Medium optimization for a methanol utilizing bacterium based on chemostat theory

Summary In the productions of biomass and vitamin B₁₂ using methanol as the sole carbon source, it is necessary to use a medium in which methanol is the growth limiting substrate. Other inorganic salts should be in slight excess so that the yield of cells and the intracellular content of vitamin B₁₂ do not vary. From basic principles of chemostat culture, a medium was optimized for Pseudomonas AM-1 a methanol utilizing bacterium, for the concentrations of various inorganic salts. This was done in a series of chemostat cultures at a dilution rate of 0.1 h^–1. Optimum amounts of NH₄ ⁺, PO₄ ^3- and Mg²⁺ were estimated from the minimum concentration of the salt at which methanol became growth limiting. The optimum concentrations of Ca²⁺, Fe²⁺, Mn²⁺, and Zn²⁺ as a group were determined in the same way. Cu²⁺, Mo⁶⁺, Co²⁺ and B³⁺ are required at concentrations of g/l and they were not studied as these very low level can be introduced as contaminants from other salts. The optimum medium composition (in g/l) was as follows: (NH₄)₂SO₄, 1.0; H₃PO₄, 75×10^–3; MgSO₄ · 7H₂O, 30×10^–3; CaCl₂ · 2H₂O, 3.3×10^–3; FeSO₄ · 7H₂O, 1.3×10^–3, MnSO₄ · 4H₂O, 0.13×10^–3; ZnSO₄ · 7H₂O, 0.13×10^–3; CuSO₄ · 5H₂O, 40×10^–6; Na₂MoO₄, 40×10^–6; CoCl₂ · 6H₂O, 40×10^–6; H₃BO₃, 30×10^–6 and methanol 4.     

Influence of metal ions on systems of dimethylsulfoxide-uracil-derivatives: NMR investigations

Summary The NMR spectra of uracil and some of its derivatives have been investigated using deuterated dimethylsulfoxide as a solvent. The spectral positions of the peaks depend on the electron-attracting or -releasing strength of the substituents: rel. to uracil, the peaks were shifted upfleld in the case of the electron-releasing substituents andvice versa. A plot of the electron affinityvs. the H-3 position exhibits a straigth line. Addition of Cu²⁺ or other metal ions, such as Pe³⁺, Ni²⁺, Co²⁺, Zn²⁺, and Ag⁺, results in a splitting of the N-H protons, the distance of which turned out to be a linear relationship to the inverse electronegativity. K⁺, Na⁺, Mg²⁺, NH₄ ⁺, Cl^–, NO₃ ^–, SO₄ ^2– had no effect at all. The effect of Cu²⁺ and the substituents as well seems to be mediated by the hydrogen bond formed between the uracil derivative and DMSO.Dedicated to Professor Dr. Dr. h. c. mult. B. Rajewsky on the occasion of his 80th birthday.     

Solution chemistry profiles of mixed-conifer forests before and after fire

Solution chemistry profiles of mixed-conifer forests in granitic catchments of the Sierra Nevada were measured for three years before (1987–1990) and three years after (1990–1993) prescribed fire. Wet deposition, throughfall and soil solution samplers were installed in both white-fir and giant-sequoia dominated forest stands underlain by poorly developed inceptisols. Stream water chemistry was monitored as part of an ongoing study of catchment outputs. Calcium, NO ₃ ^– and Cl^– were the major ions in precipitation. Canopy leaching increased mean concentrations of all major ions, especially K⁺ and Ca²⁺. Water flux through the soil occurred largely during spring snowmelt. Forest floor leachate represented the most concentrated solutions of major ions. Interaction with the mineral soil decreased mean concentrations of most species and the average composition of soil solutions closely resembled stream water at baseflow. Bicarbonate alkalinity, Ca²⁺, Mg²⁺, and Na⁺ were enriched in stream water relative to precipitation whereas inputs of H⁺, NH ₄ ⁺ , NO ₃ ^– and SO ₄ ^2– were retained within the catchments.Burning of the forest understory and litter layer increased solute concentrations in soil solution and stream water. Mean soil solution Ca²⁺, Mg²⁺ and K⁺ concentrations increased more than 10 fold, but the relative predominance of these cations was not affected by burning. Sulfate concentration, which was very low in soil solutions of undisturbed stands (<25 mmol_c m^–3), increased more than 100 times following fire. Ammonium concentration exhibited a rapid, short-term increase and then a decrease below pre-burn levels. Changes in soil solution chemistry were reflected in catchment outputs.Corresponding author.     

Accumulation of technetium by cyanobacteria

Accumulation of pertechnetate ions (⁹⁹TcO₄ ^–) by the cyanobacterial speciesSynechocystis PCC 6803,Synechococcus PCC 6301,Plectonema boryanum,Anabaena variabilis and a redOscillatoria sp. consisted solely of a single rapid energy-independent phase (biosorption); no energy-dependent uptake was detected. Biosorption of TcO₄ ^– was concentration-dependent and could be described by a Freundlich adsorption isotherm for each cyanobacterial species examined. Decreasing pH increased the accumulation of TcO₄ ^– by all the species as did an increase in external NaCl concentration. Accumulation of TcO₄ ^– was also increased inA. variabilis, P. boryanum and the redOscillatoria by an increased external osmotic potential. Concentrations of cations affected TcO₄ ^– accumulation; K⁺ increased accumulation in all the species, Mg²⁺, Ca²⁺, Sr²⁺ and Cs⁺ increased accumulation inSynechococcus PCC 6301 and Ca²⁺ increased accumulation by the redOscillatoria. Some anions decreased TcO₄ ^– accumulation; CO₃ ^2– inA. variabilis and the redOscillatoria, SO₄ ^2– inSynechocystis PCC 6803, and HCO₃ ^– inP. boryanum. The majority of TcO₄ ^– accumulated by all the cyanobacteria was easily desorbed, with no difference in the amounts desorbed between desorption agents of different pH or cation concentration.(*author for correspondence)     

Nitrogen and base cation uptake in seedlings of Acer pseudoplatanus and Calamagrostis villosa exposed to an acidified environment

The effects of solution acidity and form of nitrogen on net nutrient uptake rates in Acer pseudoplatanus and Calamagrostis villosa seedlings were examined as part of a complex ecological study. Uptake rates were measured by the depletion method under controlled conditions (temperature 20 °C, irradiance 400 mol m^–2 s^–1 PAR) from a nutrient solution containing 1.5 mM nitrogen in the form of nitrate or ammonium or an equimolar mixture of both. The solution acidity was kept constant at pH 5.5 (control treatment), 4.5 or 3.5 (low pH treatments). Strongly acid pH decreased or stopped the uptake rates of NO₃ ^–, Mg²⁺ and Ca²⁺, but the uptake of NH₄ ⁺ was not changed in both species. Ammonium ions reduced the uptake rate of NO₃ ^– in Acer but increased the uptake rate in Calamagrostis. Ammonium as the sole source of nitrogen had a strong negative impact on the uptake rates of calcium and magnesium and this effect was independent of the media acidification usually connected with NH₄ ⁺ uptake and assimilation. However, the negative effect of ammonium ions on the base cation uptake was more pronounced at low pH values.     

Lanthanide ions promote the hydrolysis of 2,3-Bisphosphoglycerate

The ³¹P NMR studies showed that lanthanide ions promote the site-specific hydrolysis of 2,3-Bisphosphoglycerate (BPG) at pH 7.4 by cleaving the 2 phosphomonoester bond. The effect of fourteen trivalent lanthanide ions and Sc³⁺, and Y³⁺ were compared by the percentage of hydrolysis obtained by determining the inorganic phosphate produced. All the trivalent lanthanide ions promote the hydrolysis, but Sc³⁺ not. Among them, Ce³⁺ affects the reaction mostly. This was mainly attributed to the autooxidation of Ce³⁺ to Ce⁴⁺, since the promoting effect of Ce³⁺ is related to the increasing Ce⁴⁺ amount in the solution and depressed by adding sulphite. Ce⁴⁺ promotes the hydrolysis more efficiently than Ce³⁺ do. The pseudo first-order rate constant for the hydrolysis of BPG by Ce(SO₄)₂ (18.7 mM) at pH 1 and pH 2, 37 °C is 3.1 h^–1 and 0.65 h^–1 respectively. A mechanism with a hydroxo species as reactive intermediate was proposed for the trivalent lanthanide ions. The site-specificity was explainable by this mechanism.     

Effects of aluminum,iron, oxygen and nitrate additions on phosphorus release from the sediment of a Danish softwater lake

The effects of oxygen, aluminum, iron and nitrate additions on phosphate release from the sediment were evaluated in the softwater Lake Vedsted, Denmark, by a 34-day experiment with undisturbed sediment cores. Six treatments were applied: (1) Control - O₂ (0–20% saturation), (2) O₂ (100% saturation) (3) Al³⁺ – O₂, (4) Fe³⁺ + O₂, (5) Fe³⁺ – O₂, and (6) NO₃ ^– – O₂. Al₂(SO₄)₃*18 H₂O and FeCl₃*4H₂O were added in amounts that theoretically should immobilize the exchangeable P-pool in the top 5 cm of the sediment, while sodium nitrate concentrations were increased to 5 mg N l^–1. The four treatments with metals or NO₃ ^– reduced the P efflux from the sediment significantly as compared to the suboxic control treatment. Mean accumulated P-release rates for suboxic treatments with Al³⁺, Fe³⁺, and NO₃ ^– were: –0.27 mmol m^–2 (st. dev = 0.02 mmol m^–2, N = 5), 0.58 mmol m^–2 (st. dev = 0.30 mmol m^–2, N = 5) and 1.40 mmol m^–2 (st. dev = 0.14 mmol m^–2, N = 5), respectively. The oxic treatment with Fe³⁺ had a P efflux of 0.36 mmol m^–2 (st. dev = 0.08 mmol m^–2, N = 5). The two highest P-release rates were observed in the control treatment and the treatment with O₂ (14.50 mmol m^–2 (st. dev = 3.90 mmol m^–2, N = 5) and 2.31 mmol m^–2 (st. dev = 0.80 mmol m^–2, N = 5), respectively). In order to identify changes in the P and Fe binding sites in the sediment as caused by the treatments, a sequential P extraction procedure was applied on the sediment before and after the efflux experiment. Addition of O₂, Fe³⁺ and NO₃ ^– to the sediment increased the amounts of oxidized Fe³⁺ and P_BD. Al³⁺ addition resulted in a lower fraction of P_BD but a correspondingly higher fraction of Al-bound P. Addition of Al³⁺ decreased the Fe-efflux from the suboxic sediment as well as the amount of oxidized Fe³⁺ in the sediment. This questions the use of Al compounds that contain sulfate because of the possible formation of FeS, which will restrict upward migration of Fe²⁺ and the formation of new Fe-oxides in the surface sediment. Instead, we suggest the use of AlCl₃ for lake restoration purposes.     

Removal of inorganic ions from wastewaters by immobilized microalgae

Anabaena doliolum and Chlorella vulgaris immobilized on chitosan were more efficient at removing NO₃ ^–, NO₂ ^p–, PO₄ ^3– and CR₂O₇ ^2– from wastewaters than cells immobilized on agar, alginate, carrageenan or even free cells. Carrageenan-immobilized cells, however, were better at removing NH₄ ⁺ and Ni²⁺. The PO₄ ^3– uptake capacity was significantly increased in cells starved of PO₄ ^3– for 24 h. Agar-immobilized cells, though having good metal and nutrient uptake efficiency, had only a slow growth rate. Chitosan is recommended as an algal support for wastewater detoxification.The authors are with the Laboratory of Algal Biology, Department of Botany, Banaras Hindu University, Varanasi-221005, India     

Localization of inorganic ions by precipitative freeze dissolution

Summary A new method for localization of inorganic diffusible ions in tissue is introduced. It has been applied to localization of Tl⁺ and Rb⁺ in barley roots and is probably also suited for Cs⁺, Ca²⁺, Cl^–, Br^–, PO ₄ ^3– and perhaps K⁺. Its principle consists of dissolution of the ice from frozen tissue in a concentrated aqueous solution of a precipitating agent that is kept at a temperature just above its melting point.     

Branchial and renal ion fluxes and transepithelial electrical potential differences in rainbow trout,Oncorhynchus mykiss: effects of aluminium at low pH

Synopsis Adult rainbow trout, Oncorhynchus mykiss, were acutely exposed for 4 hours to low pH (4.4) and elevated Al-concentrations (300 µgI^–1) in soft water (Ca²⁺ + Mg²⁺ = 25 µmolI^–1). Comparison of branchial and renal ion fluxes (Na⁺, Cl^–, Mg²⁺, Ca²⁺ and NH₄ ⁺) gave evidence that pH and Al effects were primarily localized at the gill site. The negative whole body ion balance seemed to be caused by stimulatory effects on Na and Cl efflux especially under Al stress and to a lesser extent by inhibition of influx. Measurements of gill potentials indicated positive shifts, which were similar in response to increasing levels of H⁺ ions and Al. It is suggested that Al-induced changes of branchial potentials causes high diffusable loss of ions through interference with membrane-bound Ca²⁺ at the gill site.     

The major ion chemistry of some southern African saline systems

Africa south of about 23° S has few natural athalassic lakes, saline or freshwater. South Africa, however, is rich in temporary pans, many of which are saline, while permanent saline springs occur along the coastal strip of the Namib Desert in Namibia. This paper examines the chemistry of the major ions in 67 Namibian waters, 47 of which have not previously been reported in the literature, and compares them with 66 South African waters, five of which have not previously been reported, and with saline lakes in East Africa.The highest value for total dissolved solids in South African waters was 276 g l^–1 (Koekiespan, south-western Cape) and the highest for Namibian waters were 160 g l^–1 (Hosabes, a small spring on a gypsous crust) and 302 g l^–1 (a salt pan at Oranjemund at the mouth of the Orange River). The dominant ions in fresh waters in the region are Ca²⁺ and HCO _inf3 ^– /CO _inf3 ^2– in the interior and in Namibia, and Na²⁺ and Cl⁺ on the south and east coasts. Regardless of the geochemistry of their substrata, the dominant ions in the saline waters throughout the region are Na⁺ and Cl^–. Thus differential precipitation of CaCO₃ and CaSO₄, as a result of evaporative concentration at high salinities, appears to be the determinant of the proportions of the major ions in these systems.The permanent springs on gypsous crusts along the coast of Namibia, although dominated by Na⁺ and Cl^– ions, contain considerable quantities of both Ca²⁺ and SO ₄ ^2– ions.     

A model for the regulation of brain adenylate cyclase by ionic equilibria

Multiple-equilibrium equations were solved to investigate the individual and separate effects of Mg²⁺, Mn²⁺, Ca²⁺, ATP^4–, and their complexes on the kinetics of brain adenylate cyclase. The effects of divalent metals and/or ATP^4– (in excess of their participation in complex formation) were determined and, from the corresponding apparent affinity values, the following kinetic constants were obtained:K _m(MgATP)=1.0 mM,K _i(ATP^4–)=0.27 mM,K _m(MnATP)=0.07 mM, andK _i(CaATP)=0.015 mM. MgATP, MnATP, ATP^4–, and CaATP were shown to compete for the active site of the enzyme. Hence, it is proposed that endogenous metabolites with a strong ligand activity for divalent metals, such as citrate and some amino acids, become integrated into a metabolite feedback control of the enzyme through the release of ATP^4– from MgATP. Ca²⁺ fluxes may participate in the endogenous regulation of adenylate cyclase by modifying the level of CaATP. The free divalent metals show an order of affinityK _0.5(Ca²⁺)=0.02 mM,K _0.5(Mn²⁺)=3.8 mM,K _0.5(Mg²⁺)=4.7 mM, and an order of activity Mn²⁺>Mg²⁺>Ca²⁺. The data indicate that Mn²⁺ and Mg²⁺ ions may compete for a regulatory site distinct from the active site and increaseV _m without changingK _m(MgATP),K _m(MnATP), orK _i(ATP^4–). The interactions of ATP^4– and CaATP, which act as competitive inhibitors of the reaction of the enzyme with the substrates MgATP and MnATP, and Mg²⁺ and Mn²⁺, which act as activators of the enzyme in the absence of hormones, are shown to follow the random rapid equilibrium BiBi group-transfer mechanism of Cleland with the stipulation that neither Mg²⁺ nor Mn²⁺, in excess of their respective participation in substrate formation, are obligatorily required for basal activity. ATP^4– and CaATP are involved in dead-end inhibition. For MgCl₂ saturation curves at constant total ATP concentration, the computer-generated curves based on the RARE BiBi model predict a change in the Hill cooperativityh from a basal value of 2.6, when Mg²⁺ is not obligatorily required, to 4.0 when the addition of hormones or neurotransmitters induces an obligatory requirement for Mg²⁺.Abbreviations used: Me, divalent metal; Me_T (Mg_T or Mn_T), total Me (Me²⁺ and its complexes); ATP_T, total ATP (ATP^4– and its complexes).     

Simulation study of nutrient uptake by plants from soilless cultures as affected by salinity buildup and transpiration

Soilless plant growth systems are widely used as a means to save irrigation water and to reduce groundwater contamination. While nutrient concentrations in the growth medium are depleted due to uptake by the plants, salinity and toxic substances accumulate due to transpiration. A theoretical model is suggested, to simulate nutrient uptake by plants grown in soilless cultures with recycled solutions. The model accounts for salinity accumulation with time and plant growth, and its effects on uptake of the different nutrients by means of interaction with Na and Cl ions. The sink term occurs due to uptake by a growing root system. Influx as a function of the ion concentration is according to Michaelis–Menten active mechanisms for K⁺, NO₃ ^–-N, NH₄ ⁺-N, PO₄-P, Ca²⁺, Mg²⁺ and SO₄ ^2-, whose influx parameters are affected by Na and Cl^–, but not with time (age). Sodium influx is passive above a critical concentration. Sum of cations–anions concentrations is balanced by Cl^– to maintain electro-neutrality of the growth solution. Salinity (by means of Na concentration) suppresses root and leaf growth, which further effect uptake and transpiration. The model accounts for instantaneous transpiration losses, during daytime only and its effect on uptake of nutrients and plant development due to salt accumulation. The model was tested against NO₃ ^– and K⁺ uptake by plants associated with cumulative transpiration and with different NaCl salinity levels. Deviations from observed K⁺ uptake should be attributed to the salinity tolerance of the plants. In a study with data obtained from published literature, the model indicated that nutrient depletion and salinity buildup might be completely different with fully grown-up plants (that do not grow) and plants that grow with time. Depletion of different nutrients are according to their initial concentration and plant uptake rate, but also affected by their interactions with Na and Cl ions.     

Influence of the culture medium constituents and inoculum size on the accumulation of blue pigment and cell growth of Lavandula spica

The effects of the concentration of PO₄ ^3-, NO₃ ^–, Fe ²⁺, sucrose and inoculum size on the accumulation of blue pigment and growth of suspension cultures of Lavandula spica D.C. ( L. latifolia Vill.), were studied. The combination of 2.5 mM PO₄ ^3-, 14.1 mM NO₃ ^–, 1 mM Fe ²⁺, 30 g l^–1 sucrose and 10 g l^–1fresh weight of inoculum, promoted a 7-fold enhancement on the productivity of blue pigment in comparison to the control medium. Among all the culture medium constituents tested, phosphate exerted the highest beneficial effect and Fe²⁺ showed to be essential for the accumulation of the pigment. High levels of sucrose (60 or 90 g l^–1) did not stimulate the accumulation of blue pigment. In these conditions, cell growth and cell viability were drastically affected.     

Ulva rigida (Ulvales,Chlorophyta) tank culture as biofilters for dissolved inorganic nitrogen from fishpond effluents

Ulva rigida was cultivated in 7501 tanks at different densities with direct and continuous inflow (at 2, 4, 8 and 12 volumes d^–1) of the effluents from a commercial marine fishpond (40 metric tonnes, Tm, of Sparus aurata, water exchange rate of 16 m³ Tm^–1) in order to assess the maximum and optimum dissolved inorganic nitrogen (DIN) uptake rate and the annual stability of the Ulva tank biofiltering system. Maximum yields (40 g DW m^–2 d^–1) were obtained at a density of 2.5 g FW 1^–1 and at a DIN inflow rate of 1.7 g DIN m^–2 d^–1. Maximum DIN uptake rates were obtained during summer (2.2 g DIN M^–2 d^–1), and minimum in winter (1.1 g DIN m^–2 d^–1) with a yearly average DIN uptake rate of 1.77 g DIN m^–2 d^–1 At yearly average DIN removal efficiency (2.0 g DIN m^–2 d^–1, if winter period is excluded), 153 m² of Ulva tank surface would be needed to recover 100% of the DIN produced by 1 Tm of fish.Abbreviations DIN= dissolved inorganic nitrogen (NH _inf4 ^sup+ + NO _inf3 ^sup– + NO _inf2 ^sup– ); - FW= fresh weight; - DW= dry weight; - PFD= photon flux density; - V= DIN uptake rate     

Bacterial Degradation of EDTA

Degradation of EDTA (ethylenediaminetetraacetic acid) or metal–EDTA complexes by cell suspensions of the bacterial strain DSM 9103 was studied. The activity of EDTA degradation was the highest in the phase of active cell growth and decreased considerably in the stationary phase, after substrate depletion in the medium. Exponential-phase cells were incubated in HEPES buffer (pH 7.0) with 1 mM of uncomplexed EDTA or EDTA complexes with Mg²⁺, Ca²⁺, Mn²⁺, Pb²⁺, Co²⁺, Cd²⁺, Zn²⁺, Cu²⁺, or Fe³⁺. The metal–EDTA complexes (Me–EDTA) studied could be divided into three groups according to their degradability. EDTA complexes with stability constants K below 10¹⁶ (log K < 16), such as Mg–EDTA, Ca–EDTA, and Mn–EDTA, as well as uncomplexed EDTA, were degraded by the cell suspensions at a constant rate to completion within 5–10 h of incubation. Me–EDTA complexes with log K above 16 (Zn–EDTA, Co–EDTA, Pb–EDTA, and Cu–EDTA) were not completely degraded during a 24-h incubation, which was possibly due to the toxic effect of the metal ions released. No degradation of Cd–EDTA or Fe(III)–EDTA by cell suspensions of strain DSM 9103 was observed under the conditions studied.     

Inhibition of the biosynthesis ofN-acetylneuraminic acid by metal ions and seleniumin vitro

In liver homogenate the biosynthesis ofN-acetylneuraminic acid usingN-acetylglucosamine as precursor can be followed stepwise by applying different chromatographic procedures. In this cell-free system 16 metal ions (Zn²⁺, Mn²⁺, La³⁺, Co²⁺, Cu²⁺, Hg²⁺, VO ₃ ^– , Pb²⁺, Ce³⁺, Cd²⁺, Fe²⁺, Fe³⁺, Al³⁺, Sn²⁺, Cs⁺ and Li⁺) and the selenium compounds, selenium(IV) oxide and sodium selenite, have been checked with respect to their ability to influence a single or possible several steps of the biosynthesis ofN-acetylneuraminic acid. It could be shown that the following enzymes are sensitive to these metal ions (usually applied at a concentration of 1 mmoll^–1):N-acetylglucosamine kinase (inhibited by Zn²⁺ and vandate), UDP-N-acetylglucosamine-2-epimerase (inhibited by zn²⁺, Co²⁺, Cu²⁺, Hg²⁺, VO ₃ ^– , Pb²⁺, Cd²⁺, Fe³⁺, Cs⁺, Li⁺, selenium(IV) oxide and selenite), andN-acetylmannosamine kinase (inhibited by Zn²⁺, Cu²⁺, Cd²⁺, and Co²⁺). Dose dependent measurements have shown that Zn²⁺, Cu²⁺ and selenite are more efficient inhibitors of UDP-N-acetylglucosamine-2-epimerase than vanadate. As for theN-acetylmannosamine kinase inhibition, a decreasing inhibitory effect exists in the following order Zn²⁺, Cd²⁺, Co²⁺ and Cu²⁺. In contrast, La³⁺, Al³⁺ and Mn²⁺ (1 mmoll^–1) did not interfere with the biosynthesis ofN-acetylneuraminic acid. Thus, the conclusion that the inhibitory effect of the metal ions investigated cannot be regarded as simply unspecific is justified.Dedicated to Professor Theodor Günther on the occasion of his 60th birthday     

Simultaneous determination of stable isotopically labelled l-histidine and urocanic acid in human plasma by stable isotope dilution mass spectrometry

A capillary gas chromatographic—mass spectrometric method for the simultaneous determination of stable isotopically labelled l-histidine (l-[3,3-²H₂,1′,3′-¹⁵N₂]histidine, l-His-[M + 4]) and urocanic acid ([3-²H,1′,3′-¹⁵N₂]urocanic acid, UA-[M + 3]) in human plasma was developed using dl-[2,3,3,5′-²H₄,2′-¹³C,1′,3′-¹⁵N₂]histidine (dl-His-[M + 7]) and [2,3,5′-²H₃,2′-¹³C,1′,3′-¹⁵N₂]urocanic acid (UA-[M + 6]) as internal standards. l-Histidine and urocanic acid were derivatized to ^αN-(trifluoroacetyl)-^imN-(ethoxycarbonyl)-l-histidine n-butyl ester and ^imN-(ethoxycarbonyl)urocanic acid n-butyl ester. Quantification was carried out by selected ion monitoring of the molecular ions of the respective derivatives of l-His-[M + 4], dl-His-[M + 7], UA-[M + 3] and UA-[M + 6]. The sensitivity, specificity, precision and accuracy of the method were demonstrated to be satisfactory for measuring plasma concentrations of l-His-[M + 4] and UA-[M + 3] following administration of trace amounts of l-His-[M + 4] to humans.     

Characterization of a secreted cholesterol oxidase from Rhodococcus sp.GKI (CIP 105335)

Extracellular cholesterol oxidase (COX) (EC 1.1.3.6) was produced by Rhodococcus sp. GK1 cells grown in a defined mineral salt medium containing a mixture of phytosterols (sitosterol, campesterol, stigmasterol) as the sole source of carbon and energy. In the same time, the sterols acted as enzyme inducers. The medium was enriched with yeast extract in order to stimulate enzyme secretion. COX was purified from the culture supernatants by affinity-like chromatography on a column packed with kieselguhr and cholesterol. Enzyme bound onto the column was eluted with 0.05 M phosphate buffer pH 7.0 containing Triton X-100 at 0.1% (w/v). Some properties of the purified COX were determined. Its specific activity at pH 7.0 and 30 °C, was around 5.5 units mg^–1. The molecular mass of the enzyme, as estimated by SDS-PAGE, was 59 kDa. Its isoelectrofocusing point was around pH 8.9. The C-5 double bond and the alkyl chain moiety in sterol molecules were necessary for an adequate oxidation of the sterol 3-ol. Enzyme inhibition by the ions (0.1 mM): AsO₂ ^–, Ba²⁺, Co²⁺, Cd²⁺, Cu²⁺, N₃ ^–, Ni²⁺, and Pb²⁺ was negligible (around 10%). However, COX inhibition by 0.1 mM of either Zn²⁺, 2-[(ethylmercurio)-thio]benzoic acid, or Hg²⁺ was 18%, 22% and 93% respectively. Inhibition of activity by Hg²⁺ was significant, even at 1 M. The purified COX (0.1–0.15 mg ml^–1 in 0.05 M phosphate pH 7.0) was relatively heat-stable at temperatures up to 50 °C. At this temperature, the half-life of its activity was around 70 min. However, 90% of the enzyme initial activity was lost by 20 min incubation at 60 °C. The aminoacid sequence of the COX N-terminal segment was: H2N–Ala–Pro–Pro–Val–Ala–Ser–X–Arg–Tyr–X–(Phe)– (X might be 2 Cys residues).     

Influence of inorganic microelements on the production of camptothecin with suspension cultures of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

The effects of eight microelements (I^–, BO₃ ^3–, MoO₄ ^2–, Co²⁺, Cu²⁺, Mn²⁺, Fe²⁺, Zn²⁺) on the biosynthesis of camptothecin and the growth of suspension cultures of Camptotheca acuminata were studied. The increase of I^– to 25 M l^–1, Cu²⁺ to 1 M l^–1, Co²⁺ to 2 M l^–1 and MoO₄ ^2– to 10 M l^–1 in Murashige and Skoog (MS) medium resulted in 1.66, 2.84, 2.53 and 2.04 times higher of camptothecin yield than that in standard MS medium respectively. Combined treatment of I^– (25 M l^–1), Cu²⁺ (1 M l^–1), Co²⁺ (2 M l^–1) and MoO₄ ^2– (10 M l^–1) lead to improve cell dry weight, camptothecin content, and camptothecin yield to 30.56 g l^–1, 0.0299%, and 9.15 mg l^–1, respectively, which were 20.2, 208.9 and 273.8% increment respectively when compared with those of control.