Estimation of formamide harmonic and anharmonic modes in the Kohn-Sham limit using the polarization consistent basis sets

Formamide harmonic and anharmonic frequencies of fundamental vibrations in the gas phase and in several solvents were successfully estimated in the B3LYP Kohn-Sham complete basis set limit (KS CBS). CBS results were obtained by extrapolating a power function (two-parameter formula) to the results calculated with polarization-consistent basis sets. Anharmonic corrections using the second order perturbation treatment (PT2) and hybrid B3LYP functional combined with polarization consistent pc-n (n = 0, 1, 2, 3, 4) and several Pople’s basis sets were analyzed for all fundamental formamide vibrational modes in the gas phase and solution. Solvent effects were modeled within a PCM method. The anharmonic frequency of diagnostic amide vibration C = O in the gas phase and the CCl₄ solution calculated with the VPT2 method was significantly closer to experimental data than the corresponding harmonic frequency. Both harmonic and anharmonic frequencies of C = O stretching mode decreased linearly with solvent polarity, expressed by relative environment permittivity (ε) ratio (ε − 1)/(2ε + 1). However, an unphysical behavior of solvent dependence of some low frequency anharmonic amide modes of formamide (e.g., CN stretch, NH₂ scissoring, and NH₂ in plane bend) was observed, probably due to the presence of severe anharmonicity and Fermi resonance.     

Role of Na+/ H+ exchange and HCO3 − transport in pHi recovery from intracellular acid load in cultured epithelial cells of sheep rumen

This study sought to investigate effects of short-chain fatty acids and CO₂ on intracellular pH (pH_i) and mechanisms that mediate pH_i recovery from intracellular acidification in cultured ruminal epithelial cells of sheep. pH_i was studied by spectrofluorometry using the pH-sensitive fluorescent indicator 2′,7′-bis (carboxyethyl)-5(6′)-carboxyfluorescein acetoxymethyl ester (BCECF/AM). The resting pH_i in N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)-buffered solution was 7.37 ± 0.03. In HEPES-buffered solution, a NH₄ ⁺/NH₃-prepulse (20 mM) or addition of butyrate (20 mM) led to a rapid intracellular acidification (P < 0.05). Addition of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; 10 μM) or HOE-694 (200 μM) inhibited pH_i recovery from an NH₄ ⁺/NH₃-induced acid load by 58% and 70%, respectively. pH_i recovery from acidification by butyrate was reduced by 62% and 69% in the presence of EIPA (10 μM) and HOE-694 (200 μM), respectively. Changing from HEPES- (20 mM) to CO₂/HCO₃ ⁻-buffered (5%/20 mM) solution caused a rapid decrease of pH_i (P < 0.01), followed by an effective counter-regulation. 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS; 100 μM) blocked the pH_i recovery by 88%. The results indicate that intracellular acidification by butyrate and CO₂ is effectively counter-regulated by an Na⁺/H⁺ exchanger and by DIDS-sensitive, HCO₃ ⁻-dependent mechanism(s). Considering the large amount of intraruminal weak acids in vivo, both mechanisms are of major importance for maintaining the pH_i homeostasis of ruminal epithelial cells. Accepted: 8 March 2000     

The photomechanic infrared receptor for the detection of forest fires in the beetle Melanophila acuminata (Coleoptera: Buprestidae)

We recorded from single units of individual sensilla of the thoracic infrared (IR) pit organs of Melanophila acuminata. When the organ was stimulated with a thermal radiator whose emission spectrum was similar to that of a typical forest fire, units responded phasically with up to seven spikes within 30–40 ms at a radiation power of 24 mW cm⁻². In the experiments all wavelengths shorter than 1.6 μm were excluded by a longpass IR filter. Response latencies were about 4 ms and initial impulse frequencies were up to 250 impulses per second (ips). A single spike could be generated even when stimulus duration was only 2 ms. Reduction of total radiation power from 24 mW cm⁻² to 5 mW cm⁻² resulted in increased response latencies of 5–6 ms and the occurrence of only two to three spikes. Initial impulse frequencies decreased to 125 ips. According to our physiological results and calculations, Melanophila should be able to detect a 10-hectare fire from a distance of 12 km. Mechanical stimuli also evoked responses of the IR sensilla. All present morphological and physiological findings lead to the conclusion that the IR receptors of Melanophila must function by means of a hitherto undescribed photomechanic mechanism. Accepted: 1 November 1997     

Biological nitrification–denitrification with alternating oxic and anoxic operations using aerobic granules

Efficient nitrification and denitrification of wastewater containing 1,700 mgl⁻¹ of ammonium-nitrogen was achieved using aerobic granular sludge cultivated at medium-to-high organic loading rates. The cultivated granules were tested in a sequencing batch reactor (SBR) fed with 6.4 or 10.2 kg NH₄⁺-N m⁻³ day⁻¹, a loading significantly higher than that reported in literature. With alternating 2 h oxic and 2 h anoxic operation (OA) modes, removal rate was 45.5 mg NH₄⁺-N g⁻¹ volatile suspended solids⁻¹ h⁻¹ at 6.4 kg NH₄⁺-N m⁻³ day⁻¹ loading and 41.3 ± 2.0 at 10.2 kg NH₄⁺-N m⁻³ day⁻¹ loading. Following the 60 days SBR test, granules were intact. The fluorescence in situ hybridization and confocal laser scanning microscopy results indicate that the SBR-OA granules have a distribution with nitrifers outside and heterotrophs outside that can effectively expose functional strains to surrounding substrates at high concentrations with minimal mass transfer limit. This microbial alignment combined with the smooth granule surface achieved nitrification–denitrification of wastewaters containing high-strength ammonium using aerobic granules. Conversely, the SBR continuous aeration mode yielded a distribution with nitrifers outside and heterotrophs inside with an unsatisfactory denitrification rate and floating granules as gas likely accumulated deep in the granules.     

Influences of different nitrogen sources on nitrogen- and water-use efficiency, and carbon isotope discrimination, in C3 Triticum aestivum L. and C4 Zea mays L. plants

The impacts of various nitrogen sources, i.e. NO⁻ ₃, NH₄ ⁺ or NH₄NO₃ in combination with gaseous NH₃, on nitrogen-, carbon- and water-use efficiency and ¹³C discrimination (δ¹³C) by plants of the C₃ species Triticum aestivum L. (wheat) and the C₄ species Zea mays L. (maize) were studied. Triticum aestivum and Z. mays were hydroponically grown with 2 mol · m⁻³ of N supplied as NO⁻ ₃, NH₄ ⁺ or NH₄NO₃ for 21 and 18 d, respectively, and thereafter exposed to gaseous NH₃ at 320 μg · m⁻³ or to ambient air for 7 d. In T. aestivum and Z. mays over a 7-d growth period, nitrogen-use efficiency (NUE) values were influenced by N-sources in the decreasing order NH₄NO₃-N > NO⁻ ₃-N > NH₄ ⁺-N and NO⁻ ₃-N > NH₄NO₃-N > NH₄ ⁺-N, respectively. Fumigation with NH₃ decreased the NUE values of plants grown with any of the N-forms. During 28- and 7-d growth periods, N-sources affected water-use efficiency (WUE) values in the decreasing order of NH₄ ⁺-N > NO⁻ ₃-N≈NH₄NO₃-N in non-fumigated T. aestivum, while fumigation with NH₃ increased the WUE of NO⁻ ₃-grown plants. There were insignificant effects of N-sources on WUE values of Z. mays over 25- and 7-d growth periods. Furthermore, δ¹³C values in plant tissues (leaves, stubble and roots) were higher (less negative) in NH₄ ⁺-grown plants of T. aestivum and Z. mays than in those supplied with NH₄NO₃ or NO⁻ ₃. Regardless of the N-form supplied to the roots of the plant species, exposure to NH₃ caused more-positive δ¹³C values in the plant tissues. These results indicate that the variations in N-source were associated with small but significant variations in δ¹³C values in plants of T. aestivum and Z. mays. These differences in δ¹³C values are in the direction expected from differences in WUE values over long or short growth periods and with differences in the extent of non-Rubisco (ribulose-1,5-bisphosphate carboxylase-oxygenase, EC 4.1.1.39) carboxylate contribution to net C acquisition, as a function of N-source. Received: 12 September 1997 / Accepted: 13 January 1998     

Hydroxylamine oxidation and subsequent nitrous oxide production by the heterotrophic ammonia oxidizer Alcaligenes faecalis

Nitrous oxide (N₂O), a greenhouse gas, is emitted during autotrophic and heterotrophic ammonia oxidation. This emission may result from either coupling to aerobic denitrification, or it may be formed in the oxidation of hydroxylamine (NH₂OH) to nitrite (NO₂ ⁻). Therefore, the N₂O production during NH₂OH oxidation was studied with Alcaligenes faecalis strain TUD. Continuous cultures of A. faecalis showed increased N₂O production when supplemented with increasing NH₂OH concentrations. ¹⁵N-labeling experiments showed that this N₂O production was not due to aerobic denitrification of NO₂ ⁻. Addition of ¹⁵N-labeled NH₂OH indicated that N₂O was a direct by-product of NH₂OH oxidation, which was subsequently reduced to N₂. These observations are sustained by the fact that NO₂ ⁻ production was low (0.23 mM maximum) and did not increase significantly with increasing NH₂OH concentration in the feed. The NH₂OH-oxidizing capacity increased with increasing NH₂OH concentrations. The apparent V _max and K _m were 31 nmol min⁻¹ mg dry weight⁻¹ and 1.5 mM respectively. The culture did not increase its growth yield and was not able to use NH₂OH as the sole N source. A non-haem hydroxylamine oxidoreductase was partially purified from A. faecalis strain TUD. The enzyme could only use K₃Fe(CN)₆ as an electron acceptor and reacted with antibodies raised against the hydroxylamine oxidoreductase of Thiosphaera pantotropha. Received: 1 September 1998 / Received revision: 5 November 1998 / Accepted: 7 November 1998     

Potential of active excretion of ammonia in three different haline species of crabs

Isolated perfused gills of stenohaline crabs Cancer pagurus adapted to seawater, brackish water-adapted euryhaline shore crabs Carcinus maenas and freshwater-adapted extremely euryhaline Chinese crabs Eriocheir sinensis were tested for their capacity to excrete ammonia. Gills were perfused with haemolymph-like salines and bathed with salines equal in adaptation osmolality. Applying 100 μmol · l⁻¹ NH₄Cl in the perfusion saline and concentrations of NH₄Cl in the bath that were stepwise increased from 0 to 4000 μmol · l⁻¹ allowed us to measure transbranchial fluxes of ammonia along an outwardly as well as various inwardly directed gradients. The gills of all three crab species were capable – to different extents – of active excretion of ammonia against an inwardly directed gradient. Of the three crab species, the gills of Cancer pagurus revealed the highest capacity for active excretion of ammonia, being able to excrete it from the haemolymph (100 μmol · l⁻¹ NH⁺ ₄) through the gill epithelium against ambient concentrations of up to 800 μmol · l⁻¹, i.e. against an eightfold gradient. Carcinus maenas and E. sinensis were able to actively excrete ammonia against approximately fourfold gradients. Within the three crab species, the gills of E. sinensis exhibited the greatest capacity to resist influx at very high external concentrations of up to 4000 μmol · l⁻¹. We consider the observed capacities for excretion of ammonia against the gradient as ecologically meaningful. These benthic crustaceans protect themselves by burying themselves in the sediment, where, in contrast to the water column, concentrations of ammonia have previously been reported that greatly increase haemolymph levels. Electrophysiological results indicate that the permeabilities of the gill epithelia are a clue to understanding the species-specific differences in active excretion of ammonia. During the invasion of brackish water and freshwater, the permeabilities of the body surfaces greatly decreased. The gills of marine Cancer pagurus exibited the greatest permeability (ca. 250 mS cm⁻²), thus representing practically no influx barrier for ions including NH⁺ ₄. We therefore assume that C. pagurus had to develop the strongest mechanism of active excretion of ammonia to counteract influx. On the other hand, freshwater-adapted E. sinensis exhibited the lowest ion permeability (ca. 4 mS cm⁻²) which may reduce passive NH⁺ ₄ influxes at high ambient levels. Accepted: 14 October 1998     

Actual and potential rates of hydrogen photoproduction by continuous culture of the purple non-sulphur bacterium Rhodobacter capsulatus

The influence of (NH₄)₂SO₄ concentration and dilution rate (D) on actual and potential H₂ photoproduction has been studied in ammonium-limited chemostat cultures of Rhodobacter capsulatus B10. The actual H₂ production in a photobioreactor was maximal (approx. 80 ml h⁻¹l⁻¹) at D = 0.06 h⁻¹ and 4 mM (NH₄)₂SO₄. However, it was lower than the potential H₂ evolution (calculated from hydrogen evolution rates in incubation vials), which amounted to 100–120 ml h⁻¹ l⁻¹ at D = 0.03–0.08 h⁻¹. Taking into account the fact that H₂ production in the photobioreactor under these conditions was not limited by light or lactate, another limiting (inhibiting) factor should be sought. One possibility is an inhibition of H₂ production by the H₂ accumulated in the gas phase. This is apparent from the non-linear kinetics of H₂ evolution in the vials or from its inhibition by the addition of H₂; initial rates were restored in both cases after the vials had been refilled with argon. The actual H₂ production in the photobioreactor at D = 0.06 h⁻¹ was shown to increase from approximately 80 ml h⁻¹ l⁻¹ to approximately 100 ml h⁻¹ l⁻¹ under an argon flow at 100 ml min⁻¹. Under maximal H₂ production rates in the photobioreactor, up to 30% of the lactate feedstock was utilised for H₂ production and 50% for biomass synthesis. Received: 22 April 1997 / Received revision: 14 July 1997 / Accepted: 27 July 1997     

Active excretion of ammonia across the gills of the shore crab Carcinus maenas and its relation to osmoregulatory ion uptake

The mechanism of transbranchial excretion of total ammonia of brackish-water acclimated shore crabs, Carcinus maenas was examined using isolated, perfused gills. Applying physiological gradients of NH₄Cl (100–200 μmol · l⁻¹) directed from the haemolymph space to the bath showed that the efflux of total ammonia consisted of two components. The saturable component (excretion of NH₄ ⁺) greatly exceeded the linear component (diffusion of NH₃). When an outwardly directed gradient (200 μmol · l⁻¹) was applied, total ammonia in the perfusate was reduced by more than 50% during a single passage of saline through the gill. Effluxes of ammonia along the gradient were sensitive to basolateral dinitrophenol, ouabain, and Cs⁺ and to apical amiloride. Acetazolamide (1 mmol · l⁻¹ basolateral) or Cl⁻-free conditions had no substantial effects on ammonia flux, which was thus independent of both carbonic anhydrase mediated pH regulation and osmoregulatory NaCl uptake. When an inwardly directed gradient (200 μmol · l⁻¹) was employed, influx rates were about 10-fold smaller and unaffected by basolateral ouabain (5 mmol · l⁻¹) or dinitrophenol (0.5 mmol · l⁻¹). Under symmetrical conditions (100 μmol · l⁻¹ NH₄Cl on both sides) ammonia was actively excreted against the gradient of total ammonia, which increased strongly during the experiment and against the gradient of the partial pressure of NH₃. The active excretion rate was reduced to 7% of controls by basolateral dinitrophenol (0.5 mmol · l⁻¹), to 44% by basolateral ouabain (5 mmol · l⁻¹), to 46% by Na⁺-free conditions and to 42% by basolateral Cs⁺ (10 mmol · l⁻¹), indicating basolateral membrane transport of NH₄ ⁺ via the Na⁺/K⁺-ATPase and K⁺-channels and a second active, apically located, Na⁺ independent transport mechanism of NH₄ ⁺. Anterior gills, which are less capable of active ion uptake than posterior gills, exhibited even increased rates of active excretion of ammonia. We conclude that, under physiological conditions, branchial excretion of ammonia is a directed process with a high degree of effectiveness. It even allows active extrusion against an inwardly directed gradient, if necessary. Accepted: 11 March 1998     

In vitro studies on active calcium absorption from ovine rumen

From various in vivo and in vitro studies it has been shown that the rumen represents a significant site of Ca²⁺ absorption in sheep and goats. It was the aim of the present study to further characterize the underlying mechanisms. Unidirectional flux rates of Ca²⁺ across rumen wall epithelia of sheep were measured in vitro by applying the Ussing-chamber technique in the absence of electrochemical gradients. Under these conditions, significant Ca²⁺ net flux rates (J_net) clearly indicate the presence of active mechanisms for Ca²⁺ transport. Short chain fatty acids (SCFAs) caused highest stimulation of Ca²⁺ J_net (6.3 ± 1.9 nmol · cm⁻² · h⁻¹) when used as a mixture of acetate, proprionate and butyrate in physiological proportions (36, 15, 9 mmol · l⁻¹, respectively). The effect of 30 mmol · l⁻¹ butyrate (3.2 ± 0.6 nmol · cm⁻² · h⁻¹) was higher than respective amounts of propionate and acetate (0.6 ± 0.8 nmol · cm⁻² · h⁻¹ and 0.9 ± 0.8 nmol · cm⁻² · h⁻¹, respectively). Eliminating SCFAs resulted in Ca²⁺ J_net of 0.4 ± 1.1 nmol ^. cm^−2 .h⁻¹. Addition of Ca channel blocker verapamil (mucosal 1 mmol · l⁻¹) had no significant effect on SCFA-stimulated J_net of Ca²⁺, whereas application of Na⁺/H⁺ inhibitor amiloride (mucosal 1 mmol · l⁻¹) further enhanced the Ca²⁺ J_net by >65%. The Ca²⁺-pump inhibitor vanadate had no significant effect on J_net of Ca²⁺. Dietary Ca depletion enhanced calcitriol plasma concentrations but had no effect on active Ca²⁺ absorption across the rumen wall of sheep. In addition, no effect on active Ca²⁺ absorption could be observed during early lactation. In conclusion, there is clear evidence for the rumen as a main site for active Ca²⁺ absorption in sheep. Our results suggest the presence of a Ca²⁺/H⁺ exchange mechanism in the apical membrane of rumen epithelial cells which depends on SCFA absorption and which does not seem to be under the control of calcitriol. Basolateral Ca²⁺ extrusion occurs independently from Ca²⁺-pump activity and may be accomplished via Na⁺/Ca²⁺ exchange. Accepted: 29 June 1999     

Ruthenium polypyridyl complexes and their modes of interaction with DNA: Is there a correlation between these interactions and the antitumor activity of the compounds?

Various interaction modes between a group of six ruthenium polypyridyl complexes and DNA have been studied using a number of spectroscopic techniques. Five mononuclear species were selected with formula [Ru(tpy)L₁L₂]⁽²⁻ⁿ⁾⁺, and one closely related dinuclear cation of formula [{Ru(apy)(tpy)}₂{μ-H₂N(CH₂)₆NH₂}]⁴⁺. The ligand tpy is 2,2′:6′,2″-terpyridine and the ligand L₁ is a bidentate ligand, namely, apy (2,2′-azobispyridine), 2-phenylazopyridine, or 2-phenylpyridinylmethylene amine. The ligand L₂ is a labile monodentate ligand, being Cl⁻, H₂O, or CH₃CN. All six species containing a labile L₂ were found to be able to coordinate to the DNA model base 9-ethylguanine by ¹H NMR and mass spectrometry. The dinuclear cationic species, which has no positions available for coordination to a DNA base, was studied for comparison purposes. The interactions between a selection of four representative complexes and calf-thymus DNA were studied by circular and linear dichroism. To explore a possible relation between DNA-binding ability and toxicity, all compounds were screened for anticancer activity in a variety of cancer cell lines, showing in some cases an activity which is comparable to that of cisplatin. Comparison of the details of the compound structures, their DNA binding, and their toxicity allows the exploration of structure–activity relationships that might be used to guide optimization of the activity of agents of this class of compounds. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Simultaneous nutrients and carbon removal during pretreated swine slurry degradation in a tubular biofilm photobioreactor

The biodegradation potential of an innovative enclosed tubular biofilm photobioreactor inoculated with a Chlorella sorokiniana strain and an acclimated activated sludge consortium was evaluated under continuous illumination and increasing pretreated (centrifuged) swine slurry loading rates. This photobioreactor configuration provided simultaneous and efficient carbon, nitrogen, and phosphorous treatment in a single-stage process at sustained nitrogen and phosphorous removals efficiencies ranging from 94% to 100% and 70–90%, respectively. Maximum total organic carbon (TOC), NH₄ ⁺, and PO₄ ³⁻ removal rates of 80 ± 5 g C m_r ⁻³ day⁻¹, 89 ± 5 g N m_r ⁻³ day⁻¹, and 13 ± 3 g P m_r ⁻³ day⁻¹, respectively, were recorded at the highest swine slurry loadings (TOC of 1,247 ± 62 mg L⁻¹, N–NH₄ ⁺ of 656 ± 37 mg L⁻¹, P–PO₄ ³⁺ of 117 ± 19 mg L⁻¹, and 7 days of hydraulic retention time). The unusual substrates diffusional pathways established within the phototrophic biofilm (photosynthetic O₂ and TOC/NH₄ ⁺ diffusing from opposite sides of the biofilm) allowed both the occurrence of a simultaneous denitrification/nitrification process at the highest swine slurry loading rate and the protection of microalgae from any potential inhibitory effect mediated by the combination of high pH and high NH₃ concentrations. In addition, this biofilm-based photobioreactor supported efficient biomass retention (>92% of the biomass generated during the pretreated swine slurry biodegradation).     

Role of cyclic nucleotides in pigment translocation within the freshwater shrimp, Macrobrachium potiuna, erythrophore

The participation of cyclic nucleotide-dependent intracellular signalling pathways in the pigment translocation induced by pigment-dispersing hormone (α -PDH) or pigment-concentrating hormone (PCH) was investigated in the erythrophores of the freshwater shrimp, Macrobrachium potiuna. Cholera toxin, forskolin and dibutyryl cyclic adenosine 3′5′ monophosphate (dbcAMP) were able to induce pigment dispersion with effective agonist concentrations for half maximal response (EC₅₀s) of 2.8 · 10⁻¹¹ mol · l⁻¹, 7.0 · 10⁻⁷ mol · l⁻¹ and 3.3 · 10⁻⁷ mol · l⁻¹, respectively. KT5720 (10⁻⁷ mol · l⁻¹ and 10⁻⁶ mol · l⁻¹) significantly shifted the dose response curve to α -PDH to the right. Dibutyryl cyclic guanosine 3′5′ monophosphate (dbcGMP) was ineffective in inducing either pigment aggregation or dispersion. 2′5′ dideoxyadenosine (DDA) and SQ22,536 essentially elicit a pigment-aggregating response in a dose-dependent manner. These effects were not due to the activation of purinergic receptors, since concentrations up to 10⁻⁴ mol · l⁻¹ of adenosine and adenosine triphosphate (ATP), and up to 10⁻³ mol · l⁻¹ of uracil triphosphate (UTP) did not elicit pigment aggregation. In order to verify if PCH decreased cyclic adenosine 3′5′ monophosphate (cAMP) levels, cumulative dose-response curves to PCH in the absence and presence of pertussis toxin and 8-MOM-IBMX were determined. However, neither drug significantly affected PCH activity. The levels of cAMP in the integument cells of M. potiuna were significantly increased (P < 0.05) by α -PDH (10⁻⁷ mol · l⁻¹) and forskolin (10⁻⁶ mol · l⁻¹), but were not affected by PCH (10⁻⁷ or 10⁻¹⁰ mol · l⁻¹). In conclusion, α -PDH seems to elicit pigment dispersion through the activation of a Gs-protein coupled receptor resulting in cAMP increase and cAMP-dependent protein kinase (PKA) activation. Furthermore, although a decrease in cAMP was assumed to be responsible in turn for the action of PCH, such a decrease could not be directly demonstrated. Accepted: 11 August 1998     

Exchanges of oxygen, carbon dioxide, nitrogen and water in the caecilian Dermophis mexicanus

Oxygen consumption was measured in five Dermophis mexicanus and averaged (±SEM) 0.047 ± 0.004 ml O₂ g⁻¹ h⁻¹. Carbon dioxide production averaged 0.053 ± 0.005 ml CO₂ g⁻¹ h⁻¹ in the same five animals 1 week later. This metabolic rate is similar to metabolic rates of other Gymnophionans but lower than metabolic rates reported for Anurans and Urodeles. Total nitrogen excretion averaged 1.37 μmol N g⁻¹ h⁻¹ which is higher than that found for other amphibians. Of this, 82.5% (1.13 μmol N g⁻¹ h⁻¹) was in the form of urea while 17.5% (0.24 μmol N g⁻¹ h⁻¹) was in the form of NH₃ + NH⁺ ₄. Such ureotelism is typical of terrestrial amphibians like D. mexicanus. Osmotic water flux averaged 0.0193 ml g⁻¹ h⁻¹ in control (sham injected) animals and was not significantly altered by injection of either arginine vasotocin or mesotocin. This osmotic flux is similar to osmotic fluxes found for other terrestrial amphibians. The combined data suggest that metabolism in D. mexicanus is, like most other Gymnophionans, lower than other amphibians. The high rates of nitrogen (especially urea) excretion suggests that this fossorial animal accumulates urea like other burrowing amphibians. Accepted: 27 June 2000     

Cis-diammineplatinum(II) forms a macrochelate with 2′-deoxycytidine 5′-monophosphate (dCMP2–)! Reactivity and acid-base properties of cis-Pt(NH3)2(dCMP)

 The synthesis of cis-Pt(NH₃)₂(dCMP) is reported and by various physico-chemical methods it is demonstrated that it is a macrochelate in which Pt(II) is bound simultaneously to the N3 site of cytosine in dCMP^2– and to a phosphate-oxygen atom. According to the NOESY spectra (cross-peaks between cytosine H6 and H2′ and H3′) the cytosine ring adopts an anti orientation. Highly unusual is the significant (1 ppm) downfield shift of the sugar proton H5″ in the ¹H-NMR spectrum and the sensitivity of the cytosine H6 resonance on the protonation state of the phosphate group. Based on these three features a geometry for the macrochelate is proposed. The compound is a major product of the reaction of cis-[Pt(NH₃)₂(H₂O)₂]²⁺ with dCMP^2– at neutral pH, but it even forms at pH 5. By applying pD-dependent NMR spectroscopy (¹H, ³¹P) and potentiometric pH titration, it is demonstrated that the Pt-coordinated phosphate group can be protonated (pK _a/1=3.21±0.10 and 3.31±0.05, respectively), and ¹H- and ³¹P-NMR spectra also indicate deprotonation (pK _a/2=13.35±0.25) of the exocyclic amino group of the cytosine moiety. The metal ion binding affinity of cis-Pt(NH₃)₂(dCMP) is very small, as shown for Cu²⁺ (log K<0.6). The cis-Pt(NH₃)₂(dCMP) complex reacts with nucleosides and nucleotides (L′) by losing its chelate structure and forming mixed ligand complexes, cis-Pt(NH₃)₂(dCMP)(L′); this means that the phosphate group is released from the coordination sphere of Pt(II), indicating that the Pt(II)-O(phosphate) bond is not very strong. Received: 23 October 1997 / Accepted: 17 February 1998     

Intracellular pH regulation and buffer capacity in CO2/HCO− 3-buffered media in cultured epithelial cells from rainbow trout gills

The influence of a CO₂/HCO⁻ ₃-buffered medium on intracellular pH regulation of gill pavement cells from freshwater rainbow trout was examined in monolayers grown in primary culture on glass coverslips; intracellular pH (pH_i) was monitored by continuous spectrofluorometric recording from cells loaded with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxy-fluoroscein. When cells in HEPES-buffered medium at normal pH=7.70 were transferred to normal CO₂/HCO⁻ ₃-buffered medium {P _CO2=3.71 mmHg, [HCO⁻ ₃]= 6.1 mmol l⁻¹, extracellular pH (pH_e)=7.70}, they exhibited a brief acidosis but subsequently regulated the same pHi (∼7.41) as in HEPES. Buffer capacity (β) increased by the expected amount (5.5–8.0 slykes) based on intracellular [HCO⁻ ₃], and was unaffected by most drugs and treatments. However, after transfer to high P _CO2=11.15 mmHg, [HCO⁻ ₃]= 18.2 mmol l⁻¹ at the same pH_e=7.70, the final regulated pHi was elevated (∼7.53). The rate of correction of alkalosis caused by washout of this high P _CO2, high-HCO⁻ ₃ medium was unaffected by removal of extracellular Cl⁻. Removal of extracellular Na⁺ lowered resting pH_i and greatly inhibited the rate of pH_i recovery from acidosis. Bafilomycin A₁ (3 μmol l⁻¹) had no effect on these responses. However amiloride (0.2 mmol l⁻¹) inhibited recovery from acidosis caused by washout of an ammonia prepulse, but did not affect resting pH_i, the latter differing from the response in HEPES where amiloride also lowered resting pH_i. Similarly 4-acetamido-4′- isothiocyanatostilbene-2,2′-disulfonic acid, sodium salt (0.1 mmol l⁻¹) did not affect resting pH_i but slowed the rate of recovery from acidosis, though to a lesser extent than amiloride. Removal of extracellular Cl⁻ also slowed the rate of recovery but greatly increased β by an unknown mechanism; when this was taken into account, H⁺ extrusion rate was unaffected. These results are consistent with the presence of Na⁺-(HCO⁻ ₃)_N co-transport and/or Na⁺-dependent HCO⁻ ₃/Cl⁻ exchange, in addition to Na⁺/H⁺ exchange, as mechanisms contributing to “housekeeping” pH_i regulation in gill cells in CO₂/HCO⁻ ₃ media, whereas only Na⁺/H⁺ exchange is seen in HEPES. Both Na⁺-independent Cl⁻/HCO⁻ ₃ exchange and V-type H⁺-ATPase mechanisms appear to be absent from these cells cultured in isotonic media. Accepted: 30 November 1999     

Production of sophorolipids from whey

Sophorolipids, obtained by a two-stage process starting from deproteinized whey concentrate using Cryptococcus curvatus ATCC 20509 and Candida bombicola ATCC 22214, were compared to products from one-stage processes, using different lipidic compounds as substrates. Results showed that above all carbon source and not cultivation conditions had a distinct influence on the composition of the crude product mixture and therefore on the physicochemical and biological properties of the sophorolipids, such as, for example, surface activity, cytotoxicity and stability against hydrolases. The results were completed by corresponding data for purified mono- and diacetylated (17-hydroxyoctadecenoic)-1′,4′′-lactonized sophorolipids. Crude sophorolipid mixtures showed moderate to good surface active properties (SFT^min 39 mN m⁻¹, CMC 130 mg l⁻¹), water solubilities (2–3 g l⁻¹) and low cytotoxicities (LC₅₀ 300–700 mg l⁻¹). In contrast, purified sophorolipids were more surface active (SFT^min 36 mN m⁻¹, CMC 10 mg l⁻¹), less water soluble (max. 70 mg l⁻¹) and showed stronger cytotoxic effects (LC₅₀ 15 mg l⁻¹). Incubation of crude sophorolipid mixtures with different hydrolases demonstrated that treatment with commercially available lipases such as from Candida rugosa and Mucor miehei distinctly reduced the surface active properties of the sophorolipids, while treatment with porcine liver esterase and glycosidases had no effect. Received: 23 February 1999 / Received revision: 27 May 1999 / Accepted: 28 May 1999     

Effect of ammonia on the anaerobic degradation of protein by a mesophilic and thermophilic biowaste population

The influence of ammonia on the anaerobic degradation of peptone by mesophilic and thermophilic populations of biowaste was investigated. For peptone concentrations from 5 g l⁻¹ to 20 g l⁻¹ the mesophilic population revealed a higher rate of deamination than the thermophilic population, e.g. 552 mg l⁻¹ day⁻¹ compared to 320 mg l⁻¹ day⁻¹ at 10 g l⁻¹ peptone. The final degree of deamination of the thermophilic population was, however, higher: 102 compared to 87 mg NH₃/g peptone in the mesophilic cultures. If 0.5–6.5 g l⁻¹ ammonia was added to the mesophilic biowaste cultures, deamination of peptone, degradation of its chemical oxygen demand (COD) and formation of biogas were increasingly inhibited, but no hydrogen was formed. The thermophilic biowaste cultures were most active if around 1 g ammonia l⁻¹ was present. Deamination, COD degradation and biogas production decreased at lower and higher ammonia concentrations and hydrogen was formed in addition to methane. Studies of the inhibition by ammonia of peptone deamination, COD degradation and methane formation revealed a K _i (50%) for NH₃ of 92, 95 and 88 mg l⁻¹ at 37 °C and 251, 274 and 297 mg l⁻¹ at 55 °C respectively. This indicated that the thermophilic flora tolerated significantly more NH₃ than the mesophilic flora. In the mesophilic reactor effluent 4.6 × 10⁸ peptone-degrading colony-forming units (cfu)/ml were culturable, whereas in the thermophilic reactor effluent growth of only 5.6 × 10⁷ cfu/ml was observed. Received: 24 April 1998 / Received revision: 26 June 1998 / Accepted: 27 June 1998     

Spin polymerization of DNA/RNA nucleotides

The Car-Parrinello Molecular Dynamics (CPMD) has been used to study the ion-radical (IR) polymerization (triplet (T) and singlet (S/T₀) states) of adenine mononucleotides upon interaction with Mg²⁺(H₂O)₂-ATP⁴⁻. It has been found that the IR polymerization occurs only upon Mg²⁺(H₂O)₂-ATP⁴⁻ excitation into a T state (the Franck-Condon or femtosecond laser excitation); it naturally occurs in the dark with DNA polymerase or another Mg-holoenzyme upon interaction of Mg with two Asp residues. The IR path affects only the HO-C_3′ group of ribose, leaving the HO-C_2′ group inactive. The IR polymerization starts with the homolytic removal of the hydrogen atom from the HO-C_3′ group and its transfer onto the hydroxyl radical ·OH, a product of the ATP cleavage, which yields a water molecule. A further progress of the reaction involves interaction between two ion-radicals ·ATP. The reaction is sensitive to the recombination of ·OH and ·ATP. It is mostly suppressed by the appearance of identically directed electron spins on both radicals (the radical pair in the T₀ state) in the vicinity of the HO-C_3′ group and not suppressed in the vicinity of the HO-C_2′ group (the spins in the radical pair are oppositely directed, the radical pair in the T₀ state), making the latter inert on the IR polymerization, but allowing it to be active in the ionic (hydrolytic) polymerization.