Brain Carbohydrate Metabolism in Developing Rats During Hypercapnia

Abstract: Brain glucose metabolism was studied in developing rats at ages 10 and 20 days postnatal under normal and hypercapnic conditions. Brains were removed and frozen within 1 s with a freeze-blowing apparatus. Glucose utilization was measured with [2-¹⁴C]glucose and [³H]deoxyglucose as tracers. Metabolites were determined by standard enzymatic techniques. Data from [³H]deoxyglucose phosphorylation indicated that normal brain glucose utilization increased almost threefold between the 10th and 20th postnatal days. From the relative rates of utilization of the two isotopes in the 20-day-old control group, it appeared that about 25% of ¹⁴C label derived from metabolism of [2-¹⁴C]glucose was lost from brain (probably as lactate) rather than entering the Krebs cycle. Under hypercapnic conditions (20% CO₂-21% O₂-59% N₂), rates of glucose utilization by brain were decreased by one-half at both ages and there were progressive decreases in the concentrations of many intermediary metabolites. The bases for concluding that these metabolites were used to supplement glucose as a fuel for respiration, rather than being lost by leakage into blood, are discussed. Despite the differences in brain glucose metabolism between 10-day-old and 20-day-old rats, their responses to hypercapnia are remarkably similar: Rates of glucose utilization are reduced to approximately the same proportion of the original rate by 20% CO₂, and endogenous metabolites (particularly glutamate and lactate) appear to be oxidized as replacement fuels.     

N2(C2H2)-fixation in early stages of a primary succession on a reclaimed salt marsh

Nitrogen fixation activity was determined for Lotus tenuis. Medicago lupulina and Trifolium pratense . The three species grew in clones in grassland in an area reclaimed from brackish water in the 1940s. The N₂[C₂H₂]-fixation was measured in soil cores throughout 1974 and 1975. From cores taken in dense and uniform stands of the species, the yearly N₂[C₂H₂]-fixation at maximum cover was estimated. L. tenuis fixed about 4 g N m⁻² yr⁻¹ (area with max. cover 130%), i.e. 30–56% of its requirement. Both M. lupulina and T. pratense fixed about 7 g N m⁻² yr⁻¹ (maximum cover 37% and 80%) i.e. 67% of their N-requirement. Average N₂[C₂H₂]-fixation for the whole area was 0.4 g N m⁻² yr⁻¹, considerably less than the N-addition through rainfall.     

ELECTROCONVULSIVE SEIZURE: AN INVESTIGATION INTO THE VALIDITY OF CALCULATING THE CYTOPLASMIC FREE [NAD+]/[NADH] [H+] RATIO FROM SUBSTRATE CONCENTRATIONS OF BRAIN

Abstract— This study is an investigation into the applicability of redox calculations to brain. At six intervals following electroconvulsive scizure, multiple metabolites were measured in freeze-blown brain from unanesthetized rats. From substrate ratios, the time course of the rapid changes in the cytoplasmic free [NAD⁺]/ [NADH] [H⁺] ratio was calculated from the reactions of lactate dehydrogenase [EC 1.1.1.27], malate dehydrogenase [EC 1.1.1.37] and glycerolphosphate dehydrogenase [EC 1.1.1.8], The pattern of the redox ratios in the control animals was also compared with the same ratios determined in freeze-clamped liver, a relatively homogeneous tissue. Though some evidence for effects of compartmentation are present in the results from brain, these effects are relatively minor. There was found to be very good agreement in the direction and magnitude of change of the redox ratios calculated from lactate dehydrogenase and malate dehydrogenase, and even from glycerolphosphate dehydrogenase at points of low flux. In spite of rapid changes of metabolites, the reaction catalyzed by glutamateoxaloacetate transaminase remained very near its equilibrium position at all time periods. From the results it has been concluded that in spite of the obvious structural heterogeneity of brain, meaningful calculations of the cytoplamic redox state in brain are possible.     

Carbon dioxide assimilation during postharvest removal of astringency from persimmon fruit

Application of anaerobic conditions with CO₂ or N₂ atmospheres to remove astringency from harvested persimmon fruit ( Diospryros kaki L. cv. Triumph), caused production of more acetaldehyde under CO₂ than under N₂, ¹⁴CO₂ applied in a 100% CO₂ atmosphere, for 48 h to astringent persimmon fruits was incorporated mainly into malate and very little into other metabolites, such as carbohydrate or amino acids. Application of malate or pyruvate to pulp discs of astringent persimmons caused an immediate rise in acetaldehyde production. The higher levels of acetaldehyde produced by whole fruits held in a CO₂ atmosphere, than by fruits held in a N₂ atmosphere, can be explained through fixation of atmospheric CO₂ into malate, leading to acetaldehyde production.     

Neurosteroids Modulate Nicotinic Receptor Function in Mouse Striatal and Thalamic Synaptosomes

Abstract: Progesterone and its A-ring reduced metabolites are allosteric activators of GABA_A receptors. The studies reported here examined the effects of these steroids on brain nicotinic receptors using an ⁸⁶Rb⁺ efflux assay that likely measures the function of α4β2-type nicotinic receptors and [³H]dopamine release, which may be modulated by an α3-containing nicotinic receptor. Both of the A-ring reduced metabolites of progesterone were noncompetitive inhibitors of both assays, whereas progesterone inhibited only the ⁸⁶Rb⁺ efflux assay. The ⁸⁶Rb⁺ efflux assay was slightly more sensitive than was the dopamine release assay to steroid inhibition. Inhibition developed slowly for both assays ( t _1/2 = 0.4 min) and was reversed even more slowly ( t _1/2 = 10–15 min). Steroid addition did not alter either the rate of association of [³H]nicotine binding to brain membranes, nor was equilibrium binding changed. These findings argue that neurosteroids are allosteric inhibitors of brain nicotinic receptors.     

Lactate Is Released and Taken Up by Isolated Rabbit Vagus Nerve During Aerobic Metabolism

Abstract: To determine if lactate is produced during aerobic metabolism in peripheral nerve, we incubated pieces of rabbit vagus nerve in oxygenated solution containing d -[U-¹⁴C]glucose while stimulating electrically. After 30 min, nearly all the radioactivity in metabolites in the nerve was in lactate, glucose 6-phosphate, glutamate, and aspartate. Much lactate was released to the bath: 8.2 pmol (µg dry wt)⁻¹ from the exogenous glucose and 14.2 pmol (µg dry wt)⁻¹ from endogenous substrates. Lactate release was not increased when bath P o ₂ was decreased, indicating that it did not come from anoxic tissue. When the bath contained [U-¹⁴C]lactate at a total concentration of 2.13 m M and 1 m M glucose, ¹⁴C was incorporated in CO₂ and glutamate. The initial rate of formation of CO₂ from bath lactate was more rapid than its formation from bath glucose. The results are most readily explained by the hypothesis that has been proposed for brain tissue in which glial cells supply lactate to neurons.     

Nitrogen use efficiency. 3. Nitrogen fixation: genes and costs

The nitrogen use efficiencies (NUE) of N₂ fixation, primary NH ₄⁺ assimilation and NO ₃⁻ assimilation are compared. The photon and water costs of the various biochemical and transport processes involved in plant growth, N-assimilation, pH regulation and osmolarity generation, per unit N assimilated are respectively likely to be around 5 and 7% greater for N₂ fixation than for a combination of NH ₄⁺ and root and shoot NO ₃⁻ assimilation as occurs with most crops. Studies on plant and rhizobial genes involved in nodulation and N₂ fixation may lead to more rapid nodulation, production of more stress-tolerant N₂ fixing systems and transfer of the hydrogenase system to rhizobium/legume symbioses which currently do not have this ability. The activity of an uptake hydrogenase is predicted to decrease the photon cost of diazotrophic plant growth by about 1%.     

Energy Metabolism in Rat Brain Synaptosomes from Nembutal-Anesthetized and Nonanesthetized Animals

Abstract: Cellular energetic parameters including the intramitochondrial and cytosolic [NAD⁺]/[NADH] ratios, the cellular [ATP]/[ADP][P_i and [creatine phosphate]/[creatine] ratios, the concentration of cytochrome c and its redox state and the respiratory rate were studied in suspensions of rat brain synapto-somes isolated from nembutal-anesthetized and nonanesthetized animals. The ratio of [3-hydroxybutyrate] to [acetoacetate] was 2.0 in synaptosomes isolated from nonanesthetized rats and 5.55 in those from anesthetized animals. The [lactate]/[pyruvate] ratio was 3.8 in the former and 10.9 in the latter preparation. The [ATP]/[ADP][P_i] was 3838 M⁻¹ in the synaptosomes from anesthetized rats and 840 M⁻¹ in those from nonanesthetized animals and the [creatine phosphate]/[creatine] ratios were 0.79 and 0.39, respectively. Cytochrome c was about 15% reduced in both preparations; however, the mitochon-drial cytochrome concentration was almost twofold higher in the synaptosomes from nonanesthetized animals. Calculations of the free energy relationships between the mitochondrial redox reactions and ATP synthesis showed that in synaptosomes isolated from the brains of nembutal-anesthetized rats the first two sites of oxidative phosphorylation were at near-equilibrium, in agreement with observations for intact cells and tissues. The energetic parameters for synaptosomes from anesthetized rats are very similar to the values for intact whole brain, whereas those for synaptosomes from nonanesthetized rats are lower and suggest that nembutal anesthesia protects against some irreversible damage to the synaptosome during isolation. It is concluded that synaptosomes isolated from brains of nembutal-anesthetized rats can be used as a convenient model system for studies of neuronal metabolism.     

Differential Effects of Hypoxia on Ligand Binding Properties of Nicotinic and Muscarinic Acetylcholine Receptors on Cultured Bovine Adrenal Chromaffin Cells

Abstract: Hypoxia is known to disturb neuronal signal transmission at the synapse. Presynaptically, hypoxia is reported to suppress the release of neurotransmitters, but its postsynaptic effects, especially on the function of neurotransmitter receptors, have not yet been elucidated. To clarify the postsynaptic effects, we used cultured bovine adrenal chromaffin cells as a model of postsynaptic neurons and examined specific binding of l -[³H]nicotine (an agonist for nicotinic acetylcholine receptors: nAChRs) and ²²Na⁺ flux under control and hypoxic conditions. Experiments were performed in media preequilibrated with a gas mixture of either 21% O₂/79% N₂ (control) or 100% N₂ (hypoxia). Scatchard analysis of the specific binding to the cells revealed that the K_D under hypoxic conditions was twice as large as that under control conditions, whereas the B _max was unchanged. When the specific [³H]nicotine binding was kinetically analyzed, the association constant ( k ₁) but not the dissociation constant ( k ₋₁) was decreased to 40% of the control value by hypoxia. When the binding assay was performed using the membrane fraction, these changes were not observed. Nicotine-evoked ²²Na⁺ flux into the cells was suppressed by hypoxia. In contrast, specific [³H]quinuclidinyl benzilate binding to the intact cells was unaffected by hypoxia. These results demonstrate that hypoxia specifically suppresses the function of nAChRs (and hence, neuronal signal transmission through nAChRs), primarily by acting intracellularly.     

Elevated atmospheric CO2 does not affect per se the preference for symbiotic nitrogen as opposed to mineral nitrogen of Trifolium repens L.

The objective of this investigation was to examine the effect of an elevated atmospheric CO₂ partial pressure ( p CO₂) on the N-sink strength and performance of symbiotic N₂ fixation in Trifolium repens L. cv. Milkanova. After initial growth under ambient p CO₂ in a nitrogen-free nutrient solution, T. repens in the exponential growth stage was exposed to ambient and elevated p CO₂ (35 and 60 Pa) and two levels of mineral N (N-free and 7·5 mol m^–3 N) for 36 d in single pots filled with silica sand in growth chambers. Elevated p CO₂ evoked a significant increase in biomass production from day 12 after the start of CO₂ enrichment. For plants supplied with 7·5 mol m^–3 N, the relative contribution of symbiotically fixed N (%N_sym) as opposed to N assimilated from mineral sources (¹⁵N-isotope-dilution method), dropped to 40%. However, in the presence of this high level of mineral N, %N_sym was unaffected by atmospheric p CO₂ over the entire experimental period. In plants fully dependent on N₂ fixation, the increase in N yield reflects a stimulation of symbiotic N₂ fixation that was the result of the formation of more nodules rather than of higher specific N₂ fixation. These results are discussed with regard to physiological processes governing symbiotic N₂ fixation and to the response of symbiotic N₂ fixation to elevated p CO₂ in field-grown T. repens .     

Transport of Zinc-65 at the Blood-Brain Barrier During Short Cerebrovascular Perfusion in the Rat: Its Enhancement by Histidine

Abstract: Zinc-65 transport into different regions of rat brain has been measured during short vascular perfusion of one cerebral hemisphere with an oxygenated HEPES-containing physiological saline at pH 7.40. The [Zn²⁺] was buffered with either bovine serum albumin or histidine. In each case uptake was linear with time up to 90 s. ⁶⁵Zn flux into brain in the presence of albumin followed Michaelis-Menten kinetics and for parietal cortex had a K _m of 16 n M and a V _max of 44 nmol/kg/min. Increasing concentrations of l -histidine enhanced ⁶⁵Zn flux into brain at [Zn²⁺] values between 1 and 1,000 n M . The combined effect of [histidine] and [Zn²⁺] was best accounted for by a function of [ZnHis⁺], i.e., flux = 64.4 · [ZnHis⁺]/(390 + [ZnHis⁺]) + 0.00378 · [ZnHis⁺], with concentrations being nanomolar. d -Histidine had an influence similar to that of l -histidine. ⁶⁵Zn flux in the presence of 100 µ M l -histidine was not affected by either 500 µ M l -arginine or 500 µ M l -phenylalanine. The results indicate specific transport of Zn²⁺ across the plasma membranes of brain endothelium. The enhancement due to histidine has been attributed to diffusion of ZnHis⁺ across unstirred layers "ferrying" zinc to and from transport sites.     

Fixation of molecular nitrogen by Methanosarcina barkeri

Abstract Methanosarcina barkeri cells were observed in ammonia-free anaerobic acetate enrichments for sulfate-reducing bacteria. The capacity of Methanosarcina to grow diazotrophically was proved with a pure culture in mineral media with methanol. The cell yields with N₂ or NH₄⁺ ions as nitrogen source were 2.2 g and 6.1 g dry weight, respectively, per mol of methanol. Growth experiments with ¹⁵N₂ revealed that 84% of the cell nitrogen was derived from N₂. Acetylene was highly toxic to Methanosarcina and only reduced at concentrations lower than 100 μmol dissolved per 1 of medium. Assimilation of N₂ and reduction of acetylene were inhibited by NH₄⁺ ions. The experiments show that N₂ fixation occurs not only in eubacteria but also in archaebacteria. The ecological significance of diazotrophic growth of Methanosarcina is discussed.     

Seasonal patterns of nitrogen fixation in termites

1. Termite nitrogenase activity was highest in autumn and spring (≈ 3 μg N₂ fixed termite fresh mass (g)^–1 day^–1) and lowest in winter and summer (≈ 0·8 μg N₂ fixed termite fresh mass (g)^–1 day^–1).
2. The nitrogenase activity of worker termites was significantly higher than all other castes (1·58 ± 0·27 μg N₂ fixed termite fresh mass (g)^–1 day^–1).
3. Worker termites constituted the largest proportion of all the castes throughout the study period (≈ 90%).
4. The localized input of fixed nitrogen by termites may reach 15·3 mg N log^–1 day^–1 and 5·6 g N log^–1 year^–1.     

Anatomical and hnctional differences and nyctinastic leaf movements in Chenopodium album L. and Chenopodium hircinum Schrad. (Chenopodiaceae)

The anatomical structure of the leaves of Chenopodium album L. and Chenopodium hircinum Schrad. has been examined. Leaf chlorophyll content (Chl), specific leaf area (SLA), specific leaf weight (SLA^-1) and nitrogen content (N₂) were estimated in both species. Ch1, SLA and N₂ were greater in C. album than in C. hircinum. SLA^-1 data showed that C. album is more efficient than C. hircinum , because the former invests smaller quantities of dry weight to achieve a square centimetre of leaf area. The possible effects of Chl, N₂ and leaf anatomy on efficient gas exchange are discussed. The presence of nyctinastic leaf nocturnal movements was detected in both species. The meaning of this is discussed in relation to the prevention of the loss of pollen grains.     

A Chromatographic Method for Analysis of the Gaseous Phase in Shake Flask Cultures and its Application to the Study of Methane Utilization

A simple gas chromatograph with a katharometer detector is described to determine O₂, N₂, methane and CO₂ in gas samples of 0·01–2·0 ml. The apparatus is inexpensive, and can be modified to determine other gases. The sensitivity to oxygen is 3 × 10⁻⁶ g. The use of the instrument is illustrated by a study of the growth kinetics of Methylococcus capsulatus grown on methane in shake flask experiments. The ratio of O₂ uptake to methane uptake is much lower in the stationary phase than in the growth phase of the culture.     

Convergence in the relationship of CO2 and N2O exchanges between soil and atmosphere within terrestrial ecosystems

Ecosystem CO₂ and N₂O exchanges between soils and the atmosphere play an important role in climate warming and global carbon and nitrogen cycling; however, it is still not clear whether the fluxes of these two greenhouse gases are correlated at the ecosystem scale. We collected 143 pairs of ecosystem CO₂ and N₂O exchanges between soils and the atmosphere measured simultaneously in eight ecosystems around the world and developed relationships between soil CO₂ and N₂O fluxes. Significant linear regressions of soil CO₂ and N₂O fluxes were found for all eight ecosystems; the highest slope occurred in rice paddies and the lowest in temperate grasslands. We also found the dominant role of growing season on the relationship of annual CO₂ and N₂O fluxes. No significant relationship between soil CO₂ and N₂O fluxes was found across all eight ecosystem types. The estimated annual global N₂O emission based on our findings is 13.31 Tg N yr⁻¹ with a range of 8.19–18.43 Tg N yr⁻¹ for 1980–2000, of which cropland contributes nearly 30%. Our findings demonstrated that stoichiometric relationships may work on ecological functions at the ecosystem level. The relationship of soil N₂O and CO₂ fluxes developed here could be helpful in biogeochemical modeling and large-scale estimations of soil CO₂ and N₂O fluxes.     

Inositol phospholipid levels of rat forebrain obtained by freeze-blowing method

Four different techniques of handling rat brain prior to lipid extraction and assay were tested to investigate the levels of inositol phospholipids in the brain. In these four techniques, the rat forebrains were either (1) freeze-blown followed by being preserved in liquid N2, (2) subjected to microwave irradiation prior to decapitation, (3) removed and frozen in liquid N2, or (4) removed at room temperature and subjected to lipid extraction as rapidly as possible. There was little change in phosphatidylinositol levels under any of these conditions; however, higher levels of phosphatidylinositol 4-phosphate were observed in freeze-blown and microwave-irradiated samples compared to the other samples. Even more striking differences were seen in phosphatidylinositol 4,5-bisphosphate fractions. The highest level of this lipid, 763 +/- 39 nmol/g tissue, which was obtained from freeze-blown samples, was more than 2-fold higher than that of the lowest values which were obtained by extraction without prior inactivation. These results indicate that the values of phosphatidylinositol 4,5-bisphosphate in brain in situ are higher than those generally reported, and that the freeze-blowing method has an advantage for further investigation of inositol phospholipid metabolism in brain due to the rapid breakdown of these compounds.     

Effects of mycorrhizal colonization on biomass production and nitrogen fixation of black locust (Robinia pseudoacacia) seedlings grown under elevated atmospheric carbon dioxide

Interactive effects of elevated atmospheric CO₂ and arbuscular mycorrhizal (AM) fungi on biomass production and N₂ fixation were investigated using black locust ( Robinia pseudoacacia ). Seedlings were grown in growth chambers maintained at either 350 μmol mol⁻¹ or 710 μmol mol⁻¹ CO₂. Seedlings were inoculated with Rhizobium spp. and were grown with or without AM fungi. The ¹⁵N isotope dilution method was used to determine N source partitioning between N₂ fixation and inorganic fertilizer uptake. Elevated atmospheric CO₂ significantly increased the percentage of fine roots that were colonized by AM fungi. Mycorrhizal seedlings grown under elevated CO₂ had the greatest overall plant biomass production, nodulation, N and P content, and root N absorption. Additionally, elevated CO₂ levels enhanced nodule and root mass production, as well as N₂ fixation rates, of non- mycorrhizal seedlings. However, the relative response of biomass production to CO₂ enrichment was greater in non-mycorrhizal seedlings than in mycorrhizal seedlings. This study provides strong evidence that arbuscular mycorrhizal fungi play an important role in the extent to which plant nutrition of symbiotic N₂-fixing tree species is affected by enriched atmospheric CO₂.     

Zinc Inhibition of t-[3H]Butylbicycloorthobenzoate Binding to the GABAA Receptor Complex

Abstract: The effect of Zn²⁺ on t -[³H]butylbicycloorthobenzoate ([³H]TBOB) binding to the GABA_A receptor complex was studied autoradiographically in rat brain. Zn²⁺ inhibited [³H]TBOB binding in a dose-dependent manner at physiological concentrations. Saturation analysis revealed noncompetitive inhibition in various brain regions. The inhibitory effect of Zn²⁺ had regional heterogeneity; regions showing the greatest inhibition of [³H]TBOB binding were cortical laminae I–III, most areas of hippocampus, striatum, septum, and cerebellar cortex. Regions with relatively less inhibition of [³H]TBOB binding included cortical laminae V–VI, thalamus, superior colliculus, inferior colliculus, and central gray matter. The effect of Zn²⁺ and those of other GABA_A ligands, such as benzodiazepines, bicuculline, isoguvacine, and picrotoxin, on [³H]TBOB binding seemed to be additive. Ni²⁺, Cd²⁺, and Cu²⁺ also inhibited [³H]TBOB binding with a regional heterogeneity similar to that produced by Zn²⁺. These results are consistent with Zn²⁺ acting at the previously detected recognition site on the GABA_A receptor complex, distinct from the picrotoxin, GABA, and benzodiazepine sites. The regional heterogeneity of the Zn²⁺ effect may reflect differential regional distribution of GABA_A receptor subtypes among brain regions. Other divalent cations probably act at the Zn²⁺ binding site.     

Symbiotic N2 fixation in a high Alpine grassland: effects of four growing seasons of elevated CO2

1. Increasing carbon dioxide concentration (E: 680 μl CO₂ litre^–1 vs ambient, A: 355 μl CO₂ litre^–1) around late-successional Alpine sedge communities of the Swiss Central Alps (2450 m) for four growing seasons (1992–1995) had no detectable effect on symbiotic N₂ fixation in Trifolium alpinum —the sole N₂-fixing plant species in these communities (74 ± 30 mg N m^–2 year^–1, A and E plots pooled).
2. This result is based on data collected in the fourth growing season showing that elevated CO₂ had no effect on Trifolium above-ground biomass (4·4 ± 1·7 g m^–2, A and E plots pooled, n = 24) or N content per unit land area (124 ± 51 mg N m^–2, A and E pooled), or on the percentage of N Trifolium derived from the atmosphere through symbiotic N₂ fixation (%Ndfa: 61·0 ± 4·1 across A and E plots) estimated using the ¹⁵N dilution method.
3. Thus, it appears that N inputs to this ecosystem via symbiotic N₂ fixation will not be dramatically affected in the foreseeable future even as atmospheric CO₂ continues to rise.