Release of NO by a nitrosyl complex upon activation by the mitochondrial reducing power

The reaction of trans-[Ru(NH(3))(4)P(OEt)(3)NO](3+) and mitochondria was investigated through differential pulse polarography and fluorimetry. The nitrosyl complex undergoes one-electron reduction centered on the NO ligand site. The reaction between the mitochondrial reductor and trans-[Ru(NH(3))(4)P(OEt)(3)NO](3+) exhibits a second order specific rate constant calculated as k=2 x 10(1) M(-1) s(-1). The reduced species, trans-[Ru(NH(3))(4)P(OEt)(3)NO](2+), quickly releases NO, yielding trans-[Ru(NH(3))(4)P(OEt)(3)H(2)O](2+). The low toxicities of both trans-[Ru(NH(3))(4)P(OEt)(3)(NO)](2+) and trans-[Ru(NH(3))(4)P(OEt)(3)H(2)O](2+) and its ability to release NO after reductive activation in a biological medium make the nitrosyl compound a useful model of a hypotensive drug.     

trans-[Ru(NO)(NH3)4(py)](BF4)3.H2O encapsulated in PLGA microparticles for delivery of nitric oxide to B16-F10 cells: Cytotoxicity and phototoxicity

The NO donor trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3).H(2)O (py=pyridine) was loaded into poly-lactic-co-glycolic acid (PLGA) microparticles using the double emulsification technique. Scanning electron microscopy (SEM) and dynamic light scattering revealed that the particles are spherical in shape, have a diameter of 1600nm, and have low tendency to aggregate. The entrapment efficiency was 25%. SEM analysis of the melanoma cell B16-F10 in the presence of the microparticles containing the complex trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3).H(2)O (pyMP) showed that the microparticles were adhered to the cell surface after 2h of incubation. The complex with concentrations lower than 1x10(-4)M did not show toxicity in B16-F10 murine cells. The complex in solution is toxic at higher concentrations (>1x10(-3)M), with cell death attributed to NO release following the reduction of the complex. pyMP is not cytotoxic due to the lower bioavailability and availability of the entrapped complex to the medium and its reducing agents. However, pyMP is phototoxic upon light irradiation. The phototoxicity strongly suggests that cell death is due to NO release from trans-[Ru(NO)(NH(3))(4)(py)](3+). This work shows that pyMP can serve as a model for a drug delivery system carrying the NO donor trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3).H(2)O, which can release NO locally at the tumor cell by irradiation with light only.     

Microbial decomposition of wood in streams: distribution of microflora and factors affecting [C]lignocellulose mineralization

The distribution and lignocellulolytic activity of the microbial community was determined on a large log of Douglas fir (Pseudotsuga menziesii) in a Pacific Northwest stream. Scanning electron microscopy, plate counts, and degradation of [C]lignocelluloses prepared from Douglas fir and incubated with samples of wood taken from the surface and within the log revealed that most of the microbial colonization and lignocellulose-degrading activity occurred on the surface. Labeled lignocellulose and surface wood samples were incubated in vitro with nutrient supplements to determine potential limiting factors of [C]lignocellulose degradation. Incubations carried out in a nitrogenless mineral salts and trace elements solution were no more favorable to degradation than those carried out in distilled water alone. Incubations supplemented with either (NH(4))(2)SO(4) or organic nitrogen sources showed large increases in the rates of mineralization over incubations with mineral salts and trace elements alone, with the greatest effect being observed from an addition of (NH(4))(2)SO(4). Subsequent incubations with (NH(4))(2)SO(4), KNO(3), and NH(4)NO(3) revealed that KNO(3) was the most favorable for lignin degradation, whereas all three supplements were equally favorable for cellulose degradation. Supplementation with glucose repressed both lignin and cellulose mineralization. The results reported in this study indicate that nitrogen limitation of wood decomposition may exist in streams of the Pacific Northwest. The radiotracer technique was shown to be a sensitive and useful tool for assessing relative patterns of lignocellulose decay and microbial activity in wood, along with the importance of thoroughly characterizing the experimental system before its general acceptance.     

大豆根瘤菌HH103菌株培养基的筛选与优化

以快生型大豆根瘤菌HH103菌株为供试菌株,采用单因素碳氮源利用试验和正交设计试验,确定最佳培养基及其配方。结果表明：该菌株在YMA中生长良好,最佳碳源为蔗糖,最佳氮源为酵母膏,最佳培养基成分配方（g/L）：蔗糖11,酵母膏0.9,K2HPO4 0.5,MnSO4 0.005,CaCl2 0.1,KH2PO4 0.5,MgSO4 0.2,KNO30.77,（NH4）2HPO4 0.33,FeCl3 0.005,pH 7.2。     

Impact of nitrogen and phosphorus on [C]lignocellulose decomposition by stream wood microflora

Nutritional and physical factors affecting the decomposition of [C]lignocellulose prepared from Douglas fir (Pseudotsuga menziesii) were examined by incubating the labeled substrate with homogenized surface wood scrapings obtained from a Douglas fir log in a Pacific Northwest stream. Incubations were conducted in distilled water, in stream water collected from four different sources, or in a defined mineral salts solution with or without supplemental N (KNO(3)). Decomposition rates of [C]lignocellulose, as measured by CO(2) evolution, were greater in each of the four filter-sterilized sources of stream water than in distilled water alone. Decomposition experiments conducted in stream water media with the addition of defined mineral salts demonstrated that [C]cellulose decomposition was stimulated 50% by the addition of either KNO(3) or KH(2)PO(4)/K(2)HPO(4) and further enhanced (167%) by a combination of both. In contrast, [C]lignin decomposition was stimulated (65%) only by the addition of both N and P. Decomposition of [C]lignocellulose was greatest when supplemental KNO(3) was supplied in concentrations of at least 10.0 mg of N liter but not increased further by higher concentrations. The decomposition of [C]lignocellulose increased as the incubation temperature was raised and NO(3)-N supplementation further increased these rates between three-and sevenfold over the range of temperatures examined (5 to 22 degrees C). Accumulation of NH(4) (2 to 4 mg of N liter) was always observed in culture filtrates of incubations which had been supplemented with KNO(3), the quantity being independent of NO(3) concentrations >/= 10 mg of N liter. The role of supplemental NO(3) in the decomposition of [C]lignocellulose is discussed in relation to wood decomposition and the low concentrations of N found in stream ecosystems of the Pacific Northwest.     

Preparation and characterization of activated carbon produced from rice straw by (NH4)2HPO4 activation

Effects of different pretreatment protocols in (NH(4))(2)HPO(4) activation of rice straw on porous activated carbon evolution were evaluated. The pore structure, morphology and surface chemistry of obtained activated carbons were investigated by nitrogen adsorption, scanning electron microscopy and Fourier transform infrared spectroscopy. It was found that pretreatment combining impregnation with (NH(4))(2)HPO(4) and preoxidation could significantly affect the physicochemical properties of prepared activated carbons. The apparent surface area and total pore volume as high as 1154 m(2)/g and 0.670 cm(3)/g were obtained respectively, when combined process of impregnation followed by preoxidation at 200°C and activation at 700°C was carried out. Meanwhile, the activated carbon yield and maximum methylene blue adsorption capacity up to 41.14% and 129.5 mg/g were achieved, respectively. The results exhibited that (NH(4))(2)HPO(4) could be an effective activating agent for producing activated carbons from rice straw.     

Deposition of ammonium and nitrate in the roots of maize seedlings supplied with different nitrogen salts

This study measured total osmolarity and concentrations of NH(4)(+), NO(3)(-), K(+), soluble carbohydrates, and organic acids in maize seminal roots as a function of distance from the apex, and NH(4)(+) and NO(3)(-) in xylem sap for plants receiving NH(4)(+) or NO(3)(-) as a sole N-source, NH(4)(+) plus NO(3)(-), or no nitrogen at all. The disparity between net deposition rates and net exogenous influx of NH(4)(+) indicated that growing cells imported NH(4)(+) from more mature tissue, whereas more mature root tissues assimilated or translocated a portion of the NH(4)(+) absorbed. Net root NO(3)(-) influx under Ca(NO(3))(2) nutrition was adequate to account for pools found in the growth zone and provided twice as much as was deposited locally throughout the non-growing tissue. In contrast, net root NO(3)(-) influx under NH(4)NO(3) was less than the local deposition rate in the growth zone, indicating that additional NO(3)(-) was imported or metabolically produced. The profile of NO(3)(-) deposition rate in the growth zone, however, was similar for the plants receiving Ca(NO(3))(2) or NH(4)NO(3). These results suggest that NO(3)(-) may serve a major role as an osmoticant for supporting root elongation in the basal part of the growth zone and maintaining root function in the young mature tissues.     

Production of l-Glutamine by a Penicillin-Resistant Mutant of Flavobacterium rigense

To establish a practical method for the fermentative production of l-glutamine, cultural conditions for the accumulation of a large amounts of l-glutamine were investigated by using Flavobacterium rigense 703, which was previously reported by us as a l-glutamine-producing mutant. As a result, a yield of 25 mg of l-glutamine per ml was obtained after a 48-h cultivation in a medium containing glucose, yeast extract, (NH(4))(2)-fumarate, KH(2)PO(4), K(2)HPO(4), MgSO(4).7H(2)O, and CaCO(3) (pH 6.4). Accumulation of l-glutamine was dependent upon the concentration of (NH(4))(2)-fumarate, and a suboptimum growth at a relatively high concentration of (NH(4))(2)-fumarate was essential for the maximum production of l-glutamine. At the optimum conditions, glutamic acid was formed as a by-product at a concentration of less than 1 mg/ml, but accumulation of the other amino acids was negligible. The product was isolated from the culture broth and readily purified by anion-exchange chromatography. The pure crystals of l-glutamine obtained in an 80% yield were optically and chromatographically pure.     

Effect of Different Ammonium Salts on Formation of Maize Pollen Embryoid

There are more than 80% embryoids among the induced masses in anther culture; different ammonium salts produced different effects on the formation of embryoids, and the effect of NH4H2PO4 is better than the others, within the range of the concentrations used. When the combinations of KNO3 With NH4H2PO4 act as the nitrogen source the frequencies of formation of embryoids and calli increase with the increase of NO3–/NH4+ ratio, at NO3–/NH4+ = 6:1, the frequency is the highest, but at 8:1, it decreases. In the treatments of lower total nitrogen (22.5–37.5 mN/L), the induction frequency is higher (3%–12%) than those of higher total nitrogen (45–60 mN/L). It has also been discussed that the different materials responded to the same ammonium salt, that the same material responded to different NO3–/NH4+ ratio.     

NO3- and ClO3- fluxes in the chl1-5 mutant of Arabidopsis thaliana. Does the CHL1-5 gene encode a low-affinity NO3- transporter?

The CHL1 gene is considered to encode a low-affinity transport system (LATS) for NO3- in Arabidopsis thaliana (Y.-F. Tsay, J.I. Schroeder, K.A. Feldmann, N.M. Crawford [1993] Cell 72: 705-713). However, the anticipated reduced NO3- uptake by the LATS associated with loss of CHL1 gene activity in chl1-5 deletion mutants was evident only when plants were grown on NH4NO3. When KNO3 was the sole N source, NO3- accumulation and short-term tracer influx (using 13NO3- and 15NO3-) in leaves and roots of wild-type and mutant plants were essentially identical. Nevertheless, root uptake of 36CIO3- by the LATS and CIO3- accumulation in roots and shoots of mutant plants were significantly lower than in wild-type plants when grown on KNO3. One explanation for these results is that a second LATS is able to compensate for the chl1-5 deficiency in KNO3-grown plants. Growth on NH4NO3 may down-regulate the second LATS enough that the anticipated difference in NO3- uptake becomes apparent.     

The effect of nitrogen nutrition on cluster root formation and proton extrusion by Lupinus albus

Nitrogen nutrition can influence cluster root formation in many wild species, but the effect of N form on cluster root formation and root exudation by white lupin is not known. In a solution culture study, we examined the effect of N nutrition (ammonium, nitrate, both or N2 fixation) on cluster root formation and H+ extrusion by white lupin plants under deficient and adequate P supply. The number of cluster roots increased greatly when plants were supplied with I microM P compared with 50 microM P, the increase being 7.8-fold for plants treated with (NH4)2SO4, 3-fold for plants treated with KNO3 and NH4NO3, and 2-4-fold for N2-fixing plants. Under P deficiency. NH4+-N supply resulted in production of a greater number and biomass of cluster roots than other N sources. Dry weight of cluster roots was 30 % higher than that of non-cluster roots in P-deficient plants treated with (NH4)2SO4 and NH4NO3. In plants treated with sufficient P (50 microM), the weight of non-cluster roots was approx. 90 % greater than that of cluster roots. Both total (micromol per plant h(-1)) and specific (micromol g(-1) root d. wt h(-1)) H+ extrusions were greatest from roots of plants supplied with (NH4)2SO4, followed by those supplied with NH4NO3 and N2 fixation, whereas plants receiving KNO3 had negative net H+ extrusion between the third and fifth week of growth (indicating uptake of protons or release of OH- ions). The rate of proton extrusion by NH4+-N-fed plants was similar under P-deficient and P-sufficient conditions. In contrast, proton exudation by N2-fixing plants and KNO3-treated plants was ten-fold greater under P deficiency than under P sufficiency. In comparison with P deficiency, plants treated with 50 microM P had a significantly higher concentration of P in roots, shoots and youngest expanded leaves (YEL). Compared with the N2 fixation and KNO3 treatments, total N concentration was highest in roots, shoots and YEL of plants supplied with (NH4)2SO4 and NH4NO3, regardless of P supply. Under P deficiency, K concentrations in roots decreased at all N supplies, especially in plants treated with (NH4)2SO4 and NH4NO3, which coincided with the greatest H+ extrusion at these P and N supplies. In conclusion, NH4-N nutrition stimulated cluster root formation and H+ extrusion by roots of P-deficient white lupin.     

Changes in the activity and tertiary structure of beta-amylase from wheat seeds under the effect of salts]

Changes are observed of 1-anylinonaphtalene-8-sulphonate probe fluorescence intensity, connected with beta-amylase in the presence of Ca(NO3)2, Mg(NO3)2, KNO3, NH4NO3 and (NH4)2SO4 at a concentration range within 10(-4)--1 M. Considerable decrease of the fluorescence intensity was observed under the addition of all the salts mentioned at concentration of 10(-3)--10(-4) M. A quantum yield increase of probe fluorescence was noted for Mg(NO3)2 (10(-4) M). High concentrations of Ca(NO3)2 and Mg(NO3)2 (0.25--1 M) also resulted in a sharp increase of the fluorescence intensity. Changes of beta-amylase activity took place simultaneously. The changes of the enzyme activity are suggested to be due to changes in the conformation of the enzyme protein under the effect of salts.     

Production of 2-Keto-l-Gulonic Acid from d-Glucose by Two-Stage Fermentation

A practical method for the production of calcium 2-keto-l-gulonate (an intermediate in the Reichstein synthesis of l-ascorbic acid) from d-glucose has been established by using a two-stage fermentation system. d-Glucose was first converted to calcium 2,5-diketo-d-gluconate by a mutant strain of Erwinia sp. in a medium containing d-glucose, corn steep liquor, (NH(4))(2)HPO(4), and CaCO(3). After a 26-h cultivation, 328.6 mg of calcium 2,5-diketo-d-gluconate per ml was obtained, with a 94.5% yield from d-glucose. This broth was used directly for the next conversion without removal of cells by treatment with sodium dodecyl sulfate. The stereospecific reduction of calcium 2,5-diketo-d-gluconate to calcium 2-keto-l-gulonate was performed with a mutant strain of Corynebacterium sp. When the cell growth reached a maximum (about 16 h) in a medium containing d-glucose, corn steep liquor, NaNO(3), KH(2)PO(4), and trace elements, NaNO(3) was added to the culture, and then the calcium 2,5-diketo-d-gluconate broth was fed over a period of about 50 h. Since the mutant strain requires a hydrogen donor for reduction, the calcium 2,5-diketo-d-gluconate broth was mixed with d-glucose before being fed. The results of four two-stage fermentations in 10-m conventional fermentors showed that an average of 106.3 mg of calcium 2-keto-l-gulonate per ml was obtained, with a 84.6% yield from d-glucose, the starting material of calcium 2,5-diketo-d-gluconate production. Calcium 2-keto-l-gulonate was stable in the broth. Neither 2-keto-d-gluconic acid nor 5-keto-d-gluconic acid was detected in the final broth.     

Novel process for the production of cellulolytic enzymes

A novel process for the production of extracellular carboxymethylcellulase (CMCase) and xylanase by fermentation under nonaseptic or nonsterile conditions is described. The fermentation process is carried out under very acidic conditions of pH 2.0 by using a acidophilic cellulolytic fungus. Microbial contamination is avoided or minimized to an insignificant level under this acid pH condition. The culture medium for this production consists of a carbon source from cellulosics or lignocellulosics, such as Na-CMC, xylan, Avicel cellulose, cellulose powder, alpha-cellulose, sawdust, etc., or a mixture of the forementioned together with simple ingredients such as (NH(4))(2)SO(4), K(2)HPO(4), MgSO(4) and NaNO(3). The fermentation is carried out at room temperature (28-30 degrees C), under aerobic conditions, and without controlling the pH. The CMCase and xylanase produced are stable under very simple storage conditions, such as in the fresh culture medium not containing the substrate for a period of 3 days, at any temperature from 0 to 30 degrees C. These extracellular enzymes have an optimum pH around 3, with the best range of pH from 2.0 to 3.6, for any temperature between 15 and 60 degrees C. The optimum temperatures are 55 degrees C for CMCase activity and 25-50 degrees C for xylanase activity, at any pH between 2.0 and 5.2. The apparent Michaelis constants Km are 2.6 and 1.5 mg/mL for CMCase and xylanase of the culture filtrate, respectively.     

Mitochondria permeabilization by a novel polycation peptide BTM-P1

Bacillus thuringiensis subsp. medellin is known to produce the Cry11Bb protein of 94 kDa, which is toxic for mosquito larvae due to permeabilization of the plasma membrane of midgut epithelial cells. Earlier we found that a 2.8-kDa novel peptide BTM-P1, which was artificially synthesized taking into account the primary structure of Cry11Bb endotoxin, is active against several species of bacteria. In this work we show that BTM-P1 induces cyclosporin A-insensitive swelling of rat liver mitochondria in various salt solutions but not in the sucrose medium. Inorganic phosphate and Ca(2+) significantly increased this effect of the peptide. The uncoupling action of BTM-P1 on oxidative phosphorylation was stronger in the potassium-containing media and correlated with a decrease of the inner membrane potential of mitochondria. In isotonic KNO(3), KCl, or NH(4)NO(3) media, a complete drop of the inner membrane potential was observed at 1-2 microg/ml of the peptide. The peptide-induced swelling was increased by energization of mitochondria in the potassium-containing media, but it was inhibited in the NaNO(3), NH(4)NO(3), and Tris-NO(3) media. All mitochondrial effects of the peptide were completely prevented by adding a single N-terminal tryptophan residue to the peptide sequence. We suggest a mechanism of membrane permeabilization that includes a transmembrane- and surface potential-dependent insertion of the polycation peptide into the lipid bilayer and its oligomerization leading to formation of ion channels and also to the mitochondrial permeability transition pore opening in a cyclosporin A-insensitive manner.     

Degradation of polyesters by a novel marine Nocardiopsis aegyptia sp. nov.: application of Plackett-Burman experimental design for the improvement of PHB depolymerase activity

This is the first report on the degradation of poly(3-hydroxybutyrate) (PHB), and its copolymers poly(3-hydroxyvalerate) P(3HB-co-10-20% HV) by Nocardiopsis aegyptia, a new species isolated from marine seashore sediments. The strain excreted an extracellular PHB depolymerase and grew efficiently on PHB or its copolymers as the sole carbon sources. The degradation activity was detectable by the formation of a transparent clearing zone around the colony on an agar Petri plate after 25 days, or a clearing depth under the colony in test tubes within 3 weeks. The previous techniques proved that the bacterium was able to assimilate the monomeric components of the shorter alkyl groups of the polymers. Nocardiopsis aegyptia hydrolyzed copolymers 10-20% PHBV more rapidly than the homopolymer PHB. The bacterial degradation of the naturally occurring sheets of poly(3-hydroxybutyrate), and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was observed by scanning electron microscopy (SEM). The samples were degraded at the surface and proceeded to the inner part of the materials. Clear morphological alterations of the polymers were noticed, indicating the degradative capability of the bacterium. Plackett-Burman statistical experimental design has been employed to optimize culture conditions for maximal enzyme activity. The main factors that had significant positive effects on PHB depolymerase activity of Nocardiopsis aegyptia were sodium gluconate, volume of medium/flask and age of inoculum. On the other hand, MgSO4.7H2O, KH2PO4, K2HPO4 and NH4NO3 exhibited negative effects. Under optimized culture conditions, the highest activity (0.664 U/mg protein) was achieved in a medium predicted to be near optimum containing (in g/L): PHB, 0.5; C6H11O7Na, 7.5; MgSO4.7H2O, 0.35; K2HPO4, 0.35; NH4NO3, 0.5; KH2PO4, 0.35; malt extract, 0.5 and prepared with 50% seawater. The medium was inoculated with 1% (v/v) spore suspension of 7 days old culture. Complete clarity of the medium was achieved after 3 days at 30 degrees C.     

Conversion of dechlorodauricumine into chlorinated alkaloids in Menispermum dauricum root culture

(15)N-Labeled dechlorodauricumine and dechloroacutumine were isolated from Menispermum dauricum roots cultured in a chloride-deficient medium, in which nitrogen-containing macro-components K(14)NO(3) and ((14)NH(4))(2)SO(4) were replaced by K(15)NO(3) and ((15)NH(4))(2)SO(4), respectively. These (15)N-labeled substrates were supplied independently to the roots cultured in a chloride-enriched medium. LC-ESI-MS analysis of alkaloids extracted from the roots, harvested 5 and 10 days after administering the (15)N-labeled substrates, revealed that the (15)N derived from dechlorodauricumine was much more effectively incorporated into chlorinated alkaloids than that derived from dechloroacutumine. These findings suggest that dechlorodauricumine is the principal precursor of the chlorinated alkaloids produced by M. dauricum roots.     

三种鹅膏菌培养条件及所产肽类毒素的比较研究

以外生菌根菌鹅膏菌属三个种Amanita muscaria,A.pseudoporphyria和A.fritillaria为研究材料,以生长速率为评价指标,对其最适生长温度、pH值、光照、培养基、C及N源的利用等基本培养条件及所产肽类毒素进行了比较研究。研究结果表明,三种菌株最适生长温度有差异,A.pseudoporphyria和A.fritillaria的最适温度为28℃,A.muscaria的最适温度为22℃;A.muscaria菌丝体生长的pH值范围为5-7,另外两个菌株的pH值范围为3-6;24h光照、12h光暗交替和24h黑暗对鹅膏菌的生长速率影响不大;SPDM培养基和MMN培养基都适合三种菌株的生长,但对于A.muscaria来说PDM培养基更适合其生长。鹅膏菌能够利用比较广泛的C、N源,但三个种在利用的C、N源种类上有一定的差别。通过抑芽法实验和HPLC分析分别表明三种鹅膏菌所含肽类毒素在种类和含量上有所不同,但都对绿豆发芽有一定的抑制作用。A.pseudoporphyria和A.fritillaria菌丝体中α-amanitin的含量分别为35.56μg/gDCW(drycellweight细胞干重)和26.02μg/gDCW,不含有phalloidin和β-amanitin;A.muscaria菌丝体中没有检测到α-amanitin、β-amanitin和phalloidin。结果表明供试的三种鹅膏菌在基本培养条件及所产肽类毒素方面存在种水平上的差异。     

Utilization of seawater-urea as a culture medium for Spirulina maxima.

The possibilities of utilization of seawater enriched with ureas as the culture medium for a blue-green alga, Spirulina maxima, were investigated. Pretreatment by precipitation with NaHCO3 and (or) Na2CO3 was found essential to remove the excess amounts of Ca2+ and Mg2+ present in seawater prior to cultivation. A culture medium as good as the synthetic medium reported in the literature for the growth of S. maxima was obtained after treating seawater with NaHCO3 (19.2 g/L) at pH 9.2 and 35 degrees C for 2 h, filtering to remove precipitates, and enriching with K2HPO4 (0.5 g/L), NaNO3 (3.0 g/L), and FeSO4 (0.01 g/L). The same results were obtained by substituting a small amount (0.2 g/L or less) of either crystalline or polymerized urea for the NaNO3 in the above medium. Growth of S. maxima was inhibited at higher concentration of urea in the culture medium. The inhibition effect was due to the partial decomposition of urea into ammonia in alkali medium. Tests conducted on the 130-L cultivation open pond also confirmed that the seawater-urea medium supports growth of S. maxima as well as the best known synthetic medium.     

The influence of pretreatment with different cations on anaerobic nitrite production by excisedPisum sativum roots

The influence of pretreatment with some cations on anaerobic nitrite production (in an assay medium lacking nitrate) by excised primary roots of pea (Pisum sativum L., ov. Raman), detached from six-day-old seedlings germinated in distilled water, was investigated. When the excised roots were precultivated in one-salt-solutions of KNO3, then these roots produced at 9 mM and 15 mM NO3- concentrations under anaerobic conditions significantly more NO2-, than those precultivated in a nutrient solution containing besides K+ ions also Ca2+ and Mg2+ ions, and they produced nitrite for a longer time. The KNO3 dependent increase in anaerobic NO2- production was counteracted most by Ca2+ and to a lesser extent by Mg2+; Na+ was without effect. NH4+ at higher concentrations (12 and 15 mM) significantly depressed nitrite production both by roots precultivated in a solution containing besides NH4+ only K+, and by roots precultivated in a full nutrient solution containing K+, Ca2+ and Mg2+, however at lower NH4+ concentrations (0.6 and 2mMNH4+; 15mMNO3-) the decrease was more conspicuous in the KNO3 solution than in the full nutrient solution. Nitrate reductase level was not influenced by this pretreatment. When 6% and 7.5% n-propanol, which increases membrane permeability and causes mixing of storage and metabolic nitrate pools in the cells, was added to the assay medium lacking nitrate, anaerobic nitrite production increased and the differences caused by the precultivation disappeared. These results show that higher K+ concentrations in unbalanced one-salt-solutions of KNO3 can cause higher membrane permeability by accentuating Ca8+ deficiency, which results in a faster penetration of NO3- from the storage pool to the sites of its reduction and in an easier penetration of NO2- out of the roots, and that higher NH4+ concentrations can change nitrate compartmentation and diminish the metabolic NO3- pool which results in a slower nitrate reduction. Besides that, lower NH4+ concentrations in KNO3 solutions (15mMNO3-) probably partially counteract the K+ dependent increase in membrane permeability. The results obtained show that there is no simple, direct relationship between the so-called metabolic pool of nitrate (i.e. anaerobic nitrite production) and the level of nitrate reductase, but that the velocity of nitrate reduction can be influenced by nitrate compartmentation in the cell.