Formation of the enveloping layer of the chum salmon egg in isotonic salt solutions

After stimulation in a hypotonic solution (9.4 mOsm kg⁻¹), inseminated eggs of the chum salmon Oncorhynchus keta initiate cleavages in isotonic salmon Ringer's solution (267.3 mOsm kg⁻¹) containing 3.2 mM Ca²⁺ ions. Blastomeres of these eggs, however, separate from each other and the enveloping layer is not observed at the blastula stage. An increase in external divalent cations rescues the separation; the concentration of CaCl₂ in the external medium should be 25 mM or more to induce close contact of blastomeres and the formation of an enveloping layer in isotonic salt solutions. The effectiveness of Ca²⁺ ions can be substituted by Mg²⁺, Sr²⁺ and Zn²⁺ ions; the same results are obtained in isotonic MgCl₂ and SrCl₂ solutions (100 mM) or in isotonic salmon Ringer's solution containing Zn ions (6.2 mM). The close contact of blastomeres and the formation of an enveloping layer are also observed in a low Ca²⁺ concentration (< 0.1 mM) in a hypotonic salt solution (9.4 mOsm kg⁻¹). The Ca²⁺ level in the external medium to induce the enveloping layer formation seems to be correlated with the salinity of the incubation medium. It is suggested that adhesion molecules on the surface of blastomeres in the chum salmon eggs are different in properties from those found in sea urchin and other fish species.     

Control of Nitrate Uptake by Phloem-Translocated Glutamine in Zea mays L. Seedlings

Abstract: The putative role of glutamine, exported from leaves to roots, as a negative feedback signal for nitrate uptake was investigated in Zea mays L. seedlings. Glutamine (Gln) was supplied by immersion of the tip-cut leaves in a concentrated solution. Nitrate (NO₃⁻) uptake was measured by its depletion in amino acid-free medium. The treatment with Gln resulted in a strong inhibition of nitrate uptake rate, accompanied by a significant enrichment of amino compounds in root tissue. The effect of N-availability on NO₃⁻ uptake was determined in split-root cultures. The plants were subjected to complete or localized N supply. Inducible NO₃⁻ uptake systems were also induced in N-deprived roots when the opposite side of the root system was supplied with KNO₃. The inhibitory effect of Gln was unaffected by localized N supply on one side of the split-root. The potential role of Gln in the shoot-to-root control of NO₃⁻ uptake is discussed.     

Seed dormancy in Avena fatua: Interacting effects of nitrate, water and seed coat injury

In experiments conducted under controlled conditions. KNO₃ (50 or 100 m M ) promoted germination of a dormant strain (AN 474) of Avena fatua when either one or two holes were pierced in the lower (adaxial) surface of the caryopsis in contact with the nitrate solution. Germination was increased by increasing either the KNO₃ concentration or the number of holes in the seed coat. The germination response induced by the application of water to a hole pierced in the upper surface of the caryopsis was. increased by pre-treatment of the intact caryopsis with KNO₃. Treatment with either 50 or 100 m M KNO₃ caused a transient reduction in embryo water content of intact cary-opses, but increased the nitrate and amino- N content of pierced caryopses prior to germination. Supplying a 100 m M solution of KNO₃ to pierced caryopses reduced the total water potential and osmotic potential of the embryo, and increased its pressure potential by the same amount as an equimolar solution of KC1; however, while both treatments promoted germination, the KNO₃ induced more rapid germination than the KCI. Both treatments also increased the K⁺ content of the embryo, the KNO₃ again having the greater effect. These results are consistent with the hypothesis, based on our previous investigations, that KNO₃ promotes germination of dormant caryopses by accumulating in the embryo where it acts osmotically to increase water uptake. It is also postulated, that, in contrast to KCI, KNG₃ may combine an osmotic effect on water uptake with a nutritional effect on protein synthesis.     

Effect of nitrate on nitrogen fixation and nodule carbohydrate and organic acid concentrations in pea mutants deficient in nitrate reductase

Nitrate inhibits symbiotic N₂ fixation and a number of hypotheses concerned with NO₃⁻ assimilation have been suggested to explain this inhibition. These hypotheses were tested using a pea ( Pisum sativum L. cv. Juneau) with normal nitrate reductase NR; (EC 1,6,6,4) activity and two mutants of cv. Juneau, A317 and A334, with impaired NR activity. The plants were inoculated with three strains of Rhizobium leguminosarum and grown for 3 weeks in N-free medium, followed by 1 week in medium supplemented with 0, 5 or 10 m M KNO₃ before harvesting. NO₃ was taken up at comparable rates by the parent and the mutants and accumulated in leaf and stem tissue of the latter. Acetylene reduction rates were inhibited similarly in both the parent and mutants in the presence of KNO₃ but there were differences among rhizobial strains. Starch concentration of the nodules decreased by 46% in the presence of KNO₃ and there were differences among rhizobial strains but not among pea genotypes. Malate and succinate accumulated in nodules in the presence of KNO₃. These data are not consistent with the photosynthate deprivation hypothesis as a primary mechanism for NO₃ inhibition of N₂ fixation since NO₃ affected the nodule carbohydrate composition of all three pea genotypes in a similar manner. The lack of correlation between NR activity and NO₃ inhibition of N₂ fixation suggests that NO₃ assimilation may be only indirectly involved in the inhibition phenomenon.     

Anion uptake in maize roots: Interactions between chlorate and nitrate

Effects of nitrate, chloride and chlorate ions upon nitrate and chlorate uptake by roots of maize ( Zea mays L., cv. B73) seedlings were examined. Net nitrate uptake, ³⁶ClO₃⁻ influx and ³⁶Cl⁻ influx (the latter two in a background of 0.5 m M KNO₃) displayed similar pH profiles with optima at pH 5.5 and below. External, non-labeled chloride had little effect on the accumulation of ³⁶ClO₃⁻ (both in 5 h and 20 min uptake assays), while nitrate and chlorate had almost identical, marked inhibitory effects. Nitrate pretreatment caused an apparent induction of both ³⁶ClO₃⁻ and ¹⁵NO₃⁻ uptake activities. After 5 h of treatment in nitrate, the uptake activities of chloride- and chlorate-pretreated plants increased to that of nitrate-pretreated plants. During 6 h exposure to chlorate, ³⁶ClO₃⁻ uptake activity of nitrate-pretreated plants decreased to that of chlorate- and chloride-pretreated plants. The results support the existence of a shared nitrate/chlorate transport system in maize roots which is not inhibited by external chloride, and which is induced by nitrate, but not by chlorate or chloride. The suggestion is made that selection of chlorate-resistant mutants of maize can identify nitrate uptake as well as nitrate reductase mutants.     

The nitrite oxidizing system of Nitrobacter winogradskyi

Abstract Cytochrome components which participate in the oxidation of nitrite in Nitrobacter winogradskyi have been highly purified and their properties studied in detail. Cytochrome a ₁ c ₁ is an iron-sulphur molybdoenzyme which has haems a and c and acts as a nitrite-cytochrome c oxidoreductase. Cytochrome c -550 is homologous to eukaryotic cytochrome c and acts as the electron mediator between cytochrome a ₁ c ₁ and aa ₃-type cytochrome c oxidase. The oxidase is composed of two kinds of subunits, has two molecules of haem a and two atoms of copper in the molecule, and oxidizes actively eukaryotic ferrocytochrome c as well as its own ferrocytochrome c -550. Further, a flavoenzyme has been obtained which has transhydrogenase activity and catalyses reduction of NADP⁺ with benzylviologen radical. This enzyme may be responsible for production of NADPH in N. winogradskyi . The electron transfer against redox potential from NO₂⁻ to cythochrome c could be pushed through prompt removal by cytochrome aa ₃ of H⁺ formed by the dehydrogenation of NO₂⁻+ H₂O. As cytochrome c in anaerobically kept cell-free extracts is rapidly reduced on addition of NO₂⁻, a membrane potential does not seem necessary for the reduction of cytochrome c by cytochrome a ₁ c ₁ with NO₂⁻ in vivo.     

Simultaneous Measurement of Nitrite and Nitrate Levels as Indices of Nitric Oxide Release in the Cerebellum of Conscious Rats

Abstract: We examined the modulation of nitric oxide production in vivo by measuring levels of nitrite (NO₂⁻) and nitrate (NO₃⁻) in the dialysate of the cerebellum in conscious rats, by using an in vivo brain microdialysis technique. The levels of both NO₂⁻ and NO₃⁻ were decreased by the intraperitoneal injection of N ^G-nitro- l -arginine methyl ester, an inhibitor of nitric oxide synthase, whereas N ^G-nitro- d -arginine methyl ester had no effect. l -Arginine by itself increased NO₂⁻ and NO₃⁻ levels and diminished the reduction of their levels caused by N ^G-nitro- l -arginine methyl ester. Direct infusion of l -glutamate, N -methyl- d -aspartate, or KCl into the cerebellum through a dialysis probe resulted in an increase in NO₂⁻ and/or NO₃⁻ levels. The effects of N -methyl- d -aspartate and KCl were dependent on extracellular calcium. Furthermore, the stimulatory effects of l -glutamate and N -methyl- d -aspartate were inhibited by N ^G-nitro- l -arginine methyl ester and (±)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), an N -methyl- d -aspartate receptor antagonist. These results suggest that NO₂⁻ and NO₃⁻ levels may be related to nitric oxide production in vivo.     

Membrane inlet mass spectrometric analysis of N-isotope labelling for aquatic denitrification studies

Abstract: Techniques are described for measuring the isotope distribution in dissolved nitrate and N₂ using membrane inlet mass spectrometry, which allows several gases to be measured in a water sample without the need for any separation steps. The isotope distribution in dissolved nitrate was measured using denitrifying Pseudomonas nautica to reduce the nitrate to N₂ which was then measured by mass spectrometry. Pseudomonas nautica NCIMB 1967 was easily grown in nitrate-limited continuous culture minimising intra- or extracellular nitrate or nitrite pools, and the bioassay was tolerant of a range of salinities. The precision of the bioassay when measuring samples with high ¹⁵NO₃⁻ contents (0.5 μmol) was 0.05 atom%; with 0.1 μmol ¹⁵NO₃⁻, the precision was around 0.2 atom%. Differences in labelling of N₂ in preserved samples obtained from ¹⁵NO₃⁻ incubations of water-covered sediment cores were measured on parallel samples with membrane inlet MS and GC-MS. The membrane inlet technique was accurate but the precision on ratio measurements was lower than by GC-MS.     

Induction of nitrate reductase in needles of Scots pine seedlings by NOX and NO3−

The possibility to induce nitrate reductase (NR; EC 1.6.6.2) in needles of Scots pine ( Pinus sylvestris L.) seedlings was studied. The NR activity was measured by an in vivo assay. Although increased NR activities were found in the roots after application of NO₃⁻, no such increase could be detected in the needles. Detached seedlings placed in NO₃⁻ solution showed increasing NR activities with increasing NO₃⁻ concentrations. Exposure of seedlings to NO_x (70–80 ppb NO₂ and 8–12ppb NO) resulted in an increase of the NR activity from 10–20 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹ to about 400 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹. This level was reached after 2–4 days of exposure, thereafter the NR activity decreased to about 200 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹. Analyses of free amino acids showed low concentrations of arginine and glutamine in NO_x-fumigated seedlings compared to corresponding controls.     

Methane-dependent nitrate production by a microbial consortium enriched from a cultivated humisol

Abstract A consortium was enriched from a humisol incubated with 3.6 kPa CH₄ and NH₄⁺. This consortium oxidized NH₄⁺ to NO₂⁻ and NO₃⁻ (NO₃⁻/NO₂⁻ ratio about 20) with smaller amounts of N₂O. This oxidation stopped in the stationary phase after depletion of CH₄. CH₃OH or CO₂ did not support oxidation. Growth and resting cell experiments suggested that nitrification was associated with methanotrophic activity and that chemoautotrophic nitrifiers were absent.     

The inhibition of the carbon concentrating mechanism of the green alga Chlorella saccharophila by acetazolamide

The effects of the carbonic anhydrase (CA) inhibitors acetazolamide (AZ) and dextran-bound sulfonamide (DBS) on HCO₃⁻-dependent O₂ evolution in Chlorella saccharophila were evaluated. Addition of 4 μ M AZ or 0.4 mg ml⁻¹ DBS to photosynthesizing cells reduced the O₂ evolution rate at low dissolved inorganic carbon (DIC) concentration, decreased the size of the intracellular acid-labile carbon pool, and decreased the apparent affinity of the cells for DIC. Measurement of the whole-cell affinity of cells for CO₂ and HCO₃⁻ in the presence and absence of inhibitors indicated that active HCO₃⁻ transport was inhibited by AZ and DBS. The inhibition of HCO₃⁻ transport was independent of the inhibition of external and internal CA. These results suggest that the active uptake of HCO₃⁻ occurs initially by the interaction of HCO₃⁻ and a CA-like transporter.     

THE EFFECT OF HCO3− ON THE SWELLING AND ION UPTAKE OF MONKEY CEREBRAL CORTEX UNDER CONDITIONS OF RAISED EXTRACELLULAR POTASSIUM

Abstract— Increasing the HCO₃⁻ concentration of incubation media containing raised K⁺ concentrations (18-71 mm) caused increased swelling of monkey cerebral cortex slices. This swelling was mainly associated with increased intracellular levels of Na⁺ and Cl⁻ ions. It was independent of the type of buffer used and was not a result of the increased Na⁺ concentration in the media due to added HCO₃⁻ or the increased osmolarity. The levels also were unaffected by alteration of the pH in the range of 6·9- 7·8 or pCO₂ in the range of 3–81 mm Hg.
The anatomical locus of this HCO₃⁻ stimulated swelling appeared in electron micrographs to be an expanded glial compartment. The significance of these findings is discussed in terms of the transport processes involved and the role of glial cells in maintaining correct cerebro-cortical ion balances under normal and pathological conditions.     

Activation of Xenopus Eggs by an Extract of Cynops Sperm

An extract obtained from Cynops sperm induced the activation of both Cynops and Xenopus eggs with accompanying changes in the potential of the egg membrane that were quite similar to those caused by the Cynops sperm. The activation-inducing properties of the extract were abolished by treatment with proteinase K or by heating (60°C, 15 min) and were associated with a protease activity against peptidyl Arg-MCA substrates. The activation of Xenopus eggs by the extract was inhibited by those substrates, or by protease inhibitors, aprotinin or leupeptin. The protease activity was localized in the acrosomal region of Cynops sperm. The activation of Xenopus eggs by the extract was prevented when the exterior concentration of Ca²⁺ions, [Ca²⁺]₀, was reduced to 1.5 μM, but it was enhanced when [Ca²⁺]₀ was increased to 340 μM. The activation of Xenopus eggs by the extract was not affected by positive clamping when [Ca²⁺]₀ was 340 μM. These results suggest that the sperm extract contains a protease that causes an increase in the influx of Ca²⁺ions that results in voltage-insensitive activation of the egg.     

In vitro carbon dioxide excretion from erythrocytes of two species of Antarctic fishes and its inhibition by catecholamines

This study was designed to investigate whether the blood of Pagothenia borchgrevinki , exhibits a Haldane effect, and whether activation of a Na⁺/H⁺ antiporter increases transport of intracellular protons and Bohr protons out of the erythrocytes resulting in inhibition of CO₂ excretion in both P. borchgrevinki , and Dissostichus mawsoni. When carbon dioxide dissociation curves were determined from blood samples pooled from three fish under oxygenated and deoxygenated conditions a Haldane effect was observed. Using an in vitro , CO₂ excretion assay, the rate of HCO₃⁻ dehydration was determined on blood and plasma equilibrated under an N₂atmosphere then rapidly oxygenated with air in the presence of 10⁻⁵ M noradrenaline or acetazolamide (10⁰⁴M). Whole blood and plasma from P. borchgrevinki , and D. mawsoni , were equilibrated with 0·5% CO₂ in air and assayed in the presence of 10⁻⁵ M noradrenaline. Erythrocyte CO₂ excretion rates were depressed significantly by noradrenaline in both species. The whole blood HCO₃⁻ dehydration rate was depressed significantly following rapid oxygenation in the presence of acetazolamide indicating that the pathway of CO₂ excretion included activation of intracellular carbonic anhydrase and an adrenergic receptor.     

The behaviour of Enterobacteriaceae in Manchego cheese made from raw ewes' milk treated with hydrogen peroxide, potassium nitrate or potassium nitrite

Growth of Enterobacteriaceae, coliforms and faecal coliforms in Manchego cheese during the first 24 h after manufacture was retarded by treatment of milk with 0.1 g 1^-1 H₂O₂ compared to growth in control cheese made from untreated milk. Moreover, the decrease in their numbers during cheese ripening was accelerated by the H₂O₂ treatment of milk. In contrast, KNO₃ or KNO₂ addition to milk enhanced the growth of these micro-organisms during cheese manufacture and favoured their survival during ripening.     

New pathways of phagocyte activation: the coupling of receptor-linked phospholipase D and the role of tyrosine kinase in primed neutrophils

Abstract Protein kinase C (PKC) appears to have a central role in the O₂⁻ response of neutrophils following stimulation of membrane receptors. The second messenger, diacylglycerol (DG), that activates PKC is derived from membrane phospholipids via activation of phosphatidylinositol 4,5-bisphosphate (PIP₂)-phospholipase C (PLC) and phospholipase D (PLD), with the latter pathway being more prominent in primed cells. In resting cells receptor coupling of PLD is through a G-protein. Priming brings a cytoplasmic tyrosine kinase into the transducer sequence which, through protein phosphorylation, increases the efficiency of coupling between membrane receptors and PLD. Phosphatidic acid (PA), the initial product of the PLD pathway, also appears to act as a second messenger by directly activating the NADPH oxidase responsible for generating O₂⁻. Interconversion of PA and DG by phosphatidate phosphohydrolase and DG kinase determines which of these second messengers has the dominant role.     

Effect of glutamine on the induction of nitrate reductase

Nitrate reductase (NR. EC 1.6.6.1/2) is a substrate inducible enzyme that could be repressed by its end product glutamine or amino acids. To test this hypothesis, 6-day-old maize seedlings ( Zea mays cv. W64A × W182E) were grown hydroponically in a 1/10 strength Hougland's salt solution modified to contain no nitrogen. Previous experiments had established that after a 24-h induction with NO₃⁻ (5 mM KNO₃⁻) the level of NR activity and protein had reached a constant level. In the present experiments when glutamine (5 mM) was included together with NO₃⁻, there was a significant reduction in NR activity (34% of the control values). NR protein and NR mRNA accumulation in the root. In the shoot, on the other hand, glutamine additions had little or no effect on the levels of either NR activity (81% of control) or NR protein. Inhibition of glutamine synthetase by methionine sulfoximine (MSX) resulted in reduced levels of glutamine in both root and shoot tissues. Contrary in our prediction, however, it had no effect on NR activity and mRNA content in roots. In the shoot, on the other hand, there was a marked reduction of NR activity (34% of the control value) and NR protein, but no apparent effect on NR mRNA. When detached shoots were treated with MSX and other inhibitors of glutamine synthetase (tabtoxinine-β-lactam or phosphinothricin) the induction of NR activity by NO₃⁻ was also inhibited. Glutamine additions 15 or 50 mM to detached shoots had essentially no effect on the induction of NR activity (90% of control). These results demonstrate that the influence of glutamine and MSX on the induction of NR in maize root and shoot tissues, respectively, is very different.     

Properties of Intracellular Hatching enzyme in the Embryos of the Sea Urchin, Hemicentrotus pulcherrimus

The intracellular hatching enzyme was confirmed to be particulate-bound in the sea urchin, Hemicentrotus pulcherrimus. The enzyme was solubilized most effectively by sonication in buffer containing 12.5 mM CaCl₂, and 0.5 M KCl. The intracellular hatching enzyme is suggested to be activated by an antipain- or elastatinal-susceptible protease(s) on its solubilization. Since the intracellular hatching enzyme solubilized in the absence of protease inhibitors was inhibited by phenylmethylsulfonyl fluoride (PMSF) and chymostatin, the active hatching enzyme is concluded to be a chymostatin-sensitive serine protease. The enzyme required CaCl₂, and KCl or NaCl for both stability and activity. The preference of the enzyme of anions as sodium salts was as follows: Cl⁻ > NO₃⁻ > I⁻ > SCN⁻. The apparent molecular weights of the intracellular hatching enzyme (IHE) and the hatching enzyme secreted from the blastula with or without the fertilization envelope (SHE or dSHE) were estimated as 89,000, 135,000, 80,000, respectively. On incubations with isolated fertilization envelopes as an enzyme substrate, the apparent molecular weights of dSHE and IHE increased to 128,000 and 105,000, respectively.     

Impact of gaseous nitrogen deposition on plant functioning

Dry deposition of NH₃ and NO_x (NO and NO₂) can affect plant metabolism at the cellular and whole-plant level. Gaseous pollutants enter the plant mainly through the stomata, and once in the apoplast NH₃ dissolves to form NH₄⁺, whereas NO₂ dissolves to form NO₃⁻ and NO₂⁻. The latter compound can also be formed after exposure to NO. There is evidence that NH₃-N and NO_x-N can be reversibly stored in the apoplast. Temporary storage might affect processes such as absorption rate, assimilation and re-emission. Once formed, NO₃⁻ and NO₂⁻ can be reduced, and NH₄⁺ can be assimilated via the normal enzymatic pathways, nitrate reductase (NR), nitrite reductase and the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle. Fumigation with low concentrations of atmospheric NH₃ increases in vitro glutamine synthetase activity, but whether this involves both or only one of the GS isoforms is still an open question. There seems to be no correlation between fumigation with low concentrations of NH₃ and in vitro GDH activity. The contribution of atmospheric NH₃ and NO₂ deposition to the N budget of the whole plant has been calculated for various atmospheric pollutant concentrations and relative growth rates ( RGRs ). It is concluded that at current ambient atmospheric N concentrations the direct impact of gaseous N uptake by foliage on plant growth is generally small.     

Reduction of nitrate in the perennial tissues of aerial parts of Alnus glutinosa

In vivo nitrate reductase (NR, EC 1.6.6.1.) activity was measured in leaves, branches and trunk of field-grown Alnus glutinosa (L.) Gaertn. All of the assayed tissues enzymatically reduced nitrate with a decreasing activity [μmol NO₂⁻ (g dry weight)⁻¹ h⁻¹] in the order: leaves > branch bark > inner branch tissues > trunk xylem. The NR activity of the various tissues of excised branches was inhibited by tungstate added to the transpiration stream. Part of the nitrate added to the feeding solution (0.2, 0.5 or 1 m M KNO₃) of excised branches disappeared during its transport via the transpiration stream in the perennial tissues. This disappearance was enzymatic since it was decreased by tungstate.
No evidence was obtained for the presence of nitrate in natural xylem sap nor for a significant correlation between nitrate content of soil and leaf NR activity. These results indicate that in the field-grown black alder, the nitrate not reduced in the roots could be reduced in the perennial tissues of aerial parts. Since the leaf NR activity does not reflect the actual in situ nitrate reduction, the existence of a constitutive NR activity in Alnus leaves is suggested.