Seasonal variability of inorganic and organic nitrogen in the North Sea

This study considers the cycling of nitrogen in the waters of the North Sea, particularly focussing on organic nitrogen. Dissolved inorganic nitrogen (DIN), dissolved organic nitrogen (DON) and particulate organic nitrogen (PON) were measured in the North Sea over a one-year period (autumn 2004–summer 2005). The surface water concentrations of nitrate, ammonium, DON and PON during the present study ranged from <0.1–7.2 μM, <0.1–2.0 μM, 1.9–11.2 μM and 0.3–5.6 μM, respectively, with DON the dominant fraction of total nitrogen at all times. These nutrients concentrations were significantly lower compared to previous studies in the southern North Sea. The seasonal variations showed high mean surface concentrations of nitrate (4.7 ± 0.6 μM) and DON (8.9 ± 0.9 μM), low ammonium (<0.1 μM) and PON (0.8 ± 0.1 μM) in winter, shifting to low nitrate (0.3 ± 0.3 μM) and DON (4.2 ± 1.2 μM) in summer, with high ammonium (0.8 ± 0.4 μM) in autumn and PON (2.5 ± 1.2 μM) in spring. Highest mean surface DON concentration was measured in winter and may be due to resuspension of the organic matter from the bottom sediments. For autumn and spring, phytoplankton DON release was likely to be the most significant source of DON as shown by high concentrations of low molecular weight (LMW) DON and its positive correlation to chlorophyll a. Low total and LMW DON concentrations during summer were likely to be due to the uptake of the LMW DON fraction by phytoplankton and bacteria and the stratification of the water column. DON is therefore shown to be a potentially important source of nitrogen in shelf seas especially after the spring bloom has depleted nitrate to limiting concentrations. Handling editor: L. Naselli-Flores     

Sulphide tolerance and adaptation in the California killifish, Fundulus parvipinnis, a salt marsh resident

Hydrogen sulphide is a toxicant naturally produced in hypoxic marine sediments, hydrocarbon and brine seeps and hydrothermal vents. The California killifish, a salt marsh resident, is remarkably tolerant of sulphide. The 50% lethal concentration is 700 μM total sulphide in 96 h, and 5 mM in 8 h (determined in flow-through, oxygenated sea water). Killifish exposed to sulphide produce thiosulphate which accumulates in the blood. The cytochrome c oxidase (a major site of toxicity) of the killifish is 50% inhibited by <1 μM sulphide. Killifish liver mitochondria are poisoned by 50–75 μM sulphide but can oxidize 10–20 μM sulphide to thiosulphate. Sulphide causes sulphhaemoglobin formation (and impairment of oxygen transport) at 1–5 mM in vitro and to a small extent at 2 mM in vivo . Killifish blood neither catalyses sulphide oxidation significantly nor binds sulphide at environmental (low) sulphide concentrations. Exposure to 200 μM and 700 μM sulphide over several days causes significant increases in lactate concentrations, indicating shift to anaerobic glycolysis. However, individuals with the most lactate die. In terms of diffusible H₂S, the killifish can withstand concentrations two to three orders of magnitude greater than would poison cytochrome c oxidase. The high sulphide tolerance of the killifish, particularly of concentrations typical of salt marshes, can be explained chiefly by mitochondrial sulphide oxidation. Sulphide tolerance and mitochondrial sulphide oxidation in the killifish have a constitutive basis, i.e. do not diminish in fish held in the laboratory in sulphide-free water for 1–2 months, and are improved by prior acclimation.     

A novel membrane-bound flavocytochrome c sulfide dehydrogenase from the colourless sulfur bacterium Thiobacillus sp. W5

A novel membrane-bound sulfide-oxidizing enzyme was purified 102-fold from the neutrophilic, obligately chemolithoautotrophic Thiobacillus sp. W5 by means of a six-step procedure. Spectral analysis revealed that the enzyme contains haem c and flavin. SDS-PAGE showed the presence of two types of subunit with molecular masses of 40 and 11 kDa. The smaller subunit contains covalently bound haem c, as was shown by haem staining. A combination of spectral analysis and the pyridine haemochrome test indicated that the sulfide-oxidizing heterodimer contains one molecule of haem c and one molecule of flavin. It appeared that the sulfide-oxidizing enzyme is a member of a small class of redox proteins, the flavocytochromes c, and is structurally most related to the flavocytochrome c sulfide dehydrogenase of the green sulfur bacterium Chlorobium limicola. The pH optimum of the enzyme is 8.6. At pH 9, the V _max was 2.1 ± 0.1 μmol cytochrome c (mg protein)^–1 min^–1, and the K _m values for sulfide and cytochrome c were 1.7 ± 0.4 μM and 3.8 ± 0.8 μM, respectively. Cyanide inhibited the enzyme by the formation of an N-5 adduct with the flavin moiety of the protein. On the basis of electron transfer stoichiometry, it seems likely that sulfur is the oxidation product. Received: 15 October 1996 / Accepted: 7 January 1997     

Osmoregulation in juvenile and adult white sturgeon,Acipenser transmontanus

Synopsis Blood samples from cannulated young adult (2.5–15 kg) white sturgeon, acclimated to San Francisco Bay water (24 ppt) had plasma values of 248.8 ± 13.5 mOsm kg⁻¹ H₂O, [Na⁺] = 125 ± 8.0 mEq 1⁻¹, [K⁺] = 2.6 ± 0.8 mEq 1⁻¹ and [CL⁻] = 122 ± 3.0 mEq 1⁻¹. Freshwater acclimated sturgeon had an osmolality of 236 ± 7, [Na⁺] = 131.6 + 4.4, [K⁺] = 2.5 ± 0.7 and [CL⁻] = 110.6 ± 3.6. Freshwater acclimated fish gradually exposed to sea water (increase of 5 ppt h⁻¹) had higher plasma osmolalities than did the bay water acclimated fish. These young adult sturgeon are able to tolerate transfer from fresh water to sea water as well as gradual transfer from sea water to fresh water. Plasma electrolytes in transferred fish are regulated, but tend to differ from long term acclimated fish at the same salinities. There is a gradual increase in the upper salinity tolerance (abrupt transfer) of juvenile white sturgeon with weight: 5–10 ppt for 0.4–0.9 g fish, 10–15 ppt for 0.7–1.8 g fish, and 15 ppt for 4.9–50.0 g fish. The ability of juveniles to regulate plasma osmolality is limited. The young adult fish are able to tolerate higher salinities (35 ppt) than juvenile sturgeon but probably are also characterized by low activity of the necessary ion exchange mechanisms in the gills which permit rapid adjustment of blood electrolytes with graduate change in external salinity.     

Sulfide-quinone reductase activity in membranes of the chemotrophic bacterium Paracoccus denitrificans GB17

Reduction of exogenous ubiquinone and of cytochromes by sulfide in membranes of the chemotrophic bacterium Paracoccus denitrificans GB17 was studied. For sulfide-ubiquinone reductase activity, K _m values of 26 ± 4 and 3.1 ± 0.6 μM were determined from titrations with sulfide and decyl-ubiquinone, respectively. A maximal rate of up to 0.3 μmol decyl-ubiquinone reduced (mg protein)^–1 min^–1 was estimated. The reaction was sensitive to quinone-analogous inhibitors, but insensitive to cyanide. Reduction of cytochromes by sulfide was monitored with an LED-array spectrophotometer. Under oxic conditions, reduction rates and extents of reduction were lower than those under anoxic conditions. Reoxidation of cytochromes was oxygen-dependent and cyanide-sensitive. The multiphasic behavior of transient reduction of cytochrome b with limiting amounts of sulfide reflects that sulfide, in addition to acting as an electron donor, is a slowly binding inhibitor of cytochrome c oxidase. The initial peak of cytochrome b reduction is dependent on electron flow to an oxidant, either oxygen or ferricyanide, and is stimulated by antimycin A. This oxidant-induced reduction of cytochrome b suggests that electron transport from sulfide in P. denitrificans GB17 employs the cytochrome bc ₁ complex via the quinone pool. Received: 8 April 1998 / Accepted: 29 July 1998     

Salinity and fish effects on Salton Sea microecosystems: water chemistry and nutrient cycling

A 15 month long experiment was undertaken to document responses of the Salton Sea biota to experimentally manipulated salinity levels (30, 39, 48, 57, and 65 g l-1) in 312-liter fiberglass tanks maintained outdoors. At two salinities (39 and 57 g l-1) microcosms were set up each having one small tilapia ( Oreochromis mossambicus) in order to assess its influence on the system. To 28 tanks filled with Salton Sea water diluted to 30 g l-1, different salts (NaCl, Na2SO_4, MgSO4 · 7H2O, KCl) were added in constant proportions to produce the desired salinity levels. Salton Sea shoreline sediment was added to the bottom of each tank, and inocula of algae and invertebrates were added on several occasions. Invertebrate populations, phytoplankton, periphyton, and water chemistry were monitored at regular intervals. This article present the results concerning water chemistry and nutrient cycling. There was no apparent increase in salinity over time, though ∼ 1190 l of tapwater with a salinity of ∼ 0.65 g l-1 were added to each tank during the experiment. Ionic composition varied both among treatments and over time to some degree. Ca2 concentrations were the same at all salinities, while K1 concentrations were >3 times greater at the highest salinity than at the lowest. pH showed little consistent variation among salinities until the last few months when it was higher by ∼ 0.4 units at the two higher salinities than at the lower ones; it was unaffected by fish. Absolute oxygen concentrations were negatively correlated with salinity, and occasionally depressed by the presence of fish. PO3-4, dissolved organic phosphorus, and particulate phosphorus concentrations were often reduced by 30–80% at 65 g l-1 relative to lower salinities and by the presence of fish. Early in the experiment NO2-3 concentrations were >2 times higher at 57 and 65 g l-1 than at lower salinities, but otherwise effects of salinity on dissolved forms of nitrogen were not marked; particulate nitrogen was much lower at 65 g l-1 than at other salinities and also was reduced by up to 90% by the presence of fish. Silica concentrations increased over time at all salinities, but, relative to those at lower salinities, were reduced by 60–90% at 65 g l-1 by abundant periphytic diatoms. The TN:TP ratio (molar basis) was 24–30 initially and 35–110 at the end of the experiment; it was positively correlated with salinity and the presence of fish. Mechanisms accounting for the above patterns involve principally the biological activities of phytoplankton and periphyton, as modified by grazing by Artemia franciscana and Gammarus mucronatus, and the feeding and metabolic activities of the tilapia. The large reduction in water column TN and TP levels brought about by the fast-growing, phyto- and zooplanktivorous tilapia suggest that amelioration of the Salton Sea's hypereutrophic state might be assisted by a large scale, sustained yield fish harvesting operation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Influence of glutathione on zinc-mediated cellular toxicity

The effect of zinc on various pulmonary cell lines has been studied by measuring the depletion of total cellular glutathione after exposure to zinc(II) chloride at different concentrations. Total cellular glutathione (cGS) was measured at 31 ± 3 nmol/mg, 3.8 ± 0.6 nmol/mg, and 3.7 ±1.2 nmol/mg protein in A549, L2, and 11Lu cells, respectively. After treatment with buthionine sulfoximine (BSO), the cGS levels decreased by 20% in A549 cells and below 0.2 nmol/mg in L2 and 11Lu cells. Exposure of A549 cells to 25–200 μM ZnCl₂ for 4 h alone decreased the cGS content to 60–80%. There was little additional effect in BSO-pretreated cells. In L2 and 11Lu cells, the decrease of cGS was 70–85% following exposure to 15–150 μM ZnCl₂ for 2 h. If BSO was also used, the decrease in cGS was 85–95% in L2 cells and 75–85% in 11Lu cells. Exposure to 25–250 μM ZnCl₂ for 2 h diminished protein synthesis as determined by radiolabeled methionine incorporation, with half-maximum inhibition (EC₅₀) from 40–160 μM ZnCl₂. To attain similar EC₅₀ values in BSO-pretreated cells, only about half the zinc concentrations were required as compared to cells without pretreatment. The decrease of cGS was accompanied by an increased ratio of oxidized : reduced glutathione that was more pronounced in cells with low glutathione content.     

Direct shoot bud induction and plant regeneration in <Emphasis Type="Italic">Capsicum frutescens</Emphasis> Mill.: influence of polyamines and polarity

Direct shoot bud induction and plant regeneration was achieved in Capsicum frutescens var. KTOC. Aseptically grown seedling explants devoid of roots, apical meristem and cotyledons were inoculated in an inverted position in medium comprising of Murashige and Skoog (Physiol Plant 15:472–497, 1962) basal medium supplemented with 2-(N-morpholine) ethanesulphonic acid buffer along with 2.28 μM indole-3-acetic acid, 10 μM silver nitrate and either of 13.31–89.77 μM benzyl adenine (BA), 9.29–23.23 μM kinetin, 0.91–9.12 μM zeatin, 2.46–9.84 μM 2-isopentenyl adenine. Profuse shoot bud induction was observed only in explants grown on a media supplemented with BA (26.63 μM) as a cytokinin source and 19.4 ± 4.2 shoot buds per explant was obtained in inverted mode under continuous light. Incorporation of polyamine inhibitors in the culture medium completely inhibited shoothoot bud induction. Incorporation of exogenous polyamines improved the induction of shoot buds under 24 h photoperiod. These buds were elongated in MS medium containing 2.8 μM gibberellic acid. Transfer of these shoots to hormone-free MS medium resulted in rooting and rooted plants were transferred to fields. This protocol can be efficiently used for mass propagation and presumably also for regeneration of genetically transformed C. frutescens.     

Concentrations of nitrogen in sandy sediments of a eutrophic estuarine lagoon

Concentrations of sediment organic nitrogen, dissolved inorganic nitrogen (ammonium, nitrite and nitrate), and dissolved organic nitrogen (DON) in sediments were measured at two sites in a eutrophic estuarine lagoon. One is a shallow aerobic site where macrobenthos are abundant and the other is a deep anaerobic site devoid of macrobenthos. Four species of macrobenthos (Bivalvia: Corbicula japonica, Annelida: Notomastus sp., Neanthes japonica and Oligochaeta sp.) were found in 8 sandy sediment cores collected at a shallow site in three succcessive summers. DON (170–1500 μg atom N l-1) was the major constituent of dissolved nitrogen with 10 times greater concentration than ammonium (55–180 μg atom N l-1) and 1000 times greater than nitrate (0.14–5.9 μg atom N l-1) and nitrite (0.21–1.4 μg atom N l-1). The ammonium content in anaerobic muddy sediments at the deep site (210–350 μg atom N l-1) was higher than in aerobic sandy sediments, whereas DON was higher in aerobic sediments than anaerobic sediments (90–240 μg atom N l-1). In aerobic sediments, depth profiles of DIN were nearly constant whereas DON was mostly highest at the surface. On the other hand, the increase of DON and ammonium was observed where macrobenthos was found. The occurrence of macrobenthos and high content of DON and ammonium content in the layers of sediment may suggest the influence of macrobenthos in the partitioning of nitrogen species through their motion and excretion. This revised version was published online in July 2006 with corrections to the Cover Date.     

Impairment of mineralization by metavanadate and decavanadate solutions in a fish bone-derived cell line

Vanadium, a trace metal known to accumulate in bone and to mimic insulin, has been shown to regulate mammalian bone formation using in vitro and in vivo systems. In the present work, short- and long-term effects of metavanadate (containing monomeric, dimeric, tetrameric and pentameric vanadate species) and decavanadate (containing decameric vanadate species) solutions on the mineralization of a fish bone-derived cell line (VSa13) were studied and compared to that of insulin. After 2 h of incubation with vanadate (10 μM in monomeric vanadate), metavanadate exhibited higher accumulation rates than decavanadate (6.85 ± 0.40 versus 3.95 ± 0.10 μg V/g of protein, respectively) in fish VSa13 cells and was also shown to be less toxic when applied for short periods. In longer treatments with both metavanadate and decavanadate solutions, similar effects were promoted: stimulation of cell proliferation and strong impairment (75%) of extracellular matrix (ECM) mineralization. The effect of both vanadate solutions (5 μM in monomeric vanadate), on ECM mineralization was increased in the presence of insulin (10 nM). It is concluded that chronic treatment with both vanadate solutions stimulated fish VSa13 cells proliferation and prevented ECM mineralization. Newly developed VSa13 fish cells appeared to be appropriate in the characterization of vanadate effects on vertebrate bone formation, representing a good alternative to mammalian systems. Daniel M. Tiago and Vincent Laizé1 contributed equally to this work.     

Chemiluminescent in vitro estimation of the inhibitory constants of antioxidants ascorbic and uric acids in Fenton’s reaction in urine

The goal of this research was to measure in vitro the inhibitory constants of the antioxidants ascorbic and uric acid in urine, with lucigenin enhanced chemiluminescence (CL) in Fenton’s system. Maximum CL emission is registered in urine containing H₂O₂ (5·10⁻⁴ M), Fe²⁺ (5·10⁻⁵ M), EDTA (5·10⁻⁵ M), and chemical enhancer lucigenin (10⁻⁴ M) at pH 5.5 and 36°C. Ascorbic acid exhibits up to 4-fold stronger antioxidant effect than uric acid. The constants of antioxidant inhibition in urine were measured at concentrations 10⁻³ and 10⁻⁴ M: for ascorbic acid, 5.92 ± 0.04 and 24.05 ± 1.82 μmol·sec⁻¹; for uric acid, 1.60 ± 0.02 and 21.45 ± 0.97 μmol·sec⁻¹, respectively. Three phases of CL kinetics of urine are well observed: spontaneous CL (0–10 sec), fast flash of CL (10–50 sec), and latent period (50–300 sec). The antioxidant efficiency of ascorbic and uric acids in the final stage of catabolic processes in the body is discussed. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 8, pp. 1062–1065.     

Reflex bradycardia does not influence oxygen consumption during hypoxia in the European eel (Anguilla anguilla)

Most teleost fish reduce heart rate when exposed to acute hypoxia. This hypoxic bradycardia has been characterised for many fish species, but it remains uncertain whether this reflex contributes to the maintenance of oxygen uptake in hypoxia. Here we describe the effects of inhibiting the bradycardia on oxygen consumption (MO₂), standard metabolic rate (SMR) and the critical oxygen partial pressure for regulation of SMR in hypoxia (Pcrit) in European eels Anguilla anguilla (mean ± SEM mass 528 ± 36 g; n = 14). Eels were instrumented with a Transonic flow probe around the ventral aorta to measure cardiac output (Q) and heart rate (f _H). MO₂ was then measured by intermittent closed respirometry during sequential exposure to various levels of increasing hypoxia, to determine Pcrit. Each fish was studied before and after abolition of reflex bradycardia by intraperitoneal injection of the muscarinic antagonist atropine (5 mg kg⁻¹). In the untreated eels, f _H fell from 39.0 ± 4.3 min⁻¹ in normoxia to 14.8 ± 5.2 min⁻¹ at the deepest level of hypoxia (2 kPa), and this was associated with a decline in Q, from 7.5 ± 0.8 mL min⁻¹ kg⁻¹ to 3.3 ± 0.7 mL min⁻¹ kg⁻¹ in normoxia versus deepest hypoxia, respectively. Atropine had no effect on SMR, which was 16.0 ± 1.8 μmol O₂ kg⁻¹ min⁻¹ in control versus 16.8 ± 0.8 μmol O₂ kg⁻¹ min⁻¹ following treatment with atropine. Atropine also had no significant effect on normoxic f _H or Q in the eel, but completely abolished the bradycardia and associated decline in Q during progressive hypoxia. This pharmacological inhibition of the cardiac responses to hypoxia was, however, without affect on Pcrit, which was 11.7 ± 1.3 versus 12.5 ± 1.5 kPa in control versus atropinised eels, respectively. These results indicate, therefore, that reflex bradycardia does not contribute to maintenance of MO₂ and regulation of SMR by the European eel in hypoxia.     

Effect of copper excess in environment on soybean root viability and morphology

The effects of increase copper concentrations in medium (10–150 μM CuSO₄) on growth and viability of the roots of two-week-old soybean seedlings (Glycine max L., cv. Dorintsa) were studied. Copper excess suppressed biomass accumulation and linear plant growth; copper affected root growth much stronger than shoot growth. The presence of 10 μM CuSO₄ in medium suppressed accumulation of plant biomass by 40% and the root length by 70%; in the presence of 25 μM CuSO₄, these indices were equal to 80 and 90%, respectively. In the presence of 50 μM CuSO₄, roots ceased to grow but biomass and shoot length still increased slightly. 150 μM CuSO₄ was lethal for plants. The earliest sign of excessive copper toxicity was the accumulation of MDA, indicating activation of membrane lipid peroxidation. A significant increase in MDA content was observed at plant incubation in medium with 10 μM CuSO₄ for 1 h; in this case, the content of copper in the roots increased from 36 ±1.8 (in control) to 48 ± 2.4 μg/g dry wt. The number of dead cells (permeable for the dye Evans Blue) was doubled in the presence of 200 μg/g dry wt within the root; this occurred in 72 h of growth in medium with 10 μM CuSO₄, in 6 h at 25 μM CuSO₄, in 3 h at 50 μM CuSO₄, and 1 h at 150 μM CuSO₄. Toxicity of copper excess was manifested stronger in dividing and elongation cells of the root apex (root meristem and the zone of elongation) than in more basal root regions. Copper excess resulted in the formation of breaks in the surface cell layers of the root tips and affect root morphology. When plant grew in medium with 10 μM CuSO₄, a distance of lateral root formation zone from the root tip decreased markedly, and spherical swellings were formed on the tips of lateral roots. The higher copper concentrations (50 and 150 μM) suppressed completely the development of lateral roots.     

The Ca2+-extruding ATPase of the human platelet creates and responds to cytoplasmic pH changes,consistent with a 2 Ca2+/nH+ exchange mechanism

The Ca²⁺-extruding ATPase pump of the human platelet was studiedin situ by measuring Ca²⁺ extrusion from quin2-overloaded platelets (Johansson, J.S., Haynes, D.H. 1988.J. Membrane Biol. 104:147–163). Cytoplasmic pH (pH_cyt) was measured by BCECF fluorescence in parallel experiments. The pump was studied by raising the cytoplasmic free Ca²⁺ to 2.5 μM and monitoring active Ca²⁺ extrusion into a Ca²⁺-free medium. The pump was shown to perturb pH_cyt, to not respond to changes in membrane potential and to respond to imposed changes in pH_cyt in a manner consistent with the Ca²⁺ pump acting as a 2 Ca²⁺/nH⁺ exchanger. (i) Raising the external pH (pH_ext) from 7.40 to 7.60 lowers the V_max of the pump in basal condition (V_max,1) from 110±18 to 73±12 μM/min (=μmol/liter cell volume/min). (ii) Lowering pH_ext to 7.13 raised V_max,1 to 150±15 μM/min. (iii) In an N-methyl-d-glucamine (NMDG⁺) medium, the pump operation against high [Ca²⁺]_cyt acidifies the cytoplasm by −0.36±0.10 pH units, and the pump becomes self-inhibited. (iv) Use of nigericin to drive pH_cyt down to 6.23 reduces the V_max,1 to 18±11 μM/min. (v) Alkalinization of the cytoplasm by monensin in the presence of Na⁺ raises the V_max,1 (basal state withK _m,1=80 nM) to 136±24 μM/min, but also activates the pump fourfold (V_max,2=280±28 μM/min;K _m,2=502±36 nM). (vi) Transient elevation of pH_cyt by NH₄Cl at high [Ca²⁺]_cyt activates the pump eightfold (V_max,2≥671±350 μM/min). The large activation by alkaline pH_cyt at high [Ca²⁺]_cyt can be explained by Ca²⁺-calmodulin activation of the pump (Valant, P.A., Adjei, P.N., Haynes, D.H. 1992.J. Membrane Biol. 130:63–82) and by increased Ca²⁺ affinity of calmodulin at high pH.     

Somatic embryogenesis and plant regeneration from undeveloped ovules and stigma/style explants of sweet orange navel group [ Citrus sinensis (L.) Osb.]

Somatic embryogenesis was induced and plant regeneration was obtained in 11 different genotypes of sweet orange navel group [Citrus sinensis (L.) Osb.] from cultures of stigma/style explants and undeveloped ovules. Explants were cultured on 3 different modifications of Murashige and Skoog medium: 500 mg l-1 malt extract; 500 mg l-1 malt extract and 4.6 μM kinetin; and 500 mg l-1 malt extract and 13.3 μM 6-benzylaminopurine. Sucrose (146 mM) was used as carbon source. Somatic embryogenesis occurred 1–3 months after culture initiation from undeveloped ovule and stigma/style cultures of all the genotypes tested. Somatic embryos developed into plantlets with a high frequency (74%) after transfer to Murashige and Skoog medium supplemented with 146 mM sucrose and 500 mg l-1 malt extract. Plants were successfully transferred to soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis and plant regeneration from root segments of Psoralea corylifolia L., an endangered medicinally important plant

Summary An efficient plant regeneration protocol has been developed from root explants of Psoralea corylifolia L., an endangered medicinally important herbaceous plant species belonging to the family Fabaceae. Nodular embryogenic callus was initiated from young root segments cultured on Murashige and Skoog (MS) medium (1962) supplemented with α-naphthaleneacetic acid (NAA; 2.68–13.42 μM) or 2,4-dichlorophenoxyacetic acid (2.4-D; 2.25–11.25 μM) in combination with 6-benzylaminopurine (BA: 2.2. μM). thiamine HCl (2.9 μM), L-glutamine (342.23 μM) and sucrose (3.0% w/v). The highest frequency (95.2%) of embryogenic calluses was obtained on MS medium supplemented with the growth regulators NAA (10.74 μM) and BA (2.2 μM). Development and maturation of somatic embryos was achieved after transfer of embryogenic calluses to MS medium supplemented with 1.34 μM NAA or 1.12 μM 2,4-D and 4.4–13.2 μM BA. The maximum number (13.8±1.34) of cotyledonary stage somatic embryos was obtained on MS medium containing 1.34 μM NAA and 13.2 μM BA. Germination of somatic embryos occurred on MS medium without any growth regulators and also on MS medium enriched with BA (1.1–8.8 μM), although the maximum germination frequency (76.1%) was obtained on 4.4 μM BA plus 1.45 μM gibberellic acid (GA₃). Plant regeneration without complete somatic embryo maturation was also achieved by transferring clumps of nodular embryogenic calluses onto MSO medium or MS medium supplemented with NAA (1.34 μM) and BA (2.2–8.8 μM). The highest frequency of plant regeneration (93.3%) and mean number of plantlets (15.4±0.88) were obtained on MS medium containing 1.34 μM NAA and 4.4 μM BA. Regenerated plants with well-developed root systems were transferred to pots where they grew vigorously, attained maturity and produced fertile seeds.     

Bioremoval of hexavalent chromium from water by a salt tolerant bacterium, Exiguobacterium sp. GS1

Pollution of terrestrial surfaces and aquatic systems by hexavalent chromium, Cr(VI), is a worldwide public health problem. A chromium resistant bacterial isolate identified as Exiguobacterium sp. GS1 by 16S rRNA gene sequencing displayed high rate of removal of Cr(VI) from water. Exiguobacterium sp. GS1 is 99% identical to Exiguobacterium acetylicum. The isolate significantly removed Cr(VI) at both high and low concentrations (1–200 μg mL⁻¹) within 12 h. The Michaelis–Menten K _m and V _max for Cr(VI) bioremoval were calculated to be 141.92 μg mL⁻¹ and 13.22 μg mL⁻¹ h⁻¹, respectively. Growth of Exiguobacterium sp. GS1 was indifferent at 1–75 μg mL⁻¹ Cr(VI) in 12 h. At initial concentration of 8,000 μg L⁻¹, Exiguobacterium sp. GS1 displayed rapid bioremoval of Cr(VI) with over 50% bioremoval in 3 h and 91% bioremoval in 8 h. Kinetic analysis of Cr(VI) bioremoval rate revealed zero-order in 8 h. Exiguobacterium sp. GS1 grew and significantly reduced Cr(VI) in cultures containing 1–9% salt indicating high salt tolerance. Similarly the isolate substantially reduced Cr(VI) over a wide range of temperature (18–45  °C) and initial pH (6.0–9.0). The T _opt and initial pH_opt were 35–40  °C and 7–8, respectively. Exiguobacterium sp. GS1 displayed a great potential for bioremediation of Cr(VI) in diverse complex environments.     

Cytidine and Uridine Increase Striatal CDP-Choline Levels Without Decreasing Acetylcholine Synthesis or Release

Summary Aims: Treatments that increase acetylcholine release from brain slices decrease the synthesis of phosphatidylcholine by, and its levels in, the slices. We examined whether adding cytidine or uridine to the slice medium, which increases the utilization of choline to form phospholipids, also decreases acetylcholine levels and release. Methods: We incubated rat brain slices with or without cytidine or uridine (both 25–400 μM), and with or without choline (20–40 μM), and measured the spontaneous and potassium-evoked release of acetylcholine. Results: Striatal slices stimulated for 2 h released 2650±365 pmol of acetylcholine per mg protein when incubated without choline, or 4600±450 pmol/mg protein acetylcholine when incubated with choline (20 μM). Adding cytidine or uridine (both 25–400 μM) to the media failed to affect acetylcholine release whether or not choline was also added, even though the pyrimidines (400 μM) did enhance choline`s utilization to form CDP-choline by 89 or 61%, respectively. The pyrimidines also had no effect on acetylcholine release from hippocampal and cortical slices. Cytidine or uridine also failed to affect acetylcholine levels in striatal slices, nor choline transport into striatal synaptosomes. Conclusion: These data show that cytidine and uridine can stimulate brain phosphatide synthesis without diminishing acetylcholine synthesis or release.     

Compensatory regulation of acid–base balance during salinity transfer in rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

In seawater-acclimated rainbow trout (Oncorhynchus mykiss), base secretion into the intestine is a key component of the intestinal water absorption that offsets osmotic water loss to the marine environment. Acid–base balance is maintained by the matched excretion of acid equivalents via other routes, presumably the gill and/or kidney. The goal of the present study was to examine acid–base balance in rainbow trout upon transfer to more dilute environments, conditions under which base excretion into the intestine is predicted to fall, requiring compensatory adjustments of acid excretion at the gill and/or kidney if acid–base balance is to be maintained. Net acid excretion via the gill/kidney and rectal fluid, and blood acid–base status were monitored in seawater-acclimated rainbow trout maintained in seawater or transferred to iso-osmotic conditions. As predicted, transfer to iso-osmotic conditions significantly reduced base excretion into the rectal fluid (by ~48%). Transfer to iso-osmotic conditions also significantly reduced the excretion of titratable acidity via extra-intestinal routes from 183.4 ± 71.3 to −217.5 ± 42.7 μmol kg⁻¹ h⁻¹ (N = 7). At the same time, however, ammonia excretion increased significantly during iso-osmotic transfer (by ~72%) so that the apparent overall reduction in net acid excretion (from 419.7 ± 92.9 to 189.2 ± 76.5 μmol kg⁻¹ h⁻¹; N = 7) was not significant. Trout maintained blood acid–base status during iso-osmotic transfer, although arterial pH was significantly higher in transferred fish than in those maintained in seawater. To explore the mechanisms underlying these adjustments of acid–base regulation, the relative mRNA expression and where possible, activity of a suite of proteins involved in acid–base balance were examined in intestine, gill and kidney. At the kidney, reduced mRNA expression of carbonic anhydrase (CA; cytosolic and membrane-associated CA IV), V-type H⁺-ATPase, and Na⁺/HCO₃ ⁻ co-transporter were consistent with a reduced role in net acid excretion following iso-osmotic transfer. Changes in relative mRNA expression and/or activity at the intestine and gill were consistent with the roles of these organs in osmotic rather than acid–base regulation. Overall, the data emphasize the coordination of acid–base, osmoregulatory and ionoregulatory processes that occur with salinity transfer in a euryhaline fish.     

Salinity effects on the growth,mortality and shell strength of Balanus amphitrite from the Salton Sea,California

The Salton Sea, the largest lake in California, has a salinity of around 43 g l-1 that is increasing by about 0.4 g l-1 y-1. A 15 month microcosm experiment was conducted to determined the effects of salinity (30, 39, 48, 57, and 65 g l-1) and tilapia ( Oreochromis mossambicus) on an assemblage of benthic and planktonic Salton Sea algae and invertebrates, including the barnacle Balanus amphitrite. Eleven months after the microcosms were established, acrylic plates containing newly settled B. amphitrite collected at the Salton Sea were placed in the microcosms to determine the effects of salinity on their growth and shell strength. The Brody-Bertalanffy growth model was fitted to the B. amphitrite growth data. Growth was fastest at 48 g l-1 and slowest at 65 g l-1. B. amphitrite grown at 39–48 g l-1 were the largest and required the greatest force to break, but the strength of the barnacle shell material declined steadily as the salinity increased. However, B. amphitrite at the higher salinities were shorter and had thicker walls relative to their diameters, which may have increased their structural stability. The effects of salinity on the mortality of adult B. amphitrite was determined in laboratory aquaria set up at 43, 60, 70, 75, 80, 90, and 100 g l-1. Salinities were achieved in two ways: by salt addition and by evaporation. Calculated 12-day LC50 values were 83 g l-1 when salinities were achieved through salt addition and 89 g l-1 when salinities were achieved through evaporation. Differences in B. amphitrite mortality between the two methods illustrate the importance of producing experimental salinity levels carefully. B. amphitrite is expected to become extinct within the Salton Sea when the salinity reaches 70–80 g l-1 and to show marked declines in abundance at salinities as low as 50 g l-1. This revised version was published online in July 2006 with corrections to the Cover Date.