase

A simple, rapid, and highly sensitive radiochemical assay for measuring the activity of dopamine-β-hydroxylase in tissues and serum is described. Enzyme activity is detected by converting [1-¹⁴C]tyramine to [1-¹⁴C]octopamine which is then subjected to periodate cleavage to form [¹⁴C]form-amide. This radiolabeled product is oxidized to ¹⁴CO₂ by addition of permanganate and the ¹⁴CO₂ is trapped and counted. The assay is simple and sensitive, it can linearly detect enzyme in all tissues with a wide range of activity, it uses maximal concentration of substrate, and it requires the addition of only one concentration of EMI to block endogenous inhibitor(s) in different tissues or enzyme concentrations.     

A comparison of double vial to serum bottle radiorespirometry to measure microbial mineralization in soils

Two methods of measuring mineralization rates were compared for their ability to quantify the microbiol mineralization of organic compounds in soils. In each of three soils used, the serum bottles gave higher yields for the mineralization of both [¹⁴C]glucose and [¹⁴C]cellulose (lignocellulose) than the double vials. In two of the soils, the serum bottles also showed less variation between replicates than the double vials. Furthermore, the mineralization of glucose in the serum bottles fit a first order rate model, whereas the mineralization of glucose in the double vials showed best fit to a linear model. Results using different amounts of soils indicated that container geometry may have placed unfavorable restrictions upon the double vial system, lowering the yield of ¹⁴CO₂ from the soils. Therefore, the serum bottle method was found to be the better method for measuring mineralization rates in soils.     

Determination of the specific radioactivity of 14C-labeled glutamic acid and glutamine

A new, simple, and accurate method for the sequential determination of the specific radioactivity of [1-¹⁴C]glutamic acid and [1-¹⁴C]glutamine is described. Using this method, radioactivity in H¹⁴CO₃^?, in [¹⁴C]glutamic acid, and in [¹⁴C]-glutamine can be readily determined on a single sample of blood plasma. Radioactivity is released as ¹⁴CO₂ in a stepwise fashion, trapped in the center wells, and counted in a liquid scintillation counter. The applicability of the method is discussed.     

Why and how to estimate the cost of symbiotic N2 fixation? A progressive approach based on the use of14C and15N isotopes

The process of symbiotic nitrogen fixation, though of obvious advantage to legumes in situations in which nitrogen is limiting, results in substantial penalty to the host plant in terms of cost of maintenance, synthesis and nitrogen reduction. Accurate estimates of costs are difficult to obtain because of the lack of simple methods to measure N₂ fixation and associated energy consumption. In relation to these difficulties, a multiple-step approach involving isotopes (¹⁴CO₂–¹⁵N₂) methodologies is described.The estimation of net respiratory cost associated with the N₂ reduction activity in near-natural conditions was achieved using simultaneous¹⁴CO₂ and¹⁵N₂ labelling. It gives a minimum value of 2.5 mg C/mg N fixed. This value was corrected by the estimation of the amount of carbon saved through the process of CO₂ fixation by the PEP carboxylase of the nodules, using¹⁴CO₂ in the soil atmosphere. This gives a real respiratory cost of 4 mg C/mg N fixed.     

Measurement of CO(2) Assimilation in Soils: an Experiment for the Biological Exploration of Mars

A method is described for the measurement of ¹⁴CO₂ assimilation by microorganisms in soils. A determination involves exposing soil to ¹⁴CO₂, pyrolyzing the exposed soil, trapping the organic pyrolysis products on a column of firebrick coated with CuO, combusting the trapped organics by heating, and measuring the radioactivity in the CO₂ produced in the combustion. The detection of significant levels of ¹⁴C in the trapped organic fraction appears to be an unambiguous indication of biological activity. The ¹⁴CO₂ which is adsorbed or exchanged into soils by nonbiological processes does not interfere. The method easily detects the ¹⁴CO₂ fixed by 10² to 10³ algae after light exposure for 3 to 24 hr. Assimilation of ¹⁴C is also demonstrable in dark-exposed soils containing 10⁵ to 10⁶ heterotrophic bacteria. Possible applications of the method in the biological exploration of Mars are discussed.     

Trimethylamine and Organic Matter Additions Reverse Substrate Limitation Effects on the δ13C Values of Methane Produced in Hypersaline Microbial Mats

Methane production has been observed in a number of hypersaline environments, and it is generally thought that this methane is produced through the use of noncompetitive substrates, such as the methylamines, dimethylsulfide and methanol. Stable isotope measurements of the produced methane have also suggested that the methanogens are operating under conditions of substrate limitation. Here, substrate limitation in gypsum-hosted endoevaporite and soft-mat hypersaline environments was investigated by the addition of trimethylamine, a noncompetitive substrate for methanogenesis, and dried microbial mat, a source of natural organic matter. The δ¹³C values of the methane produced after amendments were compared to those in unamended control vials. At all hypersaline sites investigated, the δ¹³C values of the methane produced in the amended vials were statistically lower (by 10 to 71‰) than the unamended controls, supporting the hypothesis of substrate limitation at these sites. When substrates were added to the incubation vials, the methanogens within the vials fractionated carbon isotopes to a greater degree, resulting in the production of more ¹³C-depleted methane. Trimethylamine-amended samples produced lower methane δ¹³C values than the mat-amended samples. This difference in the δ¹³C values between the two types of amendments could be due to differences in isotope fractionation associated with the dominant methane production pathway (or substrate used) within the vials, with trimethylamine being the main substrate used in the trimethylamine-amended vials. It is hypothesized that increased natural organic matter in the mat-amended vials would increase fermentation rates, leading to higher H₂ concentrations and increased CO₂/H₂ methanogenesis.     

Methodological Modifications for Accurate and Efficient Determination of Contaminant Biodegradation in Unsaturated Calcareous Soils

Many techniques for quantifying microbial biodegradation of ¹⁴C-labeled compounds use soil-water slurries and trap mineralization-derived ¹⁴CO₂ in solution wells suspended within the incubation flasks. These methods are not satisfactory for studies of arid-region soils that are highly calcareous and unsaturated because (i) slurries do not simulate unsaturated conditions and (ii) the amount of CO₂ released from calcareous soils exceeds the capacity of the suspended well. This report describes simple, inexpensive methodological modifications for quantifying microbial degradation of [¹⁴C]benzene and 1,2-dichloro[U-¹⁴C]ethane in calcareous soils under unsaturated conditions. Soils at 50% water holding capacity were incubated with labeled contaminants for periods up to 10 weeks, followed by acidification of the soil and trapping of the evolved CO₂ in a separate container of 2 N NaOH. The CO₂ was transferred from the incubation flask to the trap solution by a gas transfer shunt containing activated charcoal to remove any volatilized labeled organics. The amount of ¹⁴CO₂ in the trap solution was measured by scintillation counting (disintegrations per minute). The method was tested by using two regional unamended surface soils, a sandy aridisol and a clay-rich riparian soil. The results demonstrated that both [¹⁴C]benzene and 1,2-dichloro[U-¹⁴C]ethane were mineralized to release substantial amounts of ¹⁴CO₂ within 10 weeks. Levels of mineralization varied with contaminant type, soil type, and aeration status (anaerobic vs. aerobic); no significant degradation was observed in abiotic control samples. Methodological refinements of this technique resulted in total ¹⁴CO₂ recovery efficiency of approximately 90%.     

PHYSIOLOGICAL RESPONSES TO RAINFALL IN OPUNTIA BASILARIS (CACTACEAE)

An immediate, marked response to small amounts of rainfall occurs in Opuntia basilaris, despite previous drought conditions. The effect of rainfall is upon plant water potential, which is the single most important parameter influencing stomatal opening, CO₂ assimilation, and organic acid synthesis. Nocturnal stomatal opening is initiated following rainfall, and stomata remain open during the daytime. Decreasing stomatal and mesophyll resistances correlate with increasing rates of nocturnal assimilation of ¹⁴CO₂. Photosynthetic rates of ¹⁴CO₂ assimilation are low, despite high plant water potentials and low stomatal diffusion resistances. The decreased mesophyll resistances and increased rates of nocturnal ¹⁴CO₂ assimilation correlate with the increases of nocturnal efficiency of water use and CO₂ assimilation. The diurnal efficiency of water use and CO₂ assimilation is lower than the nocturnal gas exchange efficiency values.     

Short-term growth of meadow fescue with atmospheric CO2 enrichment decreases freezing tolerance, modifies photosynthetic apparatus performance and changes the expression of some genes during cold acclimation

The objective of this study was to assess the effect of an elevated atmospheric CO₂ molar ratio on freezing tolerance, photosynthetic apparatus performance and expression of CBF6, Cor14b and LOS2 in meadow fescue (Festuca pratensis Huds.). It was shown that cold acclimation under a CO₂ molar ratio of 800 μmol mol(air)^?1 decreased the freezing tolerance of meadow fescue when compared to the ambient CO₂ level. This effect was not related either to changes observed in PSII redox state or to photosynthetic acclimation to cold, which was in fact more effective at an elevated CO₂ level. The decrease in freezing tolerance was linked to changes in the expression of CBF6 and LOS2 genes, whereas the protective effect on photosynthetic apparatus was connected with the activation of a non-photochemical mechanism of photoprotection as well as upregulation of FpCOR14b expression.