Fate of thymocytes: studies with 125I-iododeoxyuridine and 3H-thymidine in mice.

Cortical thymocytes of young adult mice were labeled in situ with radioactive DNA precursors. As a result of cell emigration and cell death, total thymic radioactivity decreased within 8 days to 10% or less of that present on day 1. Accumulation of thymic migrants in peripheral lymphoid organs was estimated by computing the net thymus-derived radioactivity in these tissues. Thymic cell death was assessed by comparing values obtained with 125I-UdR to those acquired with 3H-TdR; The results indicate that cortical thymocytes migrate to the spleen, mesenteric lymph node, femurs and intestine; nevertheless, only a small fraction of the activity originally present in the thymus was recovered in these organs; the vast majority of newly formed cortical thymocytes apparently die after a relatively short life span. Exclusive of the fraction which dies in situ, evidence for thymocyte death is seen in bone marrow; however, most migrants appear to terminate in the intestine.     

Red Light Effects on Uptake of 14C and 32P into Etiolated Corn Leaf Tissue During Photomorphogenic Leaf Opening

Red light effects on the uptake of ¹⁴C and of ³²P was studied by observing leaf sections from 8-day-old etiolated corn leaves that were placed on various substrates following a brief exposure to red radiant energy. There was a general increase of ¹⁴C uptake over dark levels into all metabolite fractions that were prepared. This is in contrast with results obtained previously in which leaf samples were first floated on substrate and then irradiated (Mitrakos et al. 1967, Price et al. 1965). The latter tests resulted in a general decrease in sugar and starch as well as ¹⁴C content of all fractions. However, under both types of experimental conditions the red light effect manifested itself as an increase in hexosemonophosphate turn-over rate and accumulation of radioactivity in the cell wall polysaccharide fraction. The present data further substantiate the previous work in that they demonstrate the regulatory influence of the hexose pool size on the intermediary metabolism and the manifestation of the phytochrome responses. From the data thus far obtained it cannot be determined as to whether or not phytochrome mediates by controlling phosphorylating activity or through control of specific enzymatic processes that lead to observable cell wall polysaccharide synthesis and leaf unrolling.     

CELL LOSS FROM SEVERAL TYPES OF SOLID MURTNE TUMOUR: COMPARISON OF 125I-IODODEOXYURIDINE AND TRITIATED THYMIDINE METHODS

The method of measuring tumour cell loss rates in situ following radioactivity loss after a single injection of ¹²⁵I-iododeoxyurudine (¹²⁵I-UdR) was tested for its accuracy in five different types of murine tumour. To achieve this the method was compared with two others: (1) using ¹²⁵I-UdR, but excising tumours before the radioactivity determinations, with or without extracting DNA; (2) using tritiated thymidine and autoradiography. A third method was used on three of the tumours, in which ¹²⁵I-UdR-labelled tumours were grown in unlabelled hosts, followed by whole body counting of the tumour-bearing mice. In two of the tumours an increase was observed in total tumour radioactivity with time after ¹²⁵I-UdR injection. This prevented the estimation of cell loss parameters in these tumours. Approximately half the increase was due to reutilization of ¹²⁵I-UdR supplied from tissues within the mouse; approximately a third to an influx of labelled inflammatory cells (probably in response to infection accompanying ulceration of overlying skin); and the remainder to an increase in non-DNA radioactivity. In these tumours cell loss rates could be obtained from the whole body counting technique in which influxes of labelled cells and reutilizable radioactivity were eliminated. A comparison of either ¹²⁵I-UdR technique with the ³H-TdR technique showed good agreement of the cell loss factors for the low cell loss tumours. However, for tumours with high cell loss factors the ¹²⁵I-UdR technique gave lower values for cell loss. This implied that reutilization of ¹²⁵I-UdR within the tumour (i.e. from internal, not external sources) occurred in the high cell loss tumours. It is concluded that equating radioactivity loss with cell loss after an injection of ¹²⁵I-UdR is reasonable for some tumours, but will result in significant underestimates in others. For high cell loss tumours the ³H-TdR technique will give the     

CELL KINETICS METHODS FOR CLINICAL USE: ASSESSMENT OF AUTORADIOGRAPHY AND 125I-IODO-DEOXYURIDINE WITH MOUSE EHRLICH TUMOUR

Superficial Ehrlich tumours in mice were used to assess how much information on cell kinetics could be obtained from only the simplest techniques of autoradiography, and in situ monitoring of ¹²⁵I-iodo-deoxyuridine. These techniques were selected as being readily applicable to clinical situations. Intradermal tumours were studied from the earliest stages of rapid growth to large slow growing tumours with necrotic cores, as well as tumours undergoing regression. For comparison, intramuscular tumours were studied with systemic injections of radioactive DNA precursors. It was found that extensive information on cell production and loss rates was obtained from a single injection of tritiated thymidine followed by a single biopsy, or alternatively in vitro labelling of one minute biopsy specimen, and measurements of only the labelling index, together with a knowledge of the tumour's growth characteristics. Following a single localized injection of ¹²⁵I-iodo-deoxyuridine, the loss rate of radioactivity measured in situ for a period of about 1 week provided an index of cell loss rate from the tumours.     

INCORPORATION OF d-[3H]GLUCOSAMINE INTO THE SIALOGLYCOPROTEIN GP-350 OF ADULT RAT BRAIN

—The incorporation of d -[³H]glucosamine into the nervous specific sialoglycoprotein GP-350 has been studied in adult rat brain. Both the 100,000 g supernatant fluid and the 12,500 g pellet were used for the investigation, since GP-350 could only be detected in the soluble cell fraction (Van Nieuw Amerongen et al., 1972) and in the synaptosomal membranes, sedimenting in the crude mitochondrial fraction (Van Nieuw Amerongen & Roukema , 1973, 1974). GP-350 was separated from the other proteins by polyacrylamide gel electrophoresis at pH 7.5 and the incorporation of radioactivity into GP-350 was measured at different time intervals, ranging from 1 to 96 h after the administration of the radioisotope. The maximal incorporation of radioactivity into the soluble GP-350 was obtained after about 2 h and into the membrane-bound GP-350 after about 3 h. After 2 h there is a very rapid decrease of the radioactivity of GP-350 from the soluble cell fraction (up to 70 per cent within 2 h). Thereafter the disappearance is more gradual and of the same order as that found for the membrane-bound fraction of GP-350. The half-life of the soluble GP-350 was estimated to be 19 h and for the membrane-bound GP-350 a value of 18 h was calculated. Compared to the total pool of brain (glyco) proteins and specific nervous (glyco) proteins GP-350 has a very rapid turnover. The rapid initial decrease of the radioactivity from the soluble GP-350 may be interpreted in terms of a rapid transport of the newly-synthesized GP-350 from the cytoplasma of the perikaryon to the membranes of the synaptic region by an axoplasmic flow.     

DIFFERENT LABELLING PATTERNS IN MOUSE LYMPHOID TISSUES WITH [3H]DEOXYCYTIDINE AND [3H]THYMIDINE

The percentages of labelled lymphocytes in smear preparations of mouse thymus were higher than those in similar preparations of mesenteric lymph nodes with either generally labelled tritiated deoxycytidine, [³H]CdR, or tritiated thymidine, [³H]TdR. Lymphocytes in the thymus cortex and in germinal centres of mesenteric lymph nodes were intensely labelled with [³H]CdR, whereas with [³H]TdR lymphocytes in the peripheral region of thymus and medullary cords of mesenteric lymph nodes were heavily labelled. The majority of lymphocytes in thymic cortex and germinal centres of mesenteric lymph nodes were labelled weakly with [³H]TdR. Thus, labelling patterns with [³H]CdR differed from those with [³H]TdR in lymphoid tissues of the mouse. Mouse lymphocytes can utilize [³H]CdR as a precursor molecule for cytosine and thymine in DNA. The ratio of radioactivity of thymine to that of cytosine was measured biochemically in DNA extracted from lymphocytes labelled with [³H]CdR. This radioactivity ratio in thymus was higher than that in mesenteric lymph nodes. These results suggest that the metabolic activities of utilizing CdR for DNA synthesis differ within lymphocyte populations in various lymphoid tissues in the mouse.     

Short term 22Na+ and 42K+ uptake in intact, mid-vegetative plants

Spergularia marina (L.) Griseb. is. a rapidly growing, annual, coastal halophyte. Because of its small size, it is suitable for isotope studies of ion transport well beyond the seedling stage. The purpose of this report is to establish the similarities and differences between ²²Na⁺ and ⁴²K⁺ uptake in S. marina and in more commonly used mesophytic crop species. Vegetative plants were used 18 days after transfer to solution culture. Plants were grown either on Na⁺-free medium or on 0.2 × sea water. ²²Na⁺ uptake was linear with time for several hours. The rate was relatively insensitive to external concentration between 1 and 180 mol Na⁺ m?³, particularly in Na⁺-free plants. Transport to the shoot accounted for 40 to 70% of the total uptake, dependent on salinity but largely independent of time. ⁴²K⁺ uptake decreased with increasing salinity in Na⁺-free plants and increased in 0.2 × sea water plants. Both uptake and transport to the shoot were non-linear with time, upward concavity suggesting recovery from a manipulative and/or osmotic injury. Steady state root contents were compared with predicted contents based on cortical cell electrical potentials using the Nernst equation. Reasonable agreement was found in all cases except Na⁺ content of 0.2 × sea water plants, in which active efflux was indicated. Uptake studies conducted in the presence of chemical modifiers (dicyclohexylcarbodiimide, dinitrophenol and fusicoccin) showed responses of ⁴²K⁺ uptake as expected from studies on agronomic species, and implied the presence of a similar active uptake here despite the appearance of equilibrium. Active Na⁺ uptake was suggested at low Na⁺ levels. We conclude that S. marina is a promising experimental system combining the rapid nutrient acquisition strategy of agionomically important annuals with a high degree of salt tolerance.     

Effect of Abscisic Acid on the Activities of Photosynthetic Enzymes and 14CO2 Fixation Products in Leaves of Pennisetum typhoides Seedlings

Abscisic acid inhibited the rate of ¹⁴CO₂ fixation in leaves of Pennisetum typhoides (Burm. f.) Stapf & Hubbard seedlings, but increased the activities of phosphoenol-pyruvate-carboxylase and malic enzyme. The leaves of the seedlings grown in the presence of abscisic acid incorporated, in comparison to the control, more radioactivity in the fraction of organic acids, but less radioactivity was recorded in the amino acid fraction. On the other hand, gibberellic acid which also inhibits photosynthetic ¹⁴CO₂ assimilation and decreases the activities of photosynthetic enzymes, favours greater incorporation in alanine, and reduces that in malate. It is deduced that bio-regulants can greatly influence the flow of ¹⁴C into individual photosynthetic products. As in growth, abscisic and gibberellic acids in combination tended to antagonize each other in their effects on enzyme activity as well as in incorporation of ¹⁴CO₂ into photosynthetic products.     

Incorporation of 14C-labelled polyunsaturated fatty acids by juvenile turbot, Scophthalmus maximus (L.) in vivo

The ability of juvenile turbot, Scophthalmus maximus (L.), to elongate and desaturate various polyunsaturated fatty acids (PUFA) was examined in relation to their lipid composition. Triacylglycerols were the most abundant lipid class present in the fish and phosphatidylcholine was the predominant phospholipid. In all lipid classes examined the levels of (n-3) PUFA exceeded that of (n-6) PUFA. 18C PUFA were minor components in comparison with 20:5(n-3) and 22:6(n-3). 20:4(n-6) was present in highest concentration in phosphatidylinositol in which it accounted for 16.9% of the fatty acids. When the fish were injected with either ¹⁴C-labelled 18:2(n-6), 18:3(n-3), 20:4(n-6), 20:5(n-3) or 22:6(n-3) the highest percentage recovery of radioactivity (69%) in body lipid was observed with 22:6(n-3). With all labelled substrates free fatty acids contained only a small proportion of the total recovered radioactivity whereas triacylglycerols were highly labelled. Phosphatidylcholine/sphingomyelin was the most highly labelled polar lipid fraction. With ¹⁴C-20:4(n-6) as injected substrate, 23.2% of the radioactivity recovered in total lipid was present in phosphatidylinositol in comparison with less than 6% with the other substrates. Only small proportions of radioactivity from ¹⁴C-18:2(n-6) and ¹⁴C-18:3(n-3) were recovered in the 20 and 22C fatty acids of triacylglycerols and total polar lipid. With ¹⁴C-20:5(n-3) as substrate, 27 and 33% of the total radioactivity recovered in the fatty acids of triacylglycerols and polar lipids respectively was present in 22C fatty acids. The corresponding values for ^l4C-20:4(n-6) as substrate were 19 and 18%. The results confirm the limited capacity of turbot to convert 18C PUFA to longer chain PUFA but demonstrate their ability to synthesize 22C PUFA from 20C PUFA. They also suggest a small but specific requirement for 20:4(n-6).     

SENSITIVITY OF EARLY EMBRYONIC THYMIC LYMPHOID DEVELOPMENT TO 3H-THYMIDINE OF HIGH SPECIFIC ACTIVITY

The pulse technique, using high specific activity ³H-TdR to selectively kill cells in cell cycle, was applied to the thymic anlagen of chick embryos. With optimal specific and total ³H-TdR activities and pulse times of 2–4 hr the subsequent lymphoid development in organ culture of the thymic anlagen of 10-day-old chick embryos could be almost completely inhibited. The most important effect of the ³H-TdR was on the lymphoid precursor cells of the anlagen. The thymic epithelium appeared more resistant to ³H-TdR and allowed a lymphoid development of pulsed anlagen grafted to the chorioallantoic membrane of chick embryos when new lymphoid precursor cells were provided. The lymphoid precursor cells of the thymic anlagen of 10-day-old chick embryos therefore appeared to be in cell cycle with short generation time. The thymic anlagen of 8-, 9- and 10-day-old but not 7-day-old embryos showed a lymphoid development in organ culture. They did not differ with respect to the sensitivity to hot pulses of ³H-TdR. Thus no evidence of a lag in the onset of lymphoid precursor cell proliferation during the development of the early embryonic chick thymus was noted.     

CYTODYNAMICS IN THE THYMUS OF YOUNG ADULT MICE:

Cell proliferation and cell loss in the thymic blast cell population were studied in young adult mice by (1) stathmokinetic methods combined with an analysis of the PLMe-curve after a pulse ³H-TdR, and (2) nigrosin-dye exclusion combined with ³H-TdR-autoradiography. It was calculated that about 17% of the blast cells do not progress into mitosis within the period of an average cell cycle. The dye exclusion studies indicated a rate of blast cell death of about 2–5 %/hr. The two methods of assessing blast cell loss (death) support each other very well. In spite of these findings scintillation countings on thymuses removed from 1 to 17 hr after ³H-TdR injection showed fairly constant levels of thymic radioactivity. This suggests a very extensive reutilization of ³H-labelled break-down products from dying blast cells. The very sparse labelling of pyknotic thymocytes strongly suggests that thymic blast cells do not become pyknotic. The rate of small thymocyte production and disappearance was studied by pulse and repeated ³H-TdR labelling techniques combined with dye exclusion studies and pyknotic counts. The data from the repeated labelling experiment were analysed by use of a model based on the assumption of first order kinetics of small viable, dead, and pyknotic thymocytes. The rate of cell production was estimated to 1–6 %/hr whereas the rates of cell loss due to disintegration, i.e. supravital stainability and nuclear pyknosis, were calculated to 0–02 %/hr and 0–0006 %/hr respectively. Cell loss due to disintegration was less than 2 % of the total loss of small thymocytes. It was concluded that pyknotic counts are a useless method of assessing the cell death in the population of thymic blast cells and small thymocytes. On the basis of a model for thymocyte proliferation, production and loss it is suggested that about 45 % of the small viable thymocytes re-enter the generative cell pool, whereas about 55% disappear by emigration.     

IN VITRO INCORPORATION OF 3H-THYMIDINE, 3H-URIDINE, 3H-LEUCINE AND 3H-MELPHALAN BY PLASMACYTOMA PLASMA CELLS

Bone marrow plasma cells from fifteen cases of multiple myeloma, immunologically typed, were incubated with different tritiated compounds. The labelling index with tritiated thymidine is generally low, while the mean grain count is fairly normal in the active cells. The labelling index of ³H-uridine and ³H-leucine was very high, while the mean grain count per cell lies within the normal range. The results obtained with ³H-phenylalanine-mustard (melphalan), which is a drug used in the treatment of the plasmacytoma, show also incorporation values roughly comparable to those of ³H-leucine. The present data seem to support the clinical use of melphalan as a compound that is actively incorporated into the plasma cells of plasmacytoma although inhibition of protein synthesis due to specific binding to protein was not demonstrated.     

Reserve lipid accumulation and translocation of 14C in the photosynthetically active and senescent shoot parts of Dicranum elongatum

Lipid metabolism of the subarctic moss Dicranum elongatum was studied by feeding the moss with 2-¹⁴C-acetate and, after extraction of the lipids, counting the ¹⁴C-content of different lipid fractions immediately after feeding or after chase periods. Translocation of ¹⁴C after ¹⁴C-feeding was studied with autoradiography. Both low temperature (+6°C) and drought (at +23°C) resulted in increased incorporation of ¹⁴C into the neutral lipid (NL) fraction and decreased incorporation of ¹⁴C into the glycolipid (GL) fraction of the green shoot part of the moss. The distribution of radioactivity between the NL classes suggests that diacylglycerols (1, 2-DAG) and common triacylglycerols (cTAG) are turned into acetylenic triacylglycerols (aTAG), which are accumulated preferentially. The decrease in the radioactivity of the GL fraction was due to two unknown fractions, whereas ¹⁴C incorporation into the chloroplast membrane lipids, monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG), was very low throughout the experiments. The phospho-lipid (PL) fraction accounted for 48–63% of total lipid radioactivity at both low and high temperatures. 2-¹⁴C-acetate feeding to the senescent moss part resulted in vigorous ¹⁴C incorporation into the lipids, especially into the reserve TAGs. Electron microscopic examination showed the presence of plastids, which explains the capability of the senescent part of the moss for lipid synthesis. The fact that transport of ¹⁴C from 2-¹⁴C-acetate took place upwards and downwards in the moss shoot, together with the capability for lipid synthesis of the senescent moss part, supports the suggestion that the senescent moss part plays a role as an energy store.     

The Effect of Dichlorophenyldimethylurea on Light-Stimulated 22Na+, 42K+ and 86Rb+ Absorption in Different Tissues of Phaseolus vulgaris Leaves

The light-stimulated absorption of ⁸⁶Rb⁺ by Phaseolus vulgaris L. leaf slices was found to be sensitive to dichlorophenyldimethylurea in air as well as in nitrogen, whereas light-stimulated ²²Na⁺ absorption in nitrogen was not sensitive to this inhibitor. The absorption of ²²Na⁺ is not affected by light in air. The absorption of ⁴²K⁺ is enhanced by a dichlorophenyldimethylurea-insensitive light effect under anaerobic conditions and further increased by light in the absence of the inhibitor. Light-enhanced ⁴²K⁺ absorption in air was also inhibited by dichlorophenyldimethylurea. Previous work showed that light-stimulated ⁸⁶Rb⁺ and ⁴²K⁺ absorption by Phaseolus vulgaris leaf slices is restricted to the guard cells. The present results are discussed with reference to the effect of light on stomatal opening.     

The Effect of X-Irradiation On Cell Loss In Five Solid Murine Tumours, As Determined By the 125Iudr Method

The rate of cell loss in irradiated RIF-1, EMT6, KHJJ, B16 and KHT tumours was studied using the ¹²⁵IUdR loss technique. Administration of ¹²⁵IUdR preceded localized tumour irradiation by 2 days. Loss of tumour radioactivity was measured for 6–8 days after irradiation. the blood flow to some tumours was occluded during, and for 30 min following, injection of the label to measure the amount of radioactivity entering the tumour as a result of reutilization of label from the gut epithelia and influx of labelled host cells. Irradiation did not significantly alter the amount of radioactivity entering these clamped tumours during the 8–10 days after injection of ¹²⁵IUdR. This permitted comparison of irradiated and control groups based on the loss of radioactivity from the non-occluded tumours. Irradiation of RIF-1, EMT6, KHJJ or B16 tumours with doses of 600, 1400, 2400 or 4400 rads produced no significant increase in the rate of loss of tumour radioactivity. This suggested that, in the population of labelled cells, cell lysis following irradiation proceeded slowly. In contrast, KHT tumours showed a significant increase in loss rate following each radiation dose, although the increase was dose-independent. In all tumour systems, the constant rate of cell loss after radiation appeared to coincide with published reports of tumour growth responses after irradiation. the present data suggest that the manner of expression of radiation-induced cell killing results from the cellular proliferative status, i.e. whether a cell is cycling or non-cycling.     

UPTAKE OF [3H-METHYL]CHOLINE BY MICROSOMAL, SYNAPTOSOMAL, MITOCHONDRIAL AND SYNAPTIC VESICLE FRACTIONS OF RAT BRAIN

Abstract— Microsomal, mitochondrial, synaptosomal and synaptic vesicle fractions of rat brain took up [³H-methyl]choline by a similar carrier-mediated transport system. The apparent K_m for the uptake of [³H-methyl]choline in these subcellular fractions was about 5 × 10^?5 M. Choline uptake was also observed in microsomal fractions prepared from liver and skeletal muscle. Virtually identical kinetic properties for [³H-methyl]choline transport were found in the synaptosomal fractions prepared from the whole brain, cerebellum or basal ganglia. Countertransport of [³H-methyl]choline from the synaptosomal fraction was demonstrated against a concentration gradient. HC-3 was a competitive inhibitor of the uptake of [³H-methyl]choline in brain microsomal, synaptosomal and mitochondria] fractions with respective values for K_i of 4.0, 2.1 and 2.3 × 10^?5 M. HC-15 was a competitive inhibitor of the transport of [³H-methyl]choline in the synaptosomal fraction, with a K_i of 1.7 × 10^?4 M. Upon entry into the microsomal fraction, 74 per cent of the radioactivity could be recovered as unaltered choline, 10 per cent as phosphorylcholine, 1.5 per cent as acetylcholine and 2.5 per cent as phospholipid. Choline acetyltransferase (EC 2.3.1.6) was assayed with [¹⁴C]acetylCoA in synaptosomal fractions prepared from basal ganglia and cerebellum, and in the 31,000 g supernatant fraction of a rat brain homogenate. Enzyme activity was 11-fold greater in the synaptosomal fraction from the basal ganglia than in that from the cerebellum. HC-3 did not inhibit choline acetyltransferase and there was no evidence for acetylation of HC-3. Our findings suggest that choline uptake is a ubiquitous property of membranes in the CNS and cannot serve to distinguish cholinergic nerve endings and their synaptic vesicles.     

Activation of pre-synaptic M-type K+ channels inhibits [3H]d-aspartate release by reducing Ca2+ entry through P/Q-type voltage-gated Ca2+channels

In this study, the functional consequences of the pharmacological modulation of the M‐current (I_KM) on cytoplasmic Ca²⁺ intracellular Ca²⁺concentration ([Ca²⁺]_i) changes and excitatory neurotransmitter release triggered by various stimuli from isolated rat cortical synaptosomes have been investigated. K_v7.2 immunoreactivity was identified in pre‐synaptic elements in cortical slices and isolated glutamatergic cortical synaptosomes. In cerebrocortical synaptosomes exposed to 20 mM [K⁺]_e, the I_KM activator retigabine (RT, 10 μM) inhibited [³H]d ‐aspartate ([³H]d ‐Asp) release and caused membrane hyperpolarization; both these effects were prevented by the I_KM blocker XE‐991 (20 μM). The I_KM activators RT (0.1–30 μM), flupirtine (10 μM) and BMS‐204352 (10 μM) inhibited 20 mM [K⁺]_e‐induced synaptosomal [Ca²⁺]_i increases; XE‐991 (20 μM) abolished RT‐induced inhibition of depolarization‐triggered [Ca²⁺]_i transients. The P/Q‐type voltage‐sensitive Ca²⁺channel (VSCC) blocker ω‐agatoxin IVA prevented RT‐induced inhibition of depolarization‐induced [Ca²⁺]_i increase and [³H]d ‐Asp release, whereas the N‐type blocker ω‐conotoxin GVIA failed to do so. Finally, 10 μM RT did not modify the increase of [Ca²⁺]_i and the resulting enhancement of [³H]d ‐Asp release induced by [Ca²⁺]_i mobilization from intracellular stores, or by store‐operated Ca²⁺channel activation. Collectively, the present data reveal that the pharmacological activation of I_KM regulates depolarization‐induced [³H]d ‐Asp release from cerebrocortical synaptosomes by selectively controlling the changes of [Ca²⁺]_i occurring through P/Q‐type VSCCs.     

Soil 13C–15N dynamics in an N2-fixing clover system under long-term exposure to elevated atmospheric CO2

Reduced soil N availability under elevated CO₂ may limit the plant's capacity to increase photosynthesis and thus the potential for increased soil C input. Plant productivity and soil C input should be less constrained by available soil N in an N₂‐fixing system. We studied the effects of Trifolium repens (an N₂‐fixing legume) and Lolium perenne on soil N and C sequestration in response to 9 years of elevated CO₂ under FACE conditions. ¹⁵N‐labeled fertilizer was applied at a rate of 140 and 560 kg N ha^?1 yr^?1 and the CO₂ concentration was increased to 60 Pa pCO₂ using ¹³C‐depleted CO₂. The total soil C content was unaffected by elevated CO₂, species and rate of ¹⁵N fertilization. However, under elevated CO₂, the total amount of newly sequestered soil C was significantly higher under T. repens than under L. perenne. The fraction of fertilizer‐N (f_N) of the total soil N pool was significantly lower under T. repens than under L. perenne. The rate of N fertilization, but not elevated CO₂, had a significant effect on f_N values of the total soil N pool. The fractions of newly sequestered C (f_C) differed strongly among intra‐aggregate soil organic matter fractions, but were unaffected by plant species and the rate of N fertilization. Under elevated CO₂, the ratio of fertilizer‐N per unit of new C decreased under T. repens compared with L. perenne. The L. perenne system sequestered more ¹⁵N fertilizer than T. repens: 179 vs. 101 kg N ha^?1 for the low rate of N fertilization and 393 vs. 319 kg N ha^?1 for the high N‐fertilization rate. As the loss of fertilizer‐¹⁵N contributed to the ¹⁵N‐isotope dilution under T. repens, the input of fixed N into the soil could not be estimated. Although N₂ fixation was an important source of N in the T. repens system, there was no significant increase in total soil C compared with a non‐N₂‐fixing L. perenne system. This suggests that N₂ fixation and the availability of N are not the main factors controlling soil C sequestration in a T. repens system.     

The Fate of L-Tyrosine-UL−14C in Shoot-Forming Tobacco Callus

Tobacco callus fed L-tyrosine-UL^?14C was sampled at 3-day intervals for 15 days, homogenized and studied with respect to distribution of incorporated radioactivity. The supernatant obtained by centrifuging of the homogenates at 270 g contained the bulk of the radioactivity although significant activity was also detected in the pellet. Sucrose density gradient centrifugation of the supernatant showed over 90% of the recovered label to be associated with a fraction designated as “less dense than mitochondria”, with the remainder being found in the fraction identified as “mitochondria”. During tissue culture, virtually all of the radioactivity in the fraction “less dense than mitochondria” was recovered in the supernatant obtained by centrifugation at 100,000 g. From 4 to 18% of the labeling in the 100,000 g supernatant fraction was attributable to tyrosine-containing protein, and the rest to free tyrosine and unidentified anionic constituents. The highest proportions of radio-activity in the 270 g pellet were associated with substances extractable with NaCl, pronase, 4.6 N NaOH, and acetolyzing reagent. Low but substantial labeling characterized the extracts obtained with Triton X-100 and 1 N NaOH. The final unextractable residue contained 20% of the 270 g pellet radioactivity.     

Alterations in the Production of 14CO2 and [14C]Acetylcholine from [U-14C]Glucose in Brain Subregions Following Transient Forebrain Ischemia in the Rat

Abstract: The production of ¹⁴CO₂ and [¹⁴C]acetylcholine from [U-¹⁴C]glucose was determined in vitro using tissue prisms prepared from the dorsolateral striatum (a region developing extensive neuronal loss following ischemia) and the paramedian neocortex (an ischemia-resistant region) following 30 min of forebrain ischemia and recirculation up to 24 h. Measurements were determined under basal conditions (5 mMK⁺) and following K⁺ depolarization (31 mM K⁺). The production of ¹⁴CO₂ by the dorsolateral striatum was significantly reduced following 30 min of ischemia for measurements in either 5 or 31 mM K⁺ but recovered toward preischemic control values during the first hour of recirculation. Further recirculation resulted in ¹⁴CO₂ production again being reduced relative to control values but with larger differences (20–27% reductions) detectable under depolarized conditions at recirculation times up to 6 h. Samples from the paramedian neocortex showed no significant changes from control values at all time points examined. [¹⁴C]Acetylcholine synthesis, a marker of cholinergic terminals that is sensitive to changes in glucose metabolism in these structures, was again significantly reduced only in the dorsolateral striatum. However, even in this tissue, only small (nonstatistically significant) differences were seen during the first 6 h of recirculation, a finding suggesting that changes in glucose oxidation during this period were not uniform within all tissue components. The results of this study provide evidence that in a region susceptible to ischemic damage there were specific changes during early recirculation in the metabolic response to depolarization. This apparent inability to respond appropriately to an increased need for energy production could contribute to the further deterioration of cell function in vivo and ultimately to the death of some cells.