Ecotoxicological impacts of exposure to copper oxide nanoparticles on the gill of the Swan mussel,Anodonta cygnea (Linnaeus, 1758)

This study was conducted to investigate the ecotoxicological effects of exposure to copper oxide nanoparticles (CuO NPs) on the gill of the swan mussel Anodonta cygnea using several approaches including qualitative and quantitative histopathology, ultra-morphology (scanning electron microscopy [SEM]) and measures of clearance rate (CR) and bioaccumulation of CuO NPs. Histological alterations in mussels exposed to 0.25 (T1), 2.5 (T2) and 25.0?µg L^?1 (T3) CuO NPs for 12 days include changes in the length and form of gill lamellae, changes in inter-lamellar spaces, epithelial hyperplasia, atrophy and tissue rupture. Ultra-morphological changes following CuO NP exposure included epithelial hyperplasia and hypertrophy, epithelial lifting, tissue rupture (water channel fusion) and extensive necrosis of the gill surfaces. I_Gill (gill damage severity) index values for both histopathological and ultra-morphological data were significantly (P?0.05) higher in T3. The CR of mussels was significantly (P??1 g^?1 dry weight]) in comparison to controls (CR?=?108?±?47.14 [L min^?1 g^?1 dry weight]). CuO NPs accumulated in exposed mussels at all exposure concentrations until day 4, but there was no further change in accumulation levels by the end of the exposure period. The accumulated content of CuO NPs was significantly (P??1 exposure concentration. Based on these results, significant accumulation of CuO NPs in the gills of swan mussel could affect histological and ultra-structural characteristics of this organ and consequently have deleterious impacts on its filtration activity.     

Intracellular location of ATP citrate lyase in leaves of Pisum sativum L.

The aim of this work was to discover if there is enough ATP citrate lyase (EC 4.1.3.8) in the cytosol of the leaves of Pisum sativum L. to catalyse the synthesis of the acetyl CoA needed for terpenoid synthesis. Estimates of the maximum catalytic activity of the enzyme in leaves of 7-d-old peas gave values of 113 nmol min^-1 g^-1 fresh weight. The rate of carotenoid accumulation in these leaves corresponded to a requirement for acetyl CoA of 0.7 nmol min^-1 g^-1 fresh weight. The distribution of marker enzymes during fractionation of homogenates of leaves from 7 to 10-d-old peas showed that differential centrifugation led to the isolation in reasonable yields of chloroplasts, mitochondria, peroxisomes and the endomembrane system. None of the above components of the leaf contained appreciable detectable activity of ATP citrate lyase, the distribution of which closely paralleled that of the cytosolic marker. It was concluded that in young leaves of pea most of the ATP citrate lyase is in the cytosol.     

Distribution of natural radionuclide40K in biotic and abiotic components of the Cauvery river system,Tiruchirapalli, India

Abiotic components like water and sediment, and biotic components such as mussels, fish and grass collected from Cauvery river at Tiruchirapalli were analysed for⁴⁰K activity. The highest level of⁴⁰K activity was found in the sediment (342 mBq g^-1 dry weight) and the lowest activity was found in water (2·209 mBq ml⁻¹). In the freshwater musselParreysia favidens (Benson)⁴⁰K activity was estimated in the total soft tissues and shells of mussels belonging to three different size groups. In all the size groups⁴⁰K activity was two times higher in shells (68–39 mBq g^-1 fresh weight) than in the total soft tissues (25–17 mBq g^-1 fresh weight). The results indicate that the younger mussels accumulated more⁴⁰K than the older ones. The ability of internal organs of mussels belonging to group III to accumulate⁴⁰K was in the following order: gills > digestive gland > foot > mantle. The values ranged from 47 to 18 mBq g⁻¹ fresh weight in the various organs. Concentration of⁴⁰K in the mussel was distinctly higher than in the grassEchinochloa colonum (J Koenig) (95 mBq g⁻¹ fresh weight), and the concentration of⁴⁰K in the bone of the fishCirrhina cirrhosa (Bloch) (126 mBq g⁻¹ fresh weight) was higher than to that of muscle (113 mBq g⁻¹ fresh weight)     

Dynamics of the phycotoxin domoic acid: accumulation and excretion in two commercially important bivalves

Batch cultures of the toxigenic diatomNitzschia pungens Grunow f.multiseries Hasle were fed to blue mussels (Mytilus edulis) and deep sea Atlantic scallops (Placopecten magellanicus) to elucidate conditions under which domoic acid (DA) was accumulated and excreted (depurated). Mussels accumulated the toxin to a maximum level of 13 g g^-1, at rates of 0.21 to 3.7 g h^-1 g^-1, dry weight. Accumulation efficiency (the proportion of accumulated DA to estimated net uptake) ranged from 1–5%. The highest filtration rate of 1.71 h^-1 occurred at concentrations of 4–8 × 10⁶ Nitzschia cells 1^-1 with no formation of pseudofeces. Depuration rates between fed and starved mussels over a 2 h test period were the same. The depuration rate of domoic acid was about 17% d^-1 and did not account for the low uptake efficiencies, so it is suggested that most of the DA is lost from mussels in the solution during the feeding process. Domoic acid accumulation in mussels was dependent on the amount of toxin available, which in turn was a function of the density and growth phase of theNitzschia population. Changes in filtration rate withNitzschia concentration and depuration rate with time can account for the DA levels of mussels collected during toxic episodes in Cardigan Bay, Prince Edward Island, Canada in 1988 and 1989.Scallops accumulated DA (0.39–1.3 g h^-1 g^-1, more slowly than mussels, however, accumulation efficiencies ranged from 5–100%. Filtration rates remained relatively low and constant at 0.081 h^-1. Scallops retained domoic acid longer than mussels, a fact which must be considered in the marketing of whole scallops for human consumption.     

Andreaea Hartmanii Thed.—A histological study

Abstract

Photosynthetic responses to light intensity were studied under laboratory conditions in seven bryophyte species from evergreen laurel forest, a threatened habitat, on Terceira island in the Azores. Four mosses (Andoa berthelotiana, Echinodium prolixum, Fissidens serrulatus, Myurium hochstetteri) and three liverworts (Bazzania azorica, Frullania tamarisci, Lepidozia cupressina) were selected to encompass a range of potential responses to variations in the forest light environment. Carbon dioxide exchange measurements were made, using an infra-red gas-analyser, at photosynthetic photon flux densities (PPFD) of 0-900 µmol m^-2 s^-1 and a mean temperature of 21°C in fully hydrated shoots. Most species achieved light saturation of photosynthesis below 30 µmol m^-2 s^-1, the lowest value being for A. berthelotiana (20 µmol m^-2 s^-1) and the highest for M. hochstetteri (68 µmol m^-2 s^-1). The liverwort F. tamarisci had the highest maximum photosynthetic rate (P_max, 23 µmol CO₂ g^-1 h^-1) whereas P_max was lowest in the mosses E. prolixum and M. hochstetteri (10 µmol CO₂ g^-1 h^-1). Dark respiration rate, a critical factor in toleration of shade by forest floor plants, was highest in the species with the highest values for P_max. Compensation point was extremely low (7 µmol photons m^-2 s^-1) in Fissidens serrulatus, a species found in the deep shade of forest ravines and caves, and highest in M. hochstetteri a moss restricted to better illuminated habitats within and outside the forest. No photoinhibition was detected during the relatively short exposures to high irradiances. Comparison of these responses with data on the forest light environment indicates that, despite the possession of considerable shade adaptations, during winter in the evergreen laurel forest, low light levels may often limit photosynthetic rates of the bryophytes.     

Metabolic rates in harvestmen (Arachnida, Opiliones): the influence of running activity

Abstract. Metabolic rates of adult Lophopilio palpinalis (Herbst, 1799) (Arachnida, Opiliones, Phalangioidea) and Paranemastoma quadripunctatum (Perty, 1833) (Arachnida, Opiliones, Troguloidea) are measured during rest and activity. Carbon dioxide release during rest is continuous in both species. Mean values at 20 °C are 4.2 µL min⁻¹ g⁻¹ for the males of P. quadripunctatum, 4.1 µL min⁻¹ g⁻¹ for the males of L. palpinalis and 4.7 µL min⁻¹ g⁻¹ for the females of L. palpinalis, thus being significantly higher in the egg-producing females. In L. palpinalis, respiratory quotient at rest is 0.84. Spontaneous walking activity with speeds of 15–30 cm min⁻¹ raises the metabolic rate by up to three-fold in both species. Lophopilio palpinalis is made to undertake constant running on a treadmill with speeds of 60, 72 and 96 cm min⁻¹. Enforced activity causes the animals to raise their metabolic rates by up to five-fold above resting rates. Animals reach a steady state of CO₂ release on the treadmill and show a fast t_1/2 on-response, indicating aerobic exercise. The minimum cost of locomotion is determined to be 2.5 × 10⁻³ J cm⁻¹ g⁻¹, thus fitting the predicted values for terrestrial locomotion.     

Effects of salinity,light intensity and sediment on growth,pigments, agar production and reproduction in Gracilaria tenuistipitata from Songkhla Lagoon in Thailand

The effects of salinity, light intensity and sediment on Gracilaria tenuistipitata C.F. Chang & B.M. Xia on growth, pigments, agar production, and net photosynthesis rate were examined in the laboratory under varying conditions of salinity (0, 25 and 33 psu), light intensity (150, 400, 700 and 1000 µmol photons m^?2 s^?1) and sediment (0, 0.67 and 2.28 mg L^?1). These conditions simulated field conditions, to gain some understanding of the best conditions for cultivation of G. tenuistipitata. The highest growth rate was at 25 psu, 700 µmol photons m^?2 s^?1 with no sediments, that provided a 6.7% increase in weight gain. The highest agar production (24.8 ± 3.0 %DW) was at 25 psu, 150–400 µmol photons m^?2 s^?1 and no sediment. The highest pigment contents were phycoerythrin (0.8 ± 0.5 mg g^?1FW) and phycocyanin (0.34 ± 0.05 mg g^?1 FW) produced in low light conditions, at 150 µmol photons m^?2 s^?1. The highest photosynthesis rate was 161.3 ± 32.7 mg O₂ g^?1 DW h^?1 in 25 psu, 400 µmol photons m^?2 s^?1 without sediment in the short period of cultivation, (3 days) and 60.3 ± 6.7 mg O₂ g^?1 DW h^?1 in 25 psu, 700 µmol photons m^?2 s^?1 without sediment in the long period of cultivation (20 days). The results indicated that salinity was the most crucial factor affecting G. tenuistipitata growth and production. This would help to promote the cultivation of Gracilaria cultivation back into the lagoon using these now determined baseline conditions. Extrapolation of the results from the laboratory study to field conditions indicated that it was possible to obtain two crops of Gracilaria a year in the lagoon, with good yields of agar, from mid‐January to the end of April (dry season), and from mid‐July to the end of September (first rainy season) when provided sediment was restricted.     

Dietary carotenoid levels affect carotenoid and retinoid allocation in female Chinook salmon Oncorhynchus tshawytscha

This study examined the effect of dietary carotenoid availability on carotenoid and retinoid concentrations in the flesh, plasma, skin and eggs of female Chinook salmon Oncorhynchus tshawytscha. Carotenoid concentrations in all tissues were closely related to dietary availability. Early in the breeding season, carotenoids were stored primarily in the muscle, with a flesh carotenoid concentration of 9·9 µg g^?1 in fish fed a high carotenoid diet compared with 1·9 µg g^?1 in fish fed a low carotenoid diet. During the breeding season, carotenoid reserves were mobilized predominantly to the eggs and also to the skin. By the end of the breeding season, carotenoid concentrations in the eggs were 17·9 µg g^?1 in fish fed a high carotenoid diet and 3·9 µg g^?1 in fish fed a low carotenoid diet. Conversely, egg retinoid concentrations were only c. 20% lower in fish fed a low v. high carotenoid diet, which suggests that retinoid concentrations were not limited by the availability of carotenoid precursors. Egg carotenoid concentrations were not correlated with either skin carotenoid concentration or colouration, which suggests that female carotenoid displays are not a reliable signal that males can use to evaluate egg carotenoid resources.     

Differential protein expression due to Se deficiency and Se toxicity in rat liver

There is a U-shaped dose-response between selenium (Se) status and health outcomes, but underlying metabolic processes are unclear. This study aims to identify candidate proteins in liver regulated by dietary Se, ranging from deficiency to toxic. Male rats (n=4) were fed graded Se concentrations as selenite for 28 days. Bulk Se analysis was performed by ICP-MS on both soluble and insoluble fractions. Soluble fraction samples were chromatographically separated for identification of selenocompounds by SEC-ICP-MS and protein quantification by LC-MS/MS. Bioinformatics analysis compared low-Se (0 and 0.08 µg Se g⁻¹) and high-Se (0.8, 2 and 5 µg Se g⁻¹) with adequate-Se (0.24 µg Se g⁻¹) diets. Major breakpoints for Se were seen at 0.8 and 2 µg Se g⁻¹ in the insoluble and soluble fractions, respectively. Glutathione peroxidase 1 protein abundance reached a plateau at ≥0.08 µg Se g⁻¹diet; Se bound to selenium binding protein 2 was observed with 2 and 5 µg Se g⁻¹ Se. The extreme diets presented the highest number of differentially expressed (P value <0.05, FC ≥1.2) proteins in comparison to the adequate-Se diet (0 µg Se g⁻¹: 45 proteins; 5 µg Se g⁻¹: 59 proteins); 13 proteins were commonly affected in 0 and 5 µg Se g⁻¹ treatments. Network analysis revealed that the metabolism of glutathione, xenobiotics and amino acids were enriched in both 0 and 5 µg Se g⁻¹ diets, indicating a U-shape effect of Se. This similarity is likely due to down-stream effects of lack of essential selenoproteins in Se deficiency and due to toxic effects of Se that exceeds the capacity to cope with excess Se.     

Cytokinin metabolism in autonomous and crown gall tissue cultures

When tissues ofCatharanthus roseus A6 crown gall were incubated on medium supplemented with 50 (μM N6-isopentenyladenine (i⁶Ade), endogenous i⁶Ade, N6-isopentenyladenosine (i⁶A) and i⁶A nucleotide (i⁶AXP) increased to. 6, 5 and 12 nmol g^-1, respectively, during 100 h. Whereas i⁶Ade and i⁶AXP increased rapidly during the initial 4 h and then remained relatively constant, the level of i⁶A continued to increase to 25 nmol g^-1 by 16 h and then decreased; Ribosylzeatin (io⁶A) and its nucleotide (io⁶AXP) remained constant at 1.5 and 1.7 nmol g^-1, respectively. Upon transfer to cytokininless medium, i⁶Ade and i⁶AXP declined rapidly but i⁶A increased to 10 nmol g^-1 after 4 h and then declined. Again, io⁶A and io⁶AXP were unchanged. Prolonged incubation of crown gall tissue on i⁶Ade completely inhibited growth. By contrast, nonrtransformed, autonomous tissue lines fromCalycanthus fertilis andActinidia chinensis Xarguta continued to proliferate on this medium. TheActinidia Une was shown to metabolize i⁶Ade to zeatin and to accumulate this cytokinin to levels in excess of 70 nmol g^-1.     

The content of triterpene saponins and phenolic compounds in American ginseng hairy root extracts and their antioxidant and cytotoxic properties

Panax quinquefolium is a perennial herb of the Araliaceae family native to North America. Its roots have been used in traditional and Chinese medicine. The aim of this study was to determine the phenolic profile of methanolic extracts of P. quinquefolium hairy roots cultivated in flasks and a bioreactor, as well as extracts from the roots of three-year-old field-grown plants. Additionally, the phenol and ginsenoside components of the tested extracts were identified by HPLC, and their antioxidant and cytotoxic properties were evaluated. The antioxidant effect was evaluated by FRAP (ferric reducing antioxidant power), and ABTS ([2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) cation scavenging tests, and their effect on the viability of the glioblastoma cell (T98G) line was measured using the 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The LC–MS/MS analysis revealed the presence of 16 phenolic compounds identified as phenolic acids (ten compounds) or flavonoids (six compounds). The highest phenol content was observed in the transformed roots of flask-grown P. quinquefolium (1.6 mg g^?1 d.w.), followed by these grown in the bioreactor (1.1 mg g^?1 d.w.). However, the highest ginsenoside content was found in the roots of the naturally-cultivated plants (67.6 mg g^?1 d.w.). The methanolic extracts from hairy root culture of P. quinquefolium appear to have significant antioxidant and cytotoxic potential. Such transformed American ginseng root cultures could represent a potential source of bioactive metabolites for the food or pharmaceutical industry.

     

Toxicity of Cd to signal grass (Brachiaria decumbens Stapf.) and Rhodes grass (Chloris gayana Kunth.)

Given that Cd accumulates within plant tissues to levels that are toxic to animals, it is necessary to understand the role of plants in highly Cd-contaminated systems and their subsequent impact on the health of animals. A solution culture experiment was conducted to elucidate the effects of increasing Cd²⁺ activity ({Cd²⁺}) on growth of Rhodes grass (Chloris gayana Kunth.) and signal grass (Brachiaria decumbens Stapf.). The shoot and root fresh mass of both Rhodes grass and signal grass was reduced by 50% at ca. 0.5 µM {Cd²⁺}. Elevated {Cd²⁺} resulted in a significant decrease in the tissue Mn concentration for both the shoots and roots, and caused a chlorosis of the veins in the shoots. Root hair growth was prolific even at high {Cd²⁺}, thus root hair growth appeared to be less sensitive to elevated Cd than was root growth per se. The critical shoot tissue concentrations (50% reduction in growth), 230 µg g^?1 for Rhodes grass and 80 µg g^?1 for signal grass, exceeded the maximum level of Cd tolerated in the diet of animals (ca. 5 µg g^?1). When assessing the risk associated with the revegetation of Cd-contaminated sites with Rhodes grass or signal grass, careful consideration must be given, therefore, to the transfer of toxic concentrations of Cd to grazing animals and through the wider food chain.     

The production and antiprotozoal activity of abietane diterpenes in Salvia austriaca hairy roots grown in shake flasks and bioreactor

The biomass and concentration of bioactive quinone methide-type diterpenes in hairy roots of Salvia austriaca were determined and compared with levels of these metabolites in roots of field-grown plants. The cultures were maintained in shake flasks and a nutrient sprinkle bioreactor. Diterpene production was more efficient in the shake flask root culture than the bioreactor one. Biomass and diterpene production within the shake flask culture was evaluated using Schenk and Hildebrandt (SH), Gamborg (B5), and woody plant medium (WPM), with both full- and half-strength macro and micronutrient concentrations (1/2 SH, 1/2 B5, and 1/2 WPM). Among the tested media, SH medium proved to be most effective for biomass and diterpene production. In this medium, the transformed roots accumulated the levels of taxodone (3.89?mg?g^?1 DW; equivalent to 63.3?mg?L^?1), taxodione (1.15?mg?g^?1 DW; equivalent to 17.4?mg?L^?1), 15-deoxy-fuerstione (2.15?mg?g^?1 DW; equivalent to 32.5?mg?L^?1), and 7-(2′-oxohexyl)-taxodione (0.076?mg?g^?1 DW; equivalent to 1.1?mg?L^?1). Three diterpenes were also detected in the roots of S. austriaca intact plants, but their concentrations were lower than those in hairy root culture. No 7-(2′-oxohexyl)-taxodione was found in the roots of field-grown plants. The hairy roots were able to maintain high metabolite levels even for 6 years of cultivation. Taxodone, taxodione, 15-deoxy-fuerstione, and 7-(2′-oxohexyl)-taxodione were tested for in vitro activity against Trypanosoma brucei rhodesiense, T. cruzi, and Plasmodium falciparum and their cytotoxicity was determined using L6 cells. Among these compounds, taxodione was the most active against T. brucei rhodesiense [IC₅₀?=?0.05?µM with high selectivity, selectivity index (SI)?=?38]. Taxodione was found to inhibit the growth of P. falciparum and T. cruzi by 50% at respective concentrations of 1.9 and 7.1?µM (SI values of 1.0 and 0.27). Other diterpenoids demonstrated weaker activity against tested parasites (IC₅₀ values ranging from 0.62 to 194.7?µM) and lower selectivity (SI value ranged from 0.4 to 5.0).     

Neuronal Metabolism of Branched-Chain Amino Acids: Flux Through the Aminotransferase Pathway in Synaptosomes

Abstract: The metabolism of branched-chain amino acids (BCAAs) was studied in cortical synaptosomes. With [¹⁵N]leucine (1 mM) as precursor, the cumulative appearance of ¹⁵N in [¹⁵N]glutamate and [¹⁵N]aspartate was 0.2 nmol/min/mg of protein without supplemental α-ketoglutarate and 0.3 nmol/min/mg of protein in the presence of α-ketoglutarate (0.5 mM). The BCAA aminotransferase reaction also proceeded in the “reverse” direction [α-ketoisocaproate (KIC) + glutamate → leucine + α-ketoglutarate]. This was documented by incubating synaptosomes with [¹⁵N]glutamate and measuring the formation of [¹⁵N]leucine. Without KIC in the medium, the rate of [¹⁵N]leucine production was 0.13 nmol/min/mg of protein. In the presence of 25 µM KIC the rate was 0.79 nmol/min/mg of protein and even greater (1.0 nmol/min/mg of protein) in the presence of 500 µM KIC. The reamination of KIC was two- to threefold faster with [2-¹⁵N]glutamine as precursor compared with [¹⁵N]glutamate. The ketoacid of valine, α-ketoisovalerate (KIV), was reaminated to [¹⁵N]valine at a rate comparable to that observed with respect to KIC. The BCAA transaminase mediated not only the bidirectional transfer of amino groups between leucine or valine and glutamate, but also the direct transfer of nitrogen between leucine and valine. This was ascertained in studies in which the incubation medium was supplemented with either [¹⁵N]leucine and KIV or [¹⁵N]valine and KIC (amino acids at 1 mM and ketoacids at 25 or 500 µM). The rate was faster in the direction of leucine formation at both the lower (6.1-fold) and higher (1.7-fold) KIC concentration. It is suggested that in synaptosomes the BCAA transaminase (a) functions predominantly in the direction of leucine formation and (b) maintains a constant ratio of BCAAs and ketoacids to one other.     

Exacerbation of NMDA, AMPA, and l-Glutamate Excitotoxicity by the Succinate Dehydrogenase Inhibitor Malonate

Abstract: We report that a subtoxic dose of the succinate dehydrogenase (SDH) inhibitor malonate greatly enhances the neurotoxicity of three different excitatory amino acid agonists: N-methyl-d -aspartate (NMDA), S-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (S-AMPA), and l -glutamate. In male Sprague-Dawley rats, intrastriatal stereotaxic injection of malonate alone (0.6 µmol), NMDA alone (15 nmol), S-AMPA alone (1 nmol), or glutamate alone (0.6 µmol) produced negligible toxicity as assessed by measurement of lesion volume. Coinjection of subtoxic malonate with NMDA produced a large lesion (15.2 ± 1.4 mm³), as did coinjection of malonate with S-AMPA (11.0 ± 1.0 mm³) or glutamate (12.8 ± 0.7 mm³). Administration of the noncompetitive NMDA antagonist MK-801 (5 mg/kg i.p.) completely blocked the toxicity of malonate plus NMDA (0.5 ± 0.3 mm³). This dose of MK-801 had little effect on the lesion produced by malonate plus S-AMPA (9.0 ± 0.7 mm³), but it attenuated the toxicity of malonate plus glutamate by ～40% (7.5 ± 0.9 mm³). Coinjection of the AMPA antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)-quinoxaline (NBQX; 2 nmol) had no effect on malonate plus NMDA or malonate plus glutamate toxicity (12.3 ± 1.8 and 14.0 ± 0.9 mm³, respectively) but greatly attenuated malonate plus S-AMPA toxicity (1.5 ± 0.9 mm³). Combination of the two antagonists conferred no additional neuroprotection in any paradigm. These results indicate that metabolic inhibition exacerbates both NMDA receptor- and non-NMDA receptor-mediated excitotoxicity. They also suggest that the NMDA receptor may play a major role in situations of metabolic compromise in vivo, where glutamate is the endogenous agonist. Furthermore, glutamate toxicity under conditions of metabolic compromise may not be mediated entirely by ionotropic glutamate receptors.     

Variation in seedling growth of 11 perennial legumes in response to phosphorus supply

Phosphorus (P) deficiency is a major problem for Australian agriculture. Development of new perennial pasture legumes that acquire or use P more efficiently than the current major perennial pasture legume, lucerne (Medicago sativa L.), is urgent. A glasshouse experiment compared the response of ten perennial herbaceous legume species to a series of P supplies ranging from 0 to 384 µg g^?1 soil, with lucerne as the control. Under low-P conditions, several legumes produced more biomass than lucerne. Four species (Lotononis bainesii Baker, Kennedia prorepens F.Muell, K. prostrata R.Br, Bituminaria bituminosa (L.) C.H.Stirt) achieved maximum growth at 12 µg P g^?1 soil, while other species required 24 µg P g^?1. In most tested legumes, biomass production was reduced when P supply was ≥192 µg g^?1, due to P toxicity, while L. bainesii and K. prorepens showed reduced biomass when P was ≥24 µg g^?1 and K. prostrata at ≥48 µg P g^?1 soil. B. bituminosa and Glycine canescens F.J.Herm required less soil P to achieve 0.5 g dry mass than the other species did. Lucerne performed poorly with low P supply and our results suggest that some novel perennial legumes may perform better on low-P soils.     

CARRIER MEDIATED BLOOD-BRAIN BARRIER TRANSPORT OF CHOLINE AND CERTAIN CHOLINE ANALOGS

Blood-brain barrier (BBB) transport of choline and certain choline analogs was studied in adult and suckling rats, and additionally compared in the paleocortex and neocortex of adult rats. Saturable uptake was characterized by a single kinetic system in all cases examined, and in adult rat forebrains we determined a K_m= 442 ± 60 μM and V_max= 10.0 ± 0.6 nmol min^-1 g^-1. In 14–15-day-old suckling forebrains a similar K_m (= 404 ± 88 μM) but higher V_max (= 12.5 ± 1.5 nmol min^-1 g^-1) was determined. When choline uptake was compared in two regions of the forebrain, similar Michaelis-Menten constants were determined but a higher uptake velocity was found in the neocortex (i.e. neocortex K_m= 310 ± 103 μM and V_max= 12.6 ± 2.8 nmol min^-1g^-1; paleocortex K_m= 217 ± 76 μM and V_max= 7.2 ± 1.5 nmol min^-1 g^-1). Administration of radiolabelled choline at low (5 μM) and high (100 μM) concentrations, followed by microwave fixation 60 s later and chloroform-methanol-water separations of the homogenized brain did not suggest a relationship between concentration and the appearance of label in lipid or aqueous fractions as observed in another in-vitro study elaborating two-component kinetics of choline uptake. It was observed that 60s after carotid injection 12–14% of the radiolabel in the ipsilateral cortex was found in the chloroform-soluble fraction. Hemicholinium-3 (K_i= 111 μM), dimethylaminoethanol (K_i= 42 μM), tetraethyl ammonium chloride, tetramethyl ammonium chloride, 2-hydroxyethyl triethylammonium iodide, carnitine, normal rat serum, and to a lesser extent lithium and spermidine all inhibited choline uptake in the BBB. Unsubstituted ammonium chloride and imipramine did not inhibit choline uptake. No difference was observed in blood-brain barrier choline uptake of unanesthetised, carotid artery-catheterized animals, and comparable sodium pentobarbital-anesthetized controls.     

Complementarity in the use of nitrogen forms in a temperate broad-leaved mixed forest

Background: The complementary use of different forms of soil nitrogen (N) might lead to a higher productivity of mixed forests than monocultures, but convincing evidence for temperate mixed forests is scarce.

Aims: We searched for species differences in N uptake rates and the preference for NH₄⁺, NO₃^? or glycine among five temperate broad?leaved tree species (Acer pseudoplatanus, Carpinus betulus, Fagus sylvatica, Fraxinus excelsior, Tilia cordata) in a mature mixed stand.

Methods: ¹⁵N tracer was added to the soil and its accumulation in fine root biomass was analysed after 10 min, 1 h and 1 d.

Results: The estimated root uptake rates of the species were in the range of 5–46 µg N g^?1 root h^?1 for NH₄⁺, 6–86 µg N g^?1 h^?1 for NO₃^? and 4–29 µg N g^?1 h^?1 for glycine during the first hour after tracer application. Carpinus, Tilia and Acer tended to prefer NH₄⁺ over NO₃^?, while Fraxinus showed equal preference for both N forms and Fagus seemed to prefer NO₃^?.

Conclusions: The five co-existing tree species differed in uptake rates and partly in their N form preference, but complementarity in the use of different N forms seems to be of minor importance in this forest because tree species appear to be rather flexible in their N form use.     

Chronotoxicity of a Copper (II) Complex with a Linear Tetradentate Ligand in Mice

In vitro copper (II) complex presents antimitotic effects. In this work, we have studied the in vivo seasonal toxic effects of copper (II), ligand (H₂L) and the complex [Cu(H₂L)(H₂O)₂]Cl₂·4H₂O in male Swiss mice. During spring, an i.p. injection of CuCl₂ in aqueous NaCl (9 g·l^-1) up to 0.05 µmol·kg^-1 b.w. (body weight) killed 60% of the rodents after 6 days. LD100 was up to 0.3 µmol·kg^-1; H₂L was well tolerated, while the complex was 30% lethal with 50 µmol·kg^-1. In autumn, mice were less sensitive to CuCl₂, and both ligand and complex were equally tolerated and this leads to the conclusion that, in vivo, chronotoxicities of copper (II) and complex in NaCl aqueous solutions are quite different in spring and autumn seasons.     

Enzymes of urate synthesis and catabolism in the Gecarcinid land crab Gecarcoidea natalis

This study investigated the sites of urate synthesis and catabolism in the gecarcinid land crab Gecarcoidea natalis by assaying spongy connective tissue, midgut gland, muscle and gill for xanthine oxidoreductase, the last enzyme involved in urate synthesis, and uricase and urease, the first and last enzymes involved in urate catabolism. The spongy connective tissue and midgut gland of the G. natalis contained activities of xanthine oxidoreductase and were considered to be sites of urate synthesis. The midgut gland had a high activity of xanthine oxidoreductase [(58.87±4.6 (SE) nmol urate produced g^-1 wet wt. tissue min^-1], 2.7 times the xanthine oxidoreductase activity contained within the spongy connective tissue, and was thought to be the main site of urate synthesis. Xanthine dehydrogenase (EC 1.1.1.204) was the only form of xanthine oxidoreductase detected within the tissues. Its presence means that the cost of synthesising urate de novo is relatively small (between 1 and 3 ATP). Uricase (EC 1.7.3.3) and urease (EC 3.5.1.5) activities were present in the tissues of G. natalis. Spongy connective tissue contained the highest activities of uricase [48.44±4.29 (SE) nmol urate consumed g^-1 wet wt. tissue min^-1] while the highest activities of urease [365.31±37.21 (SE) nmol urate consumed g^-1 wet wt tissue min^-1] were contained within the gills. From this evidence it is clear that G. natalis possesses the uricolytic pathway and hence the ability to catabolise urate, and urate catabolism is begun at the site of urate storage, the spongy connective tissue, and is completed at the gills. As the gills are the site of ammonia excretion in this species the ammonia produced from the catabolism of urate is probably excreted. The urate deposits within the body of G. natalis may be involved in temporary storage of nitrogenous wastes.