Continuous Labeling Experiments Research Articles

Rice rhizodeposits affect organic matter priming in paddy soil: The role of N fertilization and plant growth for enzyme activities, CO2 and CH4 emissions

Carbon dioxide (CO2) and methane (CH4) production in paddy soils play a crucial role in the global carbon (C) cycle and greenhouse gas emissions. A rhizosphere priming effect (RPE) may change these emissions, but the relationships between RPE, CH4 emission, and the effect of N fertilization are unknown. We investigated the RPE on CO2 and CH4 emissions and their dependence from N fertilization in a13CO2 continuous labelling experiment by partitioning total CO2 and CH4 derived from roots and soil organic matter (SOM). Because of plant-derived CO2, rice plants strongly increased total CO2 emission compared to that from unplanted soil. SOM-derived CO2 and CH4 increased in the presence of roots but decreased after N fertilization. The RPE for CO2 at an early growth stage (≤40 days) was negative: −1.3 and −1.9 mg C day−1 kg−1 soil without and with N fertilization, respectively. However, 52 days after transplanting, RPE for CO2 got to positive. The RPE for CH4 increased gradually up to 1.6 and 0.5 mg C day−1 kg−1 soil at the end of the experiment without and with N fertilization, respectively. Moreover, the RPE for CH4 got half of the RPE for CO2 after 64 days showing the relevance of CH4 emissions for greenhouse gases balance and C cycling in paddy ecosystems. The RPE for CO2 and CH4 emissions increased with microbial biomass content and activities of xylanase and N-acetylglucosaminidase. Supporting the results to RPE, the enzyme activities decreased with N fertilization, suggesting that reduced N limitation decreased microbial potential to mine N from SOM. In conclusion, for the first time we showed that root-microbial interactions stimulated SOM mineralization in rice paddies through rhizosphere priming effects not only for CO2 but also for CH4, but the RPE decreased with N fertilization.

The recombinant equine LHβ subunit combines divergent intracellular traits of human LHβ and CGβ subunits

The pituitary LHβ and placental CGβ subunits are products of different genes in primates. The major structural difference between the two subunits is in the carboxy-terminal region, where the short carboxyl sequence of hLHβ is replaced by a longer O-glycosylated carboxy-terminal peptide in hCGβ. In association with this structural deviation, there are marked differences in the secretion kinetics and polarized routing of the two subunits. In equids, however, the CGβ and LHβ subunits are products of the same gene expressed in the placenta and pituitary (LHβ), and both contain a carboxy-terminal peptide. This unusual expression pattern intrigued us and led to our study of eLHβ subunit secretion by transfected Chinese hamster ovary and Madin–Darby canine kidney cells. In continuous labeling and pulse-chase experiments, the secretion of the eLHβ subunit from the transfected Chinese hamster ovary cells was inefficient (medium recovery of 16%–25%) and slow (t1/2 > 6.5 hours). This indicated that, the secretion of the eLHβ subunit resembles that of hLHβ rather than hCGβ. In Madin–Darby canine kidney cells grown on Transwell filters, the eLHβ subunit was preferentially secreted from the apical side, similar to the hCGβ subunit secretory route (∼65% of the total protein secreted). Taken together, these data suggested that secretion of the eLHβ subunit integrates features of both hLHβ and hCGβ subunits. We propose that the evolution of this intracellular behavior may fulfill the physiological demands for biosynthesis of the LH and CG β-subunits in the pituitary and placenta, respectively.

A continuous labelling approach to recover photosynthetically fixed carbon in plant tissue and rhizosphere organisms of young beech trees (Fagus sylvatica L.) using 13C depleted CO2

A continuous labelling experiment using 13C-CO2 was set up in open-top chambers in order to follow fluxes of assimilates from the plant into the rhizosphere. Labelling was performed for one growing season by adding low amounts of CO2 depleted in 13C to the atmosphere of the open-top chambers, resulting in a difference of ∆ 13C 5‰ V-PDB compared to ambient conditions. The label was recovered in both plant parts and soil microbial communities, analysed via phospholipid fatty acid (PLFA) side chains. PLFA 18:2ω6,9 showed a significant incorporation of the 13C label in October, indicating that fungi utilized plant derived carbon. In bacterial PLFA no label incorporation was detected, probably due to a lower use of rhizodeposits or a preference to older carbon compounds as energy sources. This experimental setup represents a low-cost continuous labelling method for field experiments with only minor increase of CO2 concentrations.

Magnetization transfer effects on the efficiency of flow-driven adiabatic fast passage inversion of arterial blood.

Continuous arterial spin labeling experiments typically use flow-driven adiabatic fast passage inversion of the arterial blood water protons. In this article, we measure the effect of magnetization transfer in blood and how it affects the inversion label. We use modified Bloch equations to model flow-driven adiabatic inversion in the presence of magnetization transfer in blood flowing at velocities from 1 to 30 cm/s in order to explain our findings. Magnetization transfer results in a reduction of the inversion efficiency at the inversion plane of up to 3.65% in the range of velocities examined, as well as faster relaxation of the arterial label in continuous labeling experiments. The two effects combined can result in inversion efficiency reduction of up to 8.91% in the simulated range of velocities. These effects are strongly dependent on the velocity of the flowing blood, with 10 cm/s yielding the largest loss in efficiency due to magnetization transfer effects. Flowing blood phantom experiments confirmed faster relaxation of the inversion label than that predicted by T(1) decay alone.

Three sources of CO2 efflux from soil partitioned by 13C natural abundance in an incubation study

This study describes a novel approach to separate three soil carbon (C) sources by one tracer method (here 13C natural abundance). The approach is based on the combination of C3 and C4 sources in different treatments, identical decomposition of C3 and C4 substances in soil, and subsequent calculation of their contribution to the total CO2 efflux. We used the temporal dynamics of the CO2 efflux from a C3 grassland soil amended with added C3 or C4 slurry and/or C3 or C4 sugar to estimate contributions of three separate C sources: native soil organic matter (SOM), slurry and sugar, to CO2 efflux. Soil with slurry and/or sugar was incubated under controlled conditions, and concentration and delta13C values of evolved CO2 were measured over a 2-week period. The main assumption needed for separation of three C sources in CO2 efflux, i.e. identical decomposition of applied C3 and C4 sugars in soil, was investigated and proven. The relative contribution to the CO2 efflux increased, but its duration decreased with an increased microbial availability of the C source, i.e. sugar > slurry > SOM. The microorganisms used the C sources according to their availability. The contribution of sugar to the CO2 efflux was finished after 2-4 days. Separation of three CO2 sources and comparison of CO2 from different treatments tracing the changes of SOM and slurry decomposition induced by addition of sugar were investigated. During the sugar decomposition (the first 2-4 days), the SOM decomposition strongly decreased. At the same time the contribution of slurry-C to CO2 increased. The shortcomings and limitations as well as possible future applications of the suggested method including FACE (Free Air Carbon dioxide Enrichments) and continuous labelling experiments are discussed.

Improving the amplitude‐modulated control experiment for multislice continuous arterial spin labeling

The use of an amplitude-modulated radiofrequency (RF) pulse for a control experiment is a proven method to control for off-resonance effects in multislice continuous arterial spin labeling (CASL) experiments. This method is also known as double adiabatic inversion. The adiabaticity factor of a single half-pulse, beta(1/2), and a new dimensionless parameter alpha, which is obtained from the labeling parameters and the flow velocity, are introduced. This makes it possible to distinguish three distinct cases: 1) With low alpha, a double inversion occurs. 2) With alpha > or = approximately 4, the efficiency with which the longitudinal magnetization is returned to the z-axis depends on the phase of the amplitude modulation at the time the spins cross the center of the labeling plane. 3) In the intermediate region, the efficiency shows undesirable fluctuations. In a Bloch equation simulation, three optimized parameter sets are determined. Near ideal performance should always be achieved by combinations of parameters for which beta(1/2) > or = approximately 2 and alpha approximately pi/beta(1/2). The efficiency increases were realized in a volunteer study, showing the practical application of the suggested optimization.

Theoretical background for partitioning of root and rhizomicrobial respiration by δ 13C of microbial biomass

Separate estimation of sources of root-derived CO 2 efflux from the soil into actual root respiration and microbial respiration of rhizodeposits is very important for determining the carbon (C) and energy balance of soils and plants, C sources for rhizosphere microorganisms, sources of soil organic matter (SOM), etc. Besides component integration, to date, only four adequate methods based on the pulse labeling of shoots in a 14CO 2 atmosphere and subsequent monitoring of 14CO 2 efflux from the soil have been suggested: 1) the isotope dilution, 2) the model rhizodeposition technique, 3) modeling of 14CO 2 efflux dynamics, and 4) the exudate elution procedure. These methods are based on different assumptions and principles that are very difficult to check experimentally and have different results. Therefore, none of these methods can be accepted as a standard procedure allowing quantitative separate estimation of root respiration and rhizomicrobial respiration. This contribution provides an elaboration of the theoretical background of a procedure allowing quantitative separate estimation of root respiration and rhizomicrobial respiration in non-sterile soils. The method is based on 13C natural abundance by growing C 4 plant on C 3 soil or vice versa. Four δ 13C values are necessary: of the SOM, of the roots, of soil microbial biomass, and of CO 2 efflux from the soil. The advantages and assumptions of the new approach, as well as possible applications including FACE systems and continuous labeling experiments are discussed.

Hepatic secretion of apoB-100 is impaired in hypobetalipoproteinemic mice with an apoB-38.9-specifying allele

Apolipoprotein B (apoB) truncation-specifying mutations cause familial hypobetalipoproteinemia (FHBL). Lipoprotein kinetics studies have shown that production rates of apoB-100 are reduced by 70-80% in heterozygous FHBL humans, instead of the expected 50%. To develop suitable mouse models to study the underlying mechanism, apoB-38.9-only (Apob(38.9/38.9)) mice were crossbred with Apobec-1 knockout (Apobec-1(-/-)) mice or apoB-100-only (Apob(100/100)) mice to produce two lines of apoB-38.9 heterozygous mice that produce only apoB-38.9 and apoB-100, namely Apobec-1(-/-)/Apob(38.9/+) and Apob(38.9/100) mice. In vivo rates of apoB-100 secretion were measured using [35S]Met/Cys to label proteins and Triton WR-1339 to block apoB-100 VLDL lipolysis/uptake. Rates of secretion were reduced by 80%, rather than the expected 50%, in both Apobec-1(-/-)/Apob(38.9/+) and Apob(38.9/100) mice compared with those of the respective Apobec-1(-/-)/Apob(+/+) and Apob(100/100) control mice. Continuous labeling and pulse-chase experiments in primary hepatocyte cultures revealed that rates of apoB-100 synthesis by Apobec-1(-/-)/Apob(38.9/+) and Apob(38.9/100) hepatocytes were reduced to the expected 50% of those of the respective controls, but the efficiency of secretion of apoB-100 was significantly lower in apoB-38.9 heterozygous hepatocytes. The greater-than-expected decreases in apoB-100 production rates of FHBL heterozygous humans appear to be attributable to a defect in secretion rather than in the synthesis of apoB-100 from the unaffected apoB allele.

Deletion of the GPIdeAc gene alters the location and fate of glycosylphosphatidylinositol precursors in Trypanosoma brucei.

Glycosylphosphatidylinositol (GPI) membrane anchors are ubiquitous among the eukaryotes. In most organisms, the pathway of GPI biosynthesis involves inositol acylation and inositol deacylation as discrete steps at the beginning and end of the pathway, respectively. The bloodstream form of the protozoan parasite Trypanosoma brucei is unusual in that these reactions occur on multiple GPI intermediates and that it can express side chains of up to six galactose residues on its mature GPI anchors. An inositol deacylase gene, T. brucei GPIdeAc, has been identified. A null mutant was created and shown to be capable of expressing normal mature GPI anchors on its variant surface glycoprotein. Here, we show that the null mutant synthesizes galactosylated forms of the mature GPI precursor, glycolipid A, at an accelerated rate (2.8-fold compared to wild type). These free GPIs accumulate at the cell surface as metabolic end products. Using continuous and pulse-chase labeling experiments, we show that there are two pools of glycolipid A. Only one pool is competent for transfer to nascent variant surface glycoprotein and represents 38% of glycolipid A in wild-type cells. This pool rises to 75% of glycolipid A in the GPIdeAc null mutant. We present a model for the pathway of GPI biosynthesis in T. brucei that helps to explain the complex phenotype of the GPIdeAc null mutant.

Quantitative analysis of adiabatic fast passage for steady laminar and turbulent flows.

Adiabatic fast passage (AFP) is used in noninvasive quantitative perfusion experiments to invert (or label) arterial spins. Continuous arterial spin labeling (CASL) experiments conducted in vivo often assume the inversion efficiency based on the labeling field and steady flow conditions, without direct verification. In practice, the labeling field used in CASL is often amplitude- and duty cycle-limited due to hardware or specific absorption rate constraints. In this study, the effects of the labeling field amplitude and duty cycle, and flow dynamics on the inversion efficiency of AFP were examined under steady flow conditions in a saline flow phantom. The experimental results were in general agreement with models based on Zhernovoi's theory except at high labeling field amplitudes, when the spin inversion times are at least half of the duration of the labeling pulse. The nonlinear relation observed between the inversion efficiency and the labeling duty cycle implies that the practice of linear derating the inversion efficiency with the labeling duty cycle may be prone to significant error. A secondary finding was that the T1 of the flowing fluid could be calculated based on the flow dynamics after varying the flow rate.

Heterogeneous cell renewal of pancreas in mice: [(3)H]-thymidine autoradiographic investigation.

Although cell kinetics of the gastrointestinal mucosa has been extensively examined, that of the pancreas has not been fully analyzed. To determine the renewal rate of pancreatic cells directly. Postnatal proliferative activity and cellular renewal of the parenchymal cells in ICR mouse pancreas were studied by immunohistochemistry and [(3)H]-thymidine autoradiography. In the single labeling experiments, the proliferative activity of the parenchymal cells in pancreas showed peaks at a few days after birth, decreased thereafter, and reached a low level at 2 months after birth. Continuous labeling experiments revealed that, in the adult pancreas, the half lives of acinar cells, islet cells, and duct epithelial cells were approximately 70 days, 47 days, and 40 days, respectively. Moreover, in the exocrine pancreas, acinar cells of the peri-insular region proliferated more actively than those of the tele-insular region. The renewal rate of glucagon cells was more rapid than that of insulin cells or somatostatin cells. Large ducts showed a high rate of cell renewal in comparison with small ducts. The results of this study indicate that cell renewal rates of the pancreas are not homogeneous, but heterogeneous.

The Effect of Elevated [CO2] on Uptake and Allocation of 13C and 15N in Beech (Fagus sylvatica L.) during Leafing

Abstract: A continuous dual 13CO2 and 15NH415NO3 labelling experiment was undertaken to determine the effects of ambient (350μmol mol‐1) or elevated (700μmol mol‐1) atmospheric CO2 concentrations on C and N uptake and allocation within 3‐year‐old beech (Fagus sylvatica L.) during leafing. After six weeks of growth, total carbon uptake was increased by 63 % (calculated on total C content) under elevated CO2 but the carbon partitioning was not altered. 56 % of the new carbon was found in the leaves. On a dry weight basis was the content of structural biomass in leaves 10 % lower and the lignin content remained unaffected under elevated as compared to ambient [CO2]. Under ambient [CO2] 37 %, and under elevated [CO2] 51 %, of the lignin C of the leaves derived from new assimilates. For both treatments, internal N pools provided more than 90 % of the nitrogen used for leaf‐growth and the partitioning of nitrogen was not altered under elevated [CO2]. The C/N ratio was unaffected by elevated [CO2] at the whole plant level, but the C/N ratio of the new C and N uptake was increased by 32 % under elevated [CO2].

Bud formation precedes the appearance of differential cell proliferation during branching morphogenesis of mouse lung epithelium in vitro.

Cell proliferation is an essential requirement for epithelial expansion and tubular branching; however, little is known of how these events are coupled during morphogenesis. We have previously shown that, in the absence of mesenchyme, fibroblast growth factor 1 (FGF-1) elicits budding of the mouse lung epithelium cultured in a basement membrane matrix. Although bud formation seems to be the manifestation of a localized response of lung epithelial cells to FGF-1, it is unclear whether budding results from induction of differential rates of cell proliferation within the epithelium. We performed continuous labeling and pulse-chase experiments in FGF-1-treated mesenchyme-free lung epithelial cultures at distinct stages of bud induction using bromodeoxyuridine (BrdU), to determine when and to what extent cell proliferation contributes to bud formation. When explants were incubated with BrdU either before bud induction (0-18 hr in culture) or at the onset of budding (24-30 hr), labeled nuclei were found distributed throughout the entire explant. In contrast, BrdU incubation after the onset of budding (30-48 hr) resulted in labeling concentrated in the budding areas, and a decrease of labeling toward the proximal region of the explant, between buds. These results demonstrate that differential rates of cell proliferation between bud and nonbud areas do not appear until when buds are almost completely formed. Thus, in the developing lung epithelium in vitro, bud outgrowth is not triggered by induction of localized cell proliferation.

A transition probability cell cycle model simulation of bivariate DNA/bromodeoxyuridine distributions.

The transition probability cell cycle model is extended to describe both cell cycle variability and incorporation of bromodeoxyuridine (BrdUrd). The model can simulate BrdUrd uptake in both pulse-chase and continuous-labeling experiments. With the use of a random transition, variability due to cell cycle progression is distinguished from dispersion due to staining and machine errors in the generation of bivariate DNA/BrdUrd distributions. In a comparative test with a compartmental model developed by Yanagisawa et al. (Cytometry 6:550-562, 1985), the present model is shown to provide realistic simulations with fewer model parameters and with the ability to describe gradual asynchronization of cell cycle cohorts. With model predictions as the basis, a simulated experiment is performed to illustrate the difficulty in analyzing bivariate distributions. The simulated experiment illustrated that it is very easy to overestimate unlabeled cell fractions, and, as a result, matching the periodicity of the cell cycle cohort movements is more reliable in the estimation of model parameters.

Mechanism of inhibition of neointimal formation by the angiotensin-converting enzyme inhibitor cilazapril. A study in balloon catheter-injured rat carotid arteries.

We investigated the mechanism of inhibition of neointima formation by the angiotensin-covering enzyme the carotid artery. We looked for the effects of cilazapril on all phases of the response to injury, ie, on proliferation of smooth muscle cells (SMCs) in the media, their migration, their proliferation in the neointima, and their disposition of extracellular matrix in the neointima. Although treatment was discontinued after 2 weeks, the inhibitory effect of cilazapril on neointimal formation was evident even 52 weeks after injury. The amount of extracellular matrix deposited in the intima during cilazapril treatment was decreased by 20% 2 weeks after injury, but no effect was seen if tissues were analyzed at 4 or 52 weeks. [3H]Thymidine-labeled cells (pulse labeling as well as 14-day continuous labeling) showed a decrease in SMC labeling in the tunica medica by 50%, but no inhibition in the labeling indices was seen in the neointima. The fraction of unlabeled neointimal cells in the cilazapril-treated rats as judged from continuous labeling experiments was inhibited by 86%. Taken together, these data suggest an antiproliferative effect on medial SMCs and an inhibition of SMC migration into the intima by cilazapril. Since intimal extracellular matrix deposition was only delayed, the decrease in medial SMC proliferation and subsequent migration seems to be the main reason for the reduction of neointima formation.

Effects of specific dietary sugars on the incorporation of 13C label from dietary glucose into neutral sugars of rat intestine and serum glycoproteins.

Although theoretically all glycoprotein sugars can be derived from glucose, it may be hypothesized that specific dietary sugars could be preferential substrates for glycoprotein synthesis. To test this hypothesis, groups of rats received either continuously (continuous-labelling experiment) or for a single nutritional period (pulse-labelling experiment) a 13C-rich diet containing either maize starch or artificially labelled [13C]glucose. Some groups of rats were also provided during a single nutritional period with low amounts (20-200 mg/animal) of low-13C dietary sugars (mannose, galactose, fucose or fructose). If specific dietary sugars were preferentially incorporated into glycoproteins instead of glucose-derived labelled sugars, a decrease would be expected in the intestinal or serum glycoprotein-sugar 13C enrichment monitored by gas chromatography-isotope-ratio mass spectrometry (GC-IRMS). Contrary to this hypothesis the results showed no significant decrease with any of the specific dietary sugars. Furthermore, with dietary low-13C mannose or galactose, a significant increase in 13C enrichment of glycoprotein-sugars was observed compared with some other nutritional groups. Moreover, in the pulse-labelling experiment, dietary mannose and galactose induced similar patterns of 13C enrichment in intestinal and serum glycoprotein-sugars. Therefore, although specific dietary sugars do not appear to be preferential substrates for glycosylation under conditions and doses relevant to current concepts of nutrition, regulatory roles of some specific dietary sugars in relation to glycoprotein-sugar metabolism might be hypothesized. These findings could lead to similar studies using stable-isotope methodology in man which could have practical consequences, especially in parenteral nutrition where glucose is the only sugar provided to the metabolism.

Use of transfer function and compartmental analysis to quantify11C-labelled photoassimilate export from wheat leaves

Compartmental analysis of tracer loss from a leaf after pulse-labelling with carbon isotopes has often been used to infer the flow of photosynthate through the leaf. Recently, a more general approach has been suggested based upon estimation of the transfer function using data from pulse-labelling as well as continuouslabelling experiments. A comparison of these two approaches shows that with the same data set they give equivalent physiological interpretations. The measured decline of 11C activity from a wheat leaf after 11CO2 pulse-labelling was extrapolated by compartmental as well as transfer function analysis. Both methods estimated a 66.4% loss of the initially fixed 11C due to export and respiration. The advantage of transfer function analysis, however, is its applicability to continuous-labelling experiments. The model allows the use of the net photosynthetic rate as the reference (100%) value. Data from continuous-labelling experiments with wheat plants indicate diurnal variations in the export of freshly labelled assimilate of between 32.7% and 43.6% of net photosynthesis.

Proglucagon is processed to glucagon by prohormone convertase PC2 in alpha TC1-6 cells.

Proglucagon is processed differentially in the pancreatic alpha cells and the intestinal L cells to yield either glucagon or glucagon-like peptide 1, respectively, structurally related hormones with opposing metabolic actions. Here, we have studied the processing of proglucagon in alpha TC1-6 cells, an islet-cell line transformed by simian virus 40 large tumor (T) antigen, a model of the pancreatic alpha cell. We found that these cells process proglucagon at certain dibasic cleavage sites to release glucagon and only small amounts of glucagon-like peptide 1, as demonstrated by both continuous and pulse-chase labeling experiments. Both normal islet alpha cells and alpha TC1-6 cells were shown to express the prohormone convertase PC2 at high levels, but not the related protease PC3. Expression of PC2 antisense RNA in alpha TC1-6 cells inhibited both PC2 production and proglucagon processing concomitantly. We conclude that PC2 is the key endoprotease responsible for proglucagon processing in cells with the alpha-cell phenotype.

Analysis of lactase processing in rabbit

The proteolytic processing of rabbit intestinal lactase-phlorizin-hydrolase (LPH) was studied by pulse-chase and continuous labeling experiments in organ culture from 15-day-old rabbits in the presence of glycosylation and processing inhibitors. Monensin and brefeldin A inhibited the two proteolytic cleavages of the precursor indicating that they are post-Golgi events as previously reported for the unique cleavage of LPH in man [1]. The inhibition was not related to a concomitant alteration glycosylation; in fact, if trimming was blocked by MDNM the abnormal glycosylated precursor was proteolytically processed normally. Finally the use of the anti-microtubular drug colchicine strongly inhibited both cleavages and caused accumulation of the complex-glycosylated precursor form in the brush border fraction indicating that proteolytic events depend on intact microtubule (transport).

Protein stability regulates the expression of cytochrome c during the developmental cycle of Trypanosoma brucei

The expression of cytochrome c is developmentally regulated during the life cycle of Trypanosoma brucei. The level of regulation appears to be post-transcriptional since cytochrome c mRNA is present in all life stages of the parasite. We have used RNA from each life stage to prime in vitro translation systems and found that the cytochrome c mRNAs are equally translatable. Continuous labeling experiments conducted in vivo indicate that cytochrome c is synthesized at similar rates in both bloodstream and procyclic trypanosomes. Western blots, however, confirm that steady-state levels of cytochrome c are severely depressed in bloodstream forms. In a series of pulse/chase experiments we demonstrate that the half-life of cytochrome c is approximately 1 h in the bloodstream form and no detectable turnover occurred in the procyclic form. We conclude that a major step in the developmental regulation of cytochrome c expression in T. brucei occurs post-translationally due to rapid turnover of the protein in the bloodstream trypanosomes.