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Biokinetics of cadmium, selenium, and zinc in freshwater alga Scenedesmus obliquus under different phosphorus and nitrogen conditions and metal transfer to Daphnia magna.

The uptake of Cd, Se(IV) and Zn by the freshwater alga Scenedesmus obliquus and the subsequent transfer and release budget in Daphnia magna were investigated under different nutrient additions and cell incubation conditions. An increase in ambient phosphate concentrations from 0.5 micromol l(-1) to 50 micromol l(-1) significantly increased the intracellular accumulation of Cd (by 18x) and Zn (by 5x), but decreased the accumulation of Se (by 126x) in the alga. The percentage of these metals distributing in the intracellular pool of algae also increased substantially with increasing ambient P concentrations. Nitrate addition from 5.0 to 200 micromol l(-1) did not influence the uptake of any of the three metals, although a significant decrease in the intracellular Se distribution was observed. Radiolabeled algae under different nutrient manipulations (semi-continuous culture, starvation, and P-pulse treatments) were used to measure trophic transfer assimilation efficiency (AE) in Daphnia. When the algal cells were grown in a semi-continuous culture, starved for N and P, or were treated with P-pulse, the AEs of Cd and Zn were generally independent of the nutritional conditions, but the Se AE was significantly affected by different P levels. The efflux rate constants, determined during 10 d depuration following 7 days of dietary uptake, decreased significantly for Cd and Zn, but were relatively constant for Se with increasing P concentration. N-addition caused no effect on the metal efflux rate constants. P- or N-additions did not influence the release budget (including molting, neonates, excretion and feces) for all three elements in Daphnia. Our study indicated that phosphate enrichment may substantially increase metal uptake in green alga S. obliquus. Responses of trophic transfer in Daphnia to nutrient enrichment were metal specific. P-enrichment can possibly lead to considerable decrease on Se transfer from algae to zooplankton.

Animals↗

Selection of denitrifying phosphorus accumulating organisms in activated sludge.

Although anoxic phosphorus uptake has been demonstrated, the conditions that stimulate or inhibit the anoxic uptake are not fully understood. In this study, the possibility of achieving biological excess phosphorus removal in sequencing batch reactors using nitrate as an electron acceptor without including an aerobic phase has been confirmed. The use of an anaerobic/anoxic/aerobic sequencing batch reactor allows anoxic phosphorus uptake to be achieved, in spite of the low fraction of denitrifying phosphorus accumulating organisms present in the inoculum. Biomass characterisation was carried out to determine the denitrifying fraction of the phosphorus accumulating organisms in sequencing batch reactors operating at different operational conditions. With an anaerobic/anoxic/aerobic cycle, it was demonstrated that the disadvantage of denitrifying phosphorus accumulating organisms due to the lower substrate utilisation efficiency in anoxic conditions relative to aerobic conditions, is not so effective to cause the "strictly aerobic" phosphorus accumulating organisms to dominate in the system.

Biodegradation, Environmental↗

Body protein kinetics during perioperative intravenous nutritional support.

The kinetics of whole body protein metabolism in eight patients with benign disease were evaluated during perioperative isocaloric and isonitrogenous intravenous feedings. A prime infusion of 15N-glycine with determination of urinary end product enrichment and stochastic analysis was used to determine protein kinetics. Surgically induced increases in body protein turnover, synthesis and breakdown were evident on the fifth postoperative day. Abdominal operation was associated with a rise in urinary 3-methylhistidine (3MH) excretion. The postoperative alteration in the relationship between 3MH excretion and body protein breakdown suggests that even mild to moderate surgical injury induces tissue specific changes in protein metabolism. Resting energy expenditure remained unchanged from that of the preoperative levels on the fifth postoperative day. Despite significant changes in body and tissue specific protein kinetics, there is no change in the apparent efficiency of oxygen use for protein homeostasis postoperatively. The failure to observe a relationship between energy expenditure and protein metabolism suggests that only a small proportion of body oxygen uptake is directed toward protein homeostasis after elective surgical procedures. A comparison of perioperative complete intravenous feeding to hypocaloric regimens will be necessary to evaluate more fully the impact of protein and energy balance upon cellular and body protein homeostasis after injury.

Aged↗

Leaching and crop uptake of nitrogen and phosphorus from pig slurry as affected by different application rates.

The influence of increasing pig slurry applications on leaching and crop uptake of N and P by cereals was evaluated in a 3-yr study of lysimeters filled with a sandy soil. The slurry was applied at N rates of 50 (S50), 100 (S100), 150 (S150), and 200 (S200) kg ha(-1) during 2 of the 3 yr. The P rates applied with slurry were: 40 (S50), 80 (S100), 120 (S150), and 160 (S200) kg ha(-1) yr(-1). Simultaneously, NH4NO3 and Ca(H2PO4)2 were applied at rates of 100 kg N ha(-1) and 50 kg P ha(-1), respectively, to additional lysimeters (F100), while others were left unfertilized (F0). During the 3-yr period, the leaching load of total N tended to increase with increasing slurry application to, on average, 139 kg ha(-1) at the highest application rate (S200). The corresponding N leaching loads (kg ha(-1)) in the other treatments were: 75 (F0), 103 (F100), 93 (S50), 120 (S100), and 128 (S150). The loads of slurry-derived N in the S100, S150, and S200 treatments were significantly larger (P < 0.05) than those of fertilizer-derived N. In contrast, P leaching tended to decrease with increasing input of slurry, and it was lower in all treatments that received P at or above 50 kg P ha(-1) yr(-1) with slurry or fertilizer than in the unfertilized treatment. The crop use efficiency of added N and P was clearly higher when NH4NO3 and Ca(H2PO4)2 were used rather than slurry (60 vs. 35% for N, 38 vs. 6-9% for P), irrespective of slurry application rate. Therefore, from both a production and water quality point of view, inorganic fertilizers seem to have environmental benefits over pig slurry when used on sandy soils.

Animal Husbandry↗

Regulative influence of o-aminobenzoic acid on the biosynthesis of nourseothricin in cultures of Streptomyces noursei JA 3890b. IV. Bistability of metabolism and the mechanism of action of aminobenzoic acids.

Using the semi-continuous cultivation technique we could establish that specifically in Streptomyces noursei JA 3890b during growth on a medium supplied with D,L-alanine, NH4+, and maize starch there are two different phenotypes of the organism and stationary states of metabolism, respectively. The expression of either the metabolic state I with an enhanced capacity to oxidative deamination of alanine via the NAD+-dependent alanaine dehydrogenase or the metabolic state 2 which may be characterized by the preferred use of ammonium ions via the NADP+-dependent glutamate dehydrogenase was shown to depend strongly on the conditions of inoculum cultivation. When the amino acid permeases were derepressed by cultivating the inoculum cells on amino acid media, probably due to the defective mechanism of negative feedback control of amino acid influx in this strain an abnormously high uptake of alanine was observed that, consequently, was correlated to the enhanced oxidation of this amino acid as well as to the intensive production of ammonia within the cell. This overproduction of cellular NH4+ seems to bring about the subsequent repression of biosynthetic glutamate dehydrogenase and so on the accumulation of ammonia autocatalytically may rise up (metabolic state I). On the other hand, if the influx of alanine was kept low and the NADH oxidation was less efficient, respectively, or when there was high cellular activity of glutamate dehydrogenase the level of ammonia never did exceed the respressory limit and, accordingly, the expression of the metabolic state 2 was observed. Switching-over of metabolic flux from the state 2 towards the state 1 can be brought about either by increasing the level of nitrogen sources in the medium or by adding buffers pH greater than 7.5. In contrast, decrease of cellular level of NH4+ was shown to induce the transition of metabolic state 1 into the state 2. This can be achieved not only by limitation of nitrogen source but also by adding different aminobenzoic acids and, alternatively, effectors of membrane function (short-chain alcohols), inhibitors of cytochrome oxidases (sodium azide, potassium cyanide), heavy metal (Fe++)-chelating agents (catechol, 2,5'-dipyridyl, o-phenanthroline), beta-alanine, and buffers pH less than 7. This suggests that these effectors are capable of preventing the abnormously high influx of amino acids as well as its wasteful catabolism within the cell of S. noursei JA 3890b. Therefore, it seems likely that by this way the aminobenzoic acids and similar effectors can diminish the catabolite repression or inhibition of secondary metabolism by cellular excess of some nitrogen compounds in good agreement with its well-known stimulatory action on the biosynthesis of the antibiotic nourseothricin in this strain.

Alanine↗

Biosynthesis of the validamycins: identification of intermediates in the biosynthesis of validamycin A by Streptomyces hygroscopicus var. limoneus.

To study the biosynthesis of the pseudotrisaccharide antibiotic, validamycin A (1), a number of potential precursors of the antibiotic were synthesized in (2)H-, (3)H-, or (13)C-labeled form and fed to cultures of Streptomyces hygroscopicus var. limoneus. The resulting validamycin A from each of these feeding experiments was isolated, purified and analyzed by liquid scintillation counting, (2)H- or (13)C NMR or selective ion monitoring mass spectrometry (SIM-MS) techniques. The results demonstrate that 2-epi-5-epi-valiolone (9) is specifically incorporated into 1 and labels both cyclitol moieties. This suggests that 9 is the initial cyclization product generated from an open-chain C(7) precursor, D-sedoheptulose 7-phosphate (5), by a DHQ synthase-like cyclization mechanism. A more proximate precursor of 1 is valienone (11), which is also incorporated into both cyclitol moieties. The conversion of 9 into 11 involves first epimerization to 5-epi-valiolone (10), which is efficiently incorporated into 1, followed by dehydration, although a low level of incorporation of 2-epi-valienone (15) is also observed. Reduction of 11 affords validone (12), which is also incorporated specifically into 1, but labels only the reduced cyclitol moiety. The mode of introduction of the nitrogen atom linking the two pseudosaccharide moieties is not clear yet. 7-Tritiated valiolamine (8), valienamine (2), and validamine (3) were all not incorporated into 1, although each of these amines has been isolated from the fermentation, with 3 being most prevalent. Demonstration of in vivo formation of [7-(3)H]validamine ([7-(3)H]-3) from [7-(3)H]-12 suggests that 3 may be a pathway intermediate and that the nonincorporation of [7-(3)H]-3 into 1 is due to a lack of cellular uptake. We thus propose that 3, formed by amination of 12, and 11 condense to form a Schiff base, which is reduced to the pseudodisaccharide unit, validoxylamine A (13). Transfer of a D-glucose unit to the 4'-position of 13 then completes the biosynthesis of 1. Other possibilities for the mechanism of formation of the nitrogen bridge between the two pseudosaccharide units are also discussed.

Anti-Bacterial Agents↗

In Vivo Determination of Parameters of Nitrate Utilization in Wheat (Triticum aestivum L.) Seedlings Grown with Low Concentration of Nitrate in the Nutrient Solution.

Six genotypes of winter wheat (Triticum aestivum L.) differing in grain protein concentration were grown on a nutrient solution containing low concentrations of NO(3) (-) (2 millimolar). Total NO(3) (-) uptake varied between genotypes but was not related to grain protein content. An in vivo nitrate reductase assay was used to determine the affinity of the enzyme for NO(3) (-), and large phenotypic variations were observed. In vivo estimations of the concentration and size of the metabolic pool were variable. However, the three genotypes with the higher ratios of metabolic pool size to leaf total NO(3) (-) concentration were the high protein varieties. It is proposed that a high affinity of nitrate reductase for nitrate might be a biochemical marker for the capacity of the plant to continue assimilating NO(3) (-) for a longer period during the last stage of growth.The potential use of such physiological criteria as markers is discussed, in particular with respect to breeding programs for the development of plants with efficient nitrogen utilization.

Journal Article↗

Fermentable carbohydrate and digestive nitrogen excretion.

BACKGROUND: Interventions that restrict protein intake lower plasma urea concentration and may slow the progression of renal failure. The question arises whether the effect of a dietary protein restriction could be reinforced by enrichment of the diet with fermentable carbohydrate because these carbohydrates may stimulate the extra-renal route of nitrogen (N) excretion through the digestive route. METHODS: The influence of fermentable carbohydrate and moderate protein restriction on N metabolism was investigated in a rat model of renal failure with ablation of 70% of renal mass compared with control rats with intact kidneys. Animals were adapted to diets varying with respect to nondigestible fermentable carbohydrate (0% or 10% fructooligosaccharide [FOS]) and with respect to protein content (10% or 18% casein). RESULTS: Feeding FOS led to a considerable enlargement of the cecum (increase in contents, wall thickness, and blood flow). These changes resulted in a concomitant enhancement of urea N uptake into the cecum and a decrease in plasma urea concentration (-30%). The extent of urea uptake by the cecum was influenced by plasma urea level that was determined by the dietary protein level and by the renal function. Thus, compared with control rats, the rate of urea uptake by the cecum and the total N excreted by the uremic rats was greater under all nutritional conditions. It is noteworthy that, when expressed as a percentage of total N excretion, fecal N excretion nearly doubled in rats adapted to the low-protein diets containing FOS. These effects occurred in both control rats and in uremic rats, in which a 22% decrease in urinary N was recorded as a result of FOS in addition to the low-protein diet. Globally, decreasing the amount of protein in the diet and adding a fermentable carbohydrate led to a decrease in urinary N excretion of more than 65% in uremic rats. CONCLUSION: These results suggest a possible usefulness for combining fermentable carbohydrate, such as FOS, with a low-protein diet to increase N excretion through the digestive route in detriment of the renal route. This may represent an efficient preventive measure to relieve the renal function in case of renal failure.

Ammonia↗

Elevated CO2 and ozone reduce nitrogen acquisition by Pinus halepensis from its mycorrhizal symbiont.

The effects of 700 µmol mol-1 CO2 and 200 nmol mol-1 ozone on photosynthesis in Pinus halepensis seedlings and on N translocation from its mycorrhizal symbiont, Paxillus involutus, were studied under nutrient-poor conditions. After 79 days of exposure, ozone reduced and elevated CO2 increased net assimilation rate. However, the effect was dependent on daily accumulated exposure. No statistically significant differences in total plant mass accumulation were observed, although ozone-treated plants tended to be smaller. Changes in atmospheric gas concentrations induced changes in allocation of resources: under elevated ozone, shoots showed high priority over roots and had significantly elevated N concentrations. As a result of different shoot N concentration and net carbon assimilation rates, photosynthetic N use efficiency was significantly increased under elevated CO2 and decreased under ozone. The differences in photosynthesis were mirrored in the growth of the fungus in symbiosis with the pine seedlings. However, exposure to CO2 and ozone both reduced the symbiosis-mediated N uptake. The results suggest an increased carbon cost of symbiosis-mediated N uptake under elevated CO2, while under ozone, plant N acquisition is preferentially shifted towards increased root uptake.

Journal Article↗

Influence of nitrate and COD on phosphorus, nitrogen and dinitrotoluene (DNT) removals under batch anaerobic and anoxic conditions.

In this study, the effects of COD to NO(3)-N ratio in the feed on PO(4)-P removal was investigated. Maximum PO(4)-P uptake was obtained in the anoxic reactor when the COD to NO(3)-N ratios were between 2 and 3.75. With the influent COD of 800-1500 mg COD/L a total of the maximum removable PO(4)-P was 56 mg PO(4)-P/L through 20 days of anaerobic/anoxic incubation, indicating 98% P removal in the anoxic reactor. Similarly, for the COD to NO(3)-N ratios varying between 2 and 3.75 maximum denitrification was observed. Through anoxic operation the poly-P bacteria are capable of removing NO(3)-N using VFA, COD as carbon source and NO(3)-N as the electron acceptor after methanogenesis has been completed. High NO(3)-N concentrations stopped significantly the P uptake. A total of 97-99% dinitrotoluene removal efficiencies in the reactors containing COD to NO(3)-N ratio of 2 and 3.75 after 20 days of incubation period. For maximum NO(3)-N and PO(4)-P removals optimal COD to NO(3)-N ratios, COD and NO(3)-N concentrations were 2-3.75, 2000-4000 mg COD/L and, 800-1500 mg NO(3)-N/L, respectively.

Journal Article↗

Rhizobium tropici genes involved in free-living salt tolerance are required for the establishment of efficient nitrogen-fixing symbiosis with Phaseolus vulgaris.

Characterization of nine transposon-induced mutants of Rhizobium tropici with decreased salt tolerance (DST) allowed the identification of eight gene loci required for adaptation to high external NaCl. Most of the genes also were involved in adaptation to hyperosmotic media and were required to overcome the toxicity of LiCl. According to their possible functions, genes identified could be classified into three groups. The first group included two genes involved in regulation of gene expression, such as ntrY, the sensor element of the bacterial ntrY/ntrX two-component regulatory system involved in regulation of nitrogen metabolism, and greA, which encodes a transcription elongation factor. The second group included genes related to synthesis, assembly, or maturation of proteins, such as alaS coding for alanine-tRNA synthetase, dnaJ, which encodes a molecular chaperone, and a nifS homolog probably encoding a cysteine desulfurase involved in the maturation of Fe-S proteins. Genes related with cellular build-up and maintenance were in the third group, such as a noeJ-homolog, encoding a mannose-1-phosphate guanylyltransferase likely involved in lipopolysaccharide biosynthesis, and kup, specifying an inner-membrane protein involved in potassium uptake. Another gene was identified that had no homology to known genes but that could be conserved in other rhizobia. When inoculated on Phaseolus vulgaris growing under nonsaline conditions, all DST mutants displayed severe symbiotic defects: ntrY and noeJ mutants were impaired in nodulation, and the remaining mutants formed symbiosis with very reduced nitrogenase activity. The results suggest that bacterial ability to adapt to hyperosmotic and salt stress is important for the bacteroid nitrogen-fixing function inside the legume nodule and provide genetic evidence supporting the suggestion that rhizobia face severe environmental changes after their release into plant cells.

Genes, Bacterial↗

Oxidation of nitrided si(100) by gaseous atomic and molecular oxygen.

The nitridation of Si(100) by ammonia and the subsequent oxidation of the nitrided surface by both gaseous atomic and molecular oxygen was investigated under ultrahigh vacuum (UHV) conditions using X-ray photoelectron spectroscopy (XPS). Nitridation of Si(100) by the thermal decomposition of NH3 results in the formation of a subsurface nitride and a decrease in the concentration of surface dangling bond sites. On the basis of changes in the N1s spectra obtained after NH3 adsorption and decomposition, we estimate that the nitride resides about four to five layers below the vacuum-solid interface and that the concentration of surface dangling bonds after nitridation is only 59% of its value on Si(100)-(2 x 1). Oxidation of the nitrided surface is found to produce an oxide phase that remains in the outer layers of the solid and interacts only weakly with the underlying nitride for oxygen coverages up to 2.5 ML. Slight changes in the N1s spectra observed after oxidizing at 300 K are suggested to arise primarily from the introduction of strain within the nitride, and by the formation of a small amount of Si2=N-O species near the nitride-oxide interface. The nitrogen bonding environment changes negligibly after oxidizing at 800 K, which is indicative of greater phase separation at elevated surface temperature. Nitridation is also found to significantly reduce the reactivity of the Si(100) surface toward both atomic and molecular oxygen. A comparison of the oxygen uptake on the clean and nitrided surfaces shows quantitatively that the decrease in dangling bond concentration is responsible for the reduced activity of the nitrided surface toward oxidation, and therefore that dangling bonds are the initial adsorption site for both gaseous oxygen atoms and molecules. Increasing the surface temperature is found to promote the uptake of oxygen when O2 is used as the oxidant, but brings about only a small enhancement in the uptake of gaseous O-atoms. The different effects of surface temperature on the uptake of O versus O2 are interpreted in terms of the efficiency at which dangling bond pairs are regenerated on the surface at elevated temperature and the different site requirements for the adsorption of O and O2.

Journal Article↗

Reduced grazing rates in Daphnia pulex caused by contaminants: implications for trophic cascades.

Ecotoxicological endpoints based on behavioral traits (e.g., predator avoidance, feeding, and locomotion) may be more sensitive and give more insights into patterns of sublethal toxicity than survivorship tests. In this study, the density-dependent grazing rate of Daphnia pulex pre-exposed to p,p'-dichlorodiphenyldichloroethylene (DDE) (insecticide metabolite) and glyphosate (herbicide), via water or a vector, Scenedesmus spp., was assayed in laboratory experiments. The phytoplankton biomass was estimated from the chlorophyll content, and the pesticide uptake and turnover pattern in Daphnia and Scenedesmus were determined from parallel experiments with a radiolabeled source. Scenedesmus spp. relative net growth rate was inversely and linearly related to the density of the grazer. Daphnia pulex exhibited significant reductions in grazing rate: 30% for those pre-exposed to p,p'-DDE via water and 40% for D. pulex pre-exposed to glyphosate via Scenedesmus spp. Through the process of trophic cascading, this impaired grazing allowed Scenedesmus spp. to grow at higher rates, 70 and 60%, respectively. The reduced grazing efficiencies were associated with the treatments that gave the highest body burden of p,p'-DDE (70 microg/g dry wt) and the lowest of glyphosate (13 mg/g dry wt). The pattern of results suggests a toxic effect of p,p'-DDE on D. pulex and a growth enhancement of Scenedesmus spp. in response to nitrogen and phosphorus in glyphosate excreted by D. pulex.

Animals↗

Municipal wastewater treatment by periodic biofilter without excess sludge production.

The paper reports the results of an investigation aimed at evaluating the performances of a periodic biofilter (SBBR) for treating municipal wastewater. The investigation was carried out at laboratory scale on real primary effluent coming from a municipal wastewater treatment plant located in Southern Italy. The SBBR was designed for carbon and nitrogen removal through one single stage. The results have shown that even at maximum organic load (i.e., 7 kg COD/m3.d), the COD in the effluent was lower than 60 mg/L. TKN removal efficiencies resulted high (i.e. 90-95%) up to an organic load of 5.7 kg COD/m3.d corresponding to a nitrogen load of 0.8 kg TKN/m3.d. NO3-N concentration in the treated effluent was lesser than 6 mg/L although in the SBBR treatment cycle no anoxic phase was scheduled. This indicated that denitrification extensively took place in the biofilter. The process was characterized by high suspended solids removal (about 90%) and by a negligible sludge production (lower than 0.01 kgVSS/kgCODremoved). In the SBBR, biomass grew as granules and was characterised by different measurements (biomass concentration, cellular protein and biomass density). Biomass density resulted very high, i.e. 200 gTSS/Lbiomass, and this permitted to achieve a biomass concentration such high as 40 gTSS/Lbed. Such biomass concentration did not cause any decrease of biomass metabolic activity as proved by its total protein content (29% of organic matter) and maximum oxygen uptake rate value (i.e. 50 mgO2/gVSS h).

Biomass↗

Influence of carbon source on nitrate removal by nitrate-tolerant Klebsiella oxytoca CECT 4460 in batch and chemostat cultures.

The nitrate-tolerant organism Klebsiella oxytoca CECT 4460 tolerates nitrate at concentrations up to 1 M and is used to treat wastewater with high nitrate loads in industrial wastewater treatment plants. We studied the influence of the C source (glycerol or sucrose or both) on the growth rate and the efficiency of nitrate removal under laboratory conditions. With sucrose as the sole C source the maximum specific growth rate was 0.3 h-1, whereas with glycerol it was 0.45 h-1. In batch cultures K. oxytoca cells grown on sucrose or glycerol were able to immediately use sucrose as a sole C source, suggesting that sucrose uptake and metabolism were constitutive. In contrast, glycerol uptake occurred preferentially in glycerol-grown cells. Independent of the preculture conditions, when sucrose and glycerol were added simultaneously to batch cultures, the sucrose was used first, and once the supply of sucrose was exhausted, the glycerol was consumed. Utilization of nitrate as an N source occurred without nitrite or ammonium accumulation when glycerol was used, but nitrite accumulated when sucrose was used. In chemostat cultures K. oxytoca CECT 4460 efficiently removed nitrate without accumulation of nitrate or ammonium when sucrose, glycerol, or mixtures of these two C sources were used. The growth yields and the efficiencies of C and N utilization were determined at different growth rates in chemostat cultures. Regardless of the C source, yield carbon (YC) ranged between 1.3 and 1.0 g (dry weight) per g of sucrose C or glycerol C consumed. Regardless of the specific growth rate and the C source, yield nitrogen (YN) ranged from 17.2 to 12.5 g (dry weight) per g of nitrate N consumed. In contrast to batch cultures, in continuous cultures glycerol and sucrose were utilized simultaneously, although the specific rate of sucrose consumption was higher than the specific rate of glycerol consumption. In continuous cultures double-nutrient-limited growth appeared with respect to the C/N ratio of the feed medium and the dilution rate, so that for a C/N ratio between 10 and 30 and a growth rate of 0.1 h-1 the process led to simultaneous and efficient removal of the C and N sources used. At a growth rate of 0.2 h-1 the zone of double limitation was between 8 and 11. This suggests that the regimen of double limitation is influenced by the C/N ratio and the growth rate. The results of these experiments were validated by pulse assays.

Culture Media↗

Hepatic fuel metabolism during muscular work: role and regulation.

The increased fuel demands of the working muscle necessitate that metabolic processes within the liver be accelerated accordingly. The sum of changes in hepatic glycogenolysis and gluconeogenesis are closely coupled to the increase in glucose uptake by the working muscle, due to the actions of the pancreatic hormones. The exercise-induced rise in glucagon and fall in insulin interact to stimulate hepatic glycogenolysis, whereas the increase in gluconeogenesis is determined primarily by glucagon action. The increment in gluconeogenesis is caused by increases in hepatic gluconeogenic precursor delivery and fractional extraction as well as in the efficiency of intrahepatic conversion to glucose. Glucagon stimulates the latter two processes. Epinephrine may become important in the regulation of hepatic glucose production during prolonged or heavy exercise when its levels are particularly high. On the other hand, there is no evidence that hepatic innervation is essential for the rise in hepatic glucose production during exercise. Nonesterified fatty acid (NEFA) delivery to, uptake of, and oxidation by the liver are accelerated during prolonged exercise, resulting in an increase in ketogenesis. The rate of the first two of these processes is largely determined by factors that stimulate fat mobilization. The third step is regulated by both NEFA delivery to and glucagon-stimulated fat oxidation within the liver. The increase in hepatic fat oxidation produces energy that fuels gluconeogenesis. The shuttling of amino acids to the liver provides carbon-based compounds that are used for gluconeogenesis, transfers nitrogen to the liver, and supplies substrate for protein synthesis. During exercise, metabolic events within the liver, which are regulated by hormone levels and substrate supply, integrate pathways of carbohydrate, fat, and amino acid metabolism. These processes function to provide substrates for muscular energy metabolism and conserve carbon in glucose and nitrogen in protein.

Animals↗

Do spin traps also act as classical chain-breaking antioxidants? A quantitative kinetic study of phenyl tert-butylnitrone (PBN) in solution and in liposomes.

Free radical spin traps such as phenyl tert-butylnitrone (PBN) are often reported to provide protection of the central nervous system of animal models against free radical damage, and the effects are attributed to its "antioxidant activity." The effects of PBN and p-CH(3)O-PBN were compared with known antioxidants, alpha-tocopherol and 2,2,5,7,8-pentamethyl-6-hydroxychroman (PMHC), in quantitative kinetic studies of lipid peroxidation thermally initiated under controlled conditions. Results obtained on the spin traps in organic solvents and in dilinoleoyl phosphatidylcholine (DLPC) bilayers indicated that the spin traps do not act as peroxyl radical trapping antioxidants but rather act only as moderate "retarders" of oxygen uptake at relatively high concentration. At low oxygen partial pressures, e.g., 14 torr, which better reflect oxygen partial pressures in biological systems, PBN provides a more significant reduction in oxygen uptake (up to 50%) by DLPC bilayers but still did not act as a typical antioxidant. However, at low partial pressures, PBN does act cooperatively with PMHC. It is suggested that its role in biological fluids and tissues may be to extend the suppressed oxidation by natural antioxidants expected to be present. The combination of antioxidant/spin trap, alpha-(3, 5-di-tert-butyl-4-hydroxyphenyl)-N-tert-butylnitrone did not exhibit any enhanced antioxidant efficiency compared with the related hindered phenol, 2,6-di-tert-butyl-4-methoxyphenol.

Antioxidants↗

Strategies for neuroprotection against L-trans-2,4-pyrrolidine dicarboxylate-induced neuronal damage during energy impairment in vitro.

Increased levels of extracellular excitatory amino acids and failure of energy metabolism are two conditions associated with brain ischemia. In the present study we have combined the simultaneous inhibition of glutamate uptake and mitochondrial electron transport chain to simulate neuronal damage associated with brain ischemia. Results show that cerebellar granule neurons are not vulnerable to transient glutamate uptake inhibition by L-trans-pyrrolidine-2,4-dicarboxylate (PDC) despite the increase in the extracellular concentration of glutamate, unless they are simultaneously exposed to the mitochondrial toxins 3-nitropropionic acid (3-NP) or sodium azide. Cell damage was assessed by light microscopy observation, by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and by the fluorescent markers for live and dead cells, calcein and ethidium homodimer, respectively. The protective effect of alternative energy substrates, such as pyruvate, acetoacetate, and beta-hydroxybutyrate against PDC-induced neuronal death during 3-NP exposure was studied and compared to the effects of the antioxidant vitamin E, the spin trapper alpha-phenyl-N-tert-butylnitrone (PBN), voltage-dependent calcium channel antagonists, and glutamate receptor antagonists. Results show that neuronal damage can be efficiently prevented in the presence of pyruvate and the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801, whereas the non-NMDA receptor antagonist NBQX, acetoacetate, vitamin E, and PBN showed partial protection. In contrast, beta-hydroxybutyrate and voltage-dependent calcium channels blockers did not show any protective effect at the concentrations tested.

Animals↗