Sensitivity and response dynamics of elasmobranch electrosensory primary afferent neurons to near threshold fields

Elasmobranch fishes localize weak electric sources at field intensities of <5 ηV cm⁻¹, but the response dynamics of electrosensory primary afferent neurons to near threshold stimuli in situ are not well characterized. Electrosensory primary afferents in the round stingray, Urolophus halleri, have a relatively high discharge rate, a regular discharge pattern and entrain to 1-Hz sinusoidal peak electric field gradients of ≤20 ηV cm⁻¹. Peak neural discharge for units increases as a non-linear function of stimulus intensity, and unit sensitivity (gain) decreases as stimulus intensity increases. Average peak rate-intensity encoding is commonly lost when peak spike rate approximately doubles that of resting, and for many units occurs at intensities <1 μV cm⁻¹. Best neural sensitivity for nearly all units is at 1–2 Hz with a low-frequency slope of 8 dB/decade and a high-frequency slope of −23 dB/decade. The response characteristics of stingray electrosensory primary afferents indicate sensory adaptations for detection of extremely weak phasic fields near 1–2 Hz. We argue that these properties reflect evolutionary adaptations in elasmobranch fishes to enhance detection of prey, communication and social interactions, and possibly electric-mediated geomagnetic orientation. Accepted: 20 June 1997     

Microclimatic parameters influencing nitrogen fixation in the phyllosphere in a Costa Rican premontane rain forest

The acetylene reduction method was used to measure nitrogen fixation in the phyllosphere of attached leaves of different phorophytes under natural conditions in a premontane rain forest in Costa Rica. Maximum rates of nitrogen fixation (26 ng N · cm⁻² leaf area · h⁻¹) – mainly due to the activity of two species of Scytonema (Cyanobacteria) – were measured in the rainy season in bright sunlight. Rates of nitrogen fixation were correlated with the leaf area covered by Scytonema. In periods without precipitation the fixation activity decreased to zero within 2–3 days. As long as the epiphylls were sufficiently supplied with water, other microclimatic factors like temperature and light intensity also influenced nitrogen fixation rates, but to a lesser extent. Relative humidity and species of phorophyte showed no direct influence. It was concluded that the most important factor for nitrogen fixation in the phyllosphere was the availability of liquid water. Linking these results to meteorological data, the input of nitrogen by biological nitrogen fixation in the phyllosphere in the investigation area was estimated to be as much as 1.6 ± 0.8 kg N · ha⁻¹ · year⁻¹ per unit of leaf area index (LAI). For an LAI of 2 for the understory the nitrogen input would vary between 2 and 5 kg N · ha⁻¹ · year⁻¹. This work also demonstrates the importance of detailed knowledge of variation in microclimate throughout the year as a basis for extrapolation of the annual nitrogen input. Received: 21 December 1997 / Accepted: 14 June 1998     

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     

Investigation of exchange couplings in [Fe3S4]+ clusters by electron spin-lattice relaxation

3 S₄]⁺, S=1/2, composed of three, antiferromagnetically coupled high-spin ferric ions) by continuous wave (CW) and pulsed EPR techniques: Azotobacter vinelandii ferredoxin I, Desulfovibrio gigas ferredoxin II, and the 3Fe forms of Pyrococcus furiosus ferredoxin and aconitase. The 35 GHz (Q-band) CW EPR signals are simulated to yield experimental g tensors, which either had not been reported, or had been reported only at X-band microwave frequency. Pulsed X- and Q-band EPR techniques are used to determine electron spin-lattice (T ₁, longitudinal) relaxation times at several positions on the samples' EPR envelope over the temperature range 2–4.2 K. The T ₁ values vary sharply across the EPR envelope, a reflection of the fact that the envelope results from a distribution in cluster properties, as seen earlier as a distribution in g ₃ values and in ⁵⁷ Fe hyperfine interactions, as detected by electron nuclear double resonance spectroscopy. The temperature dependence of 1/T ₁ is analyzed in terms of the Orbach mechanism, with relaxation dominated by resonant two-phonon transitions to a doublet excited state at ∼20 cm⁻¹ above the doublet ground state for all four of these 3Fe proteins. The experimental EPR data are combined with previously reported ⁵⁷Fe hyperfine data to determine electronic spin exchange-coupling within the clusters, following the model of Kent et al. Their model defines the coupling parameters as follows: J ₁₃=J, J ₁₂=J(1+ε′), J ₂₃=J(1+ε), where J _ij is the isotropic exchange coupling between ferric ions i and j, and ε and ε′ are measures of coupling inequivalence. We have extended their theory to include the effects of ε′≠0 and thus derived an exact expression for the energy of the doublet excited state for any ε, ε′. This excited state energy corresponds roughly to ε J and is in the range 5–10 cm⁻¹ for each of these four 3Fe proteins. This magnitude of the product ε J, determined by our time-domain relaxation studies in the temperature range 2–4 K, is the same as that obtained from three other distinct types of study: CW EPR studies of spin relaxation in the range 5.5–50 K, NMR studies in the range 293–303 K, and static susceptibility measurements in the range 1.8–200 K. We suggest that an apparent disagreement as to the individual values of J and ε be resolved in favor of the values obtained by susceptibility and NMR (J≳200 cm⁻¹ and ε≳0.02 cm⁻¹ ), as opposed to a smaller J and larger ε as suggested in CW EPR studies. However, we note that this resolution casts doubt on the accepted theoretical model for describing the distribution in magnetic properties of 3Fe clusters. Received: 23 December 1999 / Accepted: 8 March 2000     

Intramolecular electron transfer in the oxidation of amines by methylamine oxidase from Arthrobacter P1

 The intramolecular electron-transfer rate constant for the Cu(II)–topa_NH2⇌ Cu(I)–topa_SQ equilibrium in methylamine oxidase has been measured by temperature-jump relaxation techniques. At pH 7.0 the estimated k_obs = 150±30 s^–1 for both methylamine and benzylamine; assuming the equilibrium constant is ≈0.7–1 at pH 7.0 and 296 K, this would correspond to a forward electron-transfer rate constant k_ET≈ 60–75 s^–1. Although substantially slower than the previously determined k_ET≈ 20 000 s^–1 for pea seedling amine oxidase [5] steady-state kinetics measurements established that k_ET > k_cat≈ 4–10 s^–1. Thus the Cu(I)-semiquinone state is a viable intermediate in methylamine oxidase turnover. Received: 16 August 1995 / Accepted: 21 December 1995     

Quantification of arbuscular mycorrhizal fungi activity by the glomalin concentration on hyphal traps

 Strips of horticultural film (16–32 cm²) were used to trap extraradical hyphae emanating from roots of sudangrass [Sorghum sudanense (Piper) Staph] enclosed in 40-μm mesh bags and colonized by Gigaspora rosea FL 224-1, Glomus intraradices EY 113/114, or Glomus caledonium UK 301-1. Strips of film were placed at opposite sides of 17–21 replicate sand culture pots for each isolate and were removed after 12–14 weeks of plant growth. To extract glomalin, a strip was cut into small pieces and submerged in 2 ml of 20 mM citrate, pH 7.0 and then autoclaved for 60 min. A quantitative enzyme-linked immunosorbent assay (ELISA) detected 0.005–0.04 μg glomalin in the volume of extract tested. The Bradford protein assay detected 1.25–5 μg of protein in the volume of extract tested. Both assays gave results ranging from 5–40 μg glomalin/cm² of film. Protein assay values were correlated with ELISA values (r=0.6091, P≤0.001, n=118). Analysis of variance indicated that isolates differed in Bradford protein values (P=0.001), but not ELISA values (P=0.154). Spatial variability of glomalin deposition ca. 7 cm from roots on opposite sides of pots was indicated by significant paired T tests (P<0.05) for protein values for each of the three isolates and ELISA for two isolates. These results indicate that hyphal traps, Bradford protein assay and ELISA are useful to assess hyphal activity over a growing season. Accepted: 11 October 1998     

Pelagic occurrence of the sympagic amphipod Gammarus wilkitzkii in ice-free waters of the Greenland Sea – dead end or part of life-cycle?

The sympagic (=ice-associated) amphipod Gammarus wilkitzkii usually lives attached to the underside of Arctic sea ice. During an expedition to the Greenland Sea in May/June 1997, high numbers of this species were found in pelagic Rectangular Midwater Trawl catches (0–500 m water depth) in an ice-free area, 35–42 km away from the ice edge. The amphipods seemed to have maintained position in the water column for at least 4 days. Mean biomass data (length: 2.9 cm, organic content: 73% dry mass), gut fullness (>50% in 85% of specimens) and sex ratio (females:males = 1:1.5) of these amphipods were very similar to values for under-ice populations. Due to their relatively high body density (mean: 1.134 g cm⁻³), the energy demand for swimming was assumed to be high. Measurements of oxygen consumption of swimming and resting amphipods (8.8 and 4.0 J g wet mass⁻¹ day⁻¹, respectively) suggested that, from an energetic point of view, G. wilkitzkii would maintain position in an ice-free water column for the time period. Accepted: 11 January 1999     

Osmotic water permeability of isolated vacuoles

We measured the osmotic water permeability (P _os) of vacuoles isolated from onion (Allium cepa L.), rape (Brassica napus L.), petunia (Petunia hybrida Hook.) and red beet (Beta vulgaris L.). For all the vacuolar types investigated, P _os values were in the range 200–1000 μm s⁻¹. The change in membrane surface area induced by an osmotic gradient was smaller than 2–6%. The vacuolar P _os values for red beet and onion were reduced by 1 mM HgCl₂, to 14% and 30% of the control values, respectively, but were partially restored to 51% and 76% by 5 mM β-mercaptoethanol. These results suggest that aquaporins were present in all the vacuoles tested. In HgCl₂-treated onion vacuoles, the reduced P _os (56 μm s⁻¹) had a low activation energy (approx. 6 kJ mol⁻¹), indicating that water permeation was still occurring mainly via aquaporins, and that the water permeability of the lipid part of the vacuolar membrane is probably very low. Received: 18 February 1999 / Accepted: 21 June 1999     

Molecular modeling studies of poly lactic acid initiation mechanisms

The two possible routes to synthesize poly (lactic acid) are polycondensation of the lactic acid and ring opening polymerization (ROP) of the lactide. This work involves molecular modeling of the polymerization initiation mechanisms using different initiators a) H₂SO₄ for polycondensation b) aluminum isopropoxide for coordination-insertion ROP c)methyl triflate for cationic ROP, and d) potassium methoxide for anionic ROP. For molecular modeling of PLA, we have benchmarked our approach using Ryner’s work on ROP of L-lactide using stannous (II) 2-ethylhexanoate (Sn(Oct)₂) and methanol as initiators. Our values of -15.2 kcal mol^-1 and -14.1 kcal mol^-1 for enthalpy changes in the two steps of activated complex formation match with Ryner’s. Geometric and frequency optimizations have been done on Gaussian’03 using B3LYP density functional theory along with the basis sets LANL2DZ for metal atoms and 6–31G* and 6–31G** for non metal atoms. The kinetic rate constant for each mechanism has been calculated using the values of energy of activation, change in enthalpy, Gibbs free energy, entropy and the partition functions from the Gaussian’03 output. Our polycondensation rate constant value of 1.07 × 10^–4 se^-1 compares well with 1.51 × 10^–4 se^-1 as reported by Wang. However, ROP rate constants could not be validated due to lack of experimental data. Figure Cationic Ring Opening Polymerization of L-Lactide

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Phytoplankton biomass and production during late austral spring (1991) and summer (1993) in the Bransfield Strait

Phytoplankton biomass and productivity were measured during two cruises in the Bransfield Strait in December 1991 (D91) and January/February 1993 (J93). Strong seasonal variability in productivity values was observed due to differences in the physiological response of phytoplankton. However, although the photosynthetic capacity of phytoplankton was markedly lower in D91 [P _m ^B =0.61 ± 0.25 mg C (mg Chla)⁻¹ h⁻¹] than in J93 [P _m ^B =2.18 ± 0.91 mg C (mg Chla)⁻¹ h⁻¹], average water column chlorophyll values in different areas of the strait were approximately similar in D91 (49–78 mg Chla m⁻²) and J93 (22–76 mg Chla m⁻²). The spatial distribution of chlorophyll was patchy and generally associated with the influence of the different water masses that meet together in the Bransfield Strait. No correlation was found between the mixed layer depth and either the integrated chlorophyll or the productivity. Our results suggest that major phytoplankton blooms in the Bransfield Strait are advected from the nearby Gerlache Strait or Bellingshausen Sea following the main eastward surface currents. Accepted: 5 July 1998     

Cysteine ligand vibrations are responsible for the complex resonance Raman spectrum of azurin

 In the redox center of azurin, the Cu(II) is strongly coordinated to one thiolate S from Cys 112 and two imidazole Ns from His 46 and 117. This site yields a complex resonance Raman (RR) spectrum with >20 vibrational modes between 200 and 1500 cm^–1. We have investigated the effects of ligand-selective isotope replacements on the RR spectrum of Pseudomonas aeruginosa azurin to determine the relative spectral contribution from each of the copper ligands. Growth on ³⁴S-sulfate labels the cysteine ligand and allows the identification of a cluster of bands with Cu–S(Cys) stretching character between 370 and 430 cm^–1 whose frequencies are consistent with the trigonal or distorted tetrahedral coordination in type 1 sites. In type 2 copper-cysteinate sites, the lower ν (Cu–S) frequencies between 260 and 320 cm^–1 are consistent with square-planar coordination. Addition of exogenous ¹⁵N-labeled imidazole or histidine to the His117Gly mutant generates type 1 or type 2 sites, respectively. Because neither the above nor the His46Gly mutant reconstituted with ¹⁵N-imidazole exhibits significant isotope dependence, the histidine ligands can be ruled out as important contributors to the RR spectrum. Instead, a variety of evidence, including extensive isotope shifts upon global substitution with ¹⁵N, suggests that the multiple RR modes of azurin are due principally to vibrations of the cysteine ligand. These are resonance-enhanced through kinematic coupling with the Cu–S stretch in the ground state or through an excited-state A-term mechanism involving a Cu-cysteinate chromophore that extends into the peptide backbone. Received: 29 July 1996 / Accepted: 9 November 1996     

Effects of temperature on photosynthesis of two morphologically contrasting plant species along an altitudinal gradient in the tropical high Andes

The effects of temperature on photosynthesis of a rosette plant growing at ground level, Acaena cylindrostachya R. et P., and an herb that grows 20–50 cm above ground level, Senecio formosus H.B.K., were studied along an altitudinal gradient in the Venezuelan Andes. These species were chosen in order to determine – in the field and in the laboratory – how differences in leaf temperature, determined by plant form and microenvironmental conditions, affect their photosynthetic capacity. CO₂ assimilation rates (A) for both species decreased with increasing altitude. For Acaena leaves at 2900 m, A reached maximum values above 9 μmol m⁻² s⁻¹, nearly twice as high as maximum A found at 3550 m (5.2) or at 4200 m (3.9). For Senecio leaves, maximum rates of CO₂ uptake were 7.5, 5.8 and 3.6 μmol m⁻² s⁻¹ for plants at 2900, 3550 and 4200 m, respectively. Net photosynthesis-leaf temperature relations showed differences in optimum temperature for photosynthesis (A _o.t.) for both species along the altitudinal gradient. Acaena showed similar A _o.t. for the two lower altitudes, with 19.1°C at 2900 m and 19.6°C at 3550 m, while it increased to 21.7°C at 4200 m. Maximum A for this species at each altitude was similar, between 5.5 and 6.0 μmol m⁻² s⁻¹. For the taller Senecio, A _o.t. was more closely related to air temperatures and decreased from 21.7°C at 2900 m, to 19.7°C at 3550 m and 15.5°C at 4200 m. In this species, maximum A was lower with increasing altitude (from 6.0 at 2900 m to 3.5 μmol m⁻² s⁻¹ at 4200 m). High temperature compensation points for Acaena were similar at the three altitudes, c. 35°C, but varied in Senecio from 37°C at 2900 m, to 39°C at 3550 m and 28°C at 4200 m. Our results show how photosynthetic characteristics change along the altitudinal gradient for two morphologically contrasting species influenced by soil or air temperatures. Received: 5 July 1997 / Accepted: 25 October 1997     

Azide adducts of stearoyl-ACP desaturase: a model for μ-1,2 bridging by dioxygen in the binuclear iron active site

 The stearoyl-acyl carrier protein Δ⁹ desaturase (Δ9D) uses an oxo-bridged diiron center to catalyze the NAD(P)H– and O₂–dependent desaturation of stearoyl-ACP. Δ9D, ribonucleotide reductase, and methane monooxygenase have substantial similarities in their amino acid primary sequences and the physical properties of their diiron centers. These three enzymes also appear to share common features of their reaction cycles, including the binding of O₂ to the diferrous state and the subsequent generation of transient diferric-peroxo and diferryl species. In order to investigate the coordination environment of the proposed diferric-peroxo intermediate, we have studied the binding of azide to the diiron center of Δ9D using optical, resonance Raman (RR), and transient kinetic spectroscopic methods. The addition of azide results in the appearance of new absorption bands at 325 nm and 440 nm (k _app≈3.5 s^–1 in 0.7 M NaN₃, pH 7.8). RR experiments demonstrate the existence of two different adducts: an η¹–terminal structure at pH 7.8 (¹⁴N₃ ^– asymmetric stretch at 2073 cm^–1, resolved into two bands with ¹⁵N¹⁴N₂ ^–) and a μ-1,3 bridging structure at pH<7 (¹⁴N₃ ^– asymmetric stretch at 2100 cm^–1, shifted as a single band with ¹⁵N¹⁴N₂ ^–). Both adducts also exhibit an Fe–N₃ stretching mode at ≈380 cm^–1, but no accompanying Fe–O–Fe stretching mode, presumably due to either protonation or loss of the oxo bridge. The ability to form a μ-1,3 bridging azide supports the likelihood of a μ-1,2 bridging peroxide as a catalytic intermediate in the Δ9D reaction cycle and underscores the adaptability of binuclear sites to different bridging geometries. Received: 23 August 1996 / Accepted: 4 October 1996     

Biosurfactant production by a thermophilic Bacillus subtilis strain

A strain of Bacillus subtilis was able to grow and produce a biosurfactant on 2% sucrose at 45°C. As a result of biosurfactant synthesis the surface tension of the medium was reduced from 68 dynes cm⁻¹ to 28 dynes cm⁻¹. The strain had the capacity to produce the biosurfactant at high NaCl concentrations (4%) and a wide range of pH (4.5–10.5). The biosurfactant retained its surface-active properties after heating at 100°C for 2 h and at different pH values (4.5–10.5). A maximum amount of biosurfactant was produced when urea or nitrate ions were supplied as nitrogen source. The use of the biosurfactant at high temperatures, acidic, alkaline and saline environments is discussed. As a result of its action, 62% of oil in a sand pack column could be recovered, indicating its potential application in microbiologically enhanced oil recovery. Received 28 March 1996/ Accepted in revised form 16 September 1996     

Fluorescent siderophore-based chemosensors: iron(III) quantitative determinations

 A highly sensitive and selective method is described for a rapid and easy determination of iron(III). This procedure is based on fluorimetric detection combined with the attractive properties of siderophores and biomimetic ligands, which are strong and selective ferric chelators. Azotobactin δ, a bacterial fluorescent siderophore, three fluorescent derivatives of desferriferrioxamine B with a linear structure (NBD-, MA-, NCP-desferriferrioxamine B) and one tripodal biomimetic ligand of desferriferrichrome carrying an anthracenyl fluorescent probe were examined. A very efficient static quenching mechanism by iron was observed for all the ligands considered in this work. Our results identify azotobactin δ as the most promising chemosensor of ferric traces in water, more sensitive than the NBD-desferriferrioxamine B fluorescent ligand. Under more lipophilic conditions, the anthryl-desferriferrichrome biomimetic analogue showed similar analytical potential and was found to be more sensitive than the lipophilic MA- and NCP-desferriferrioxamine B. Their detection limits were respectively 0.5 ng mL^–1 for azotobactin δ and 0.6 ng mL^–1 for the anthryl tripodal chelator. The calibration curves were linear over the range 0–95 ng mL^–1 and 0–180 ng mL^–1. Various foreign cations have been examined and only copper(II) and aluminium(III) were shown to interfere when present in similar concentrations as iron(III). The developed procedure using fluorescent siderophores or biomimetic ligands of iron(III) may be applied (1) to monitor iron(III)-dependent biological systems and (2) to determine iron(III) quantitatively in natural waters and in biological systems. Received: 12 September 1998 · Accepted: 19 January 1999     

Repair of UVB-Damaged Skin by the Antioxidant Sulphated Flavone Glycoside Thalassiolin B Isolated from the Marine Plant Thalassia testudinum Banks ex König

Daily topical application of the aqueous ethanolic extract of the marine sea grass, Thalassia testudinum, on mice skin exposed to UVB radiation resulted in a dose-dependent recovery of the skin macroscopic alterations over a 6-day period. Maximal effect (90%) occurred at a dose of 240 μg/cm², with no additional effects at higher doses. Bioassay-guided fractionation of the plant extract resulted in the isolation of thalassiolin B (1). Topical application of 1 (240 μg/cm²) markedly reduces skin UVB-induced damage. In addition, thalassiolin B scavenged 2,2-diphenyl-2-picrylhydrazyl radical with an EC₅₀ = 100 μg/ml. These results suggest that thalassiolin B is responsible for the skin-regenerating effects of the crude extract of T. testudinum. Erik L. Regalado and María Rodríguez have contributed equally to this work and should be considered as first authors.     

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     

A new manganese superoxide dismutase identified from Beauveria bassiana enhances virulence and stress tolerance when overexpressed in the fungal pathogen

A superoxide dismutase (SOD) was characterized from Beauveria bassiana, a fungal entomopathogen widely applied to insect control. This 209-aa enzyme (BbSod2) showed no more than 71% sequence identity to other fungal Mn-SODs, sharing all conserved residues with the Mn-SOD family and lacking a mitochondrial signal. The SOD activity of purified BbSod2 was significantly elevated by Mn²⁺, suppressed by Cu²⁺ and Zn²⁺ but inhibited by Fe³⁺. Overexpressing the enzyme in a BbSod2-absent B. bassiana strain enhanced its SOD activity (107.2 ± 6.1 U mg⁻¹ protein) by 4–10-fold in different transformants analyzed. The best BbSod2-transformed strain with the SOD activity of 1,157.9 ± 74.7 U mg⁻¹ was 93% and 61% more tolerant to superoxide-generating menadione in both colony growth (EC₅₀ = 2.41 ± 0.03 versus 1.25 ± 0.01 mM) and conidial germination (EC₅₀ = 0.89 ± 0.06 versus 0.55 ± 0.07 mM), and 23% more tolerant to UV-B irradiation (LD₅₀ = 0.49 ± 0.02 versus 0.39 ± 0.01 J cm⁻²). Its virulence to Spodoptera litura larvae was enhanced by 26% [LT₅₀ = 4.5 (4.2–4.8) versus 5.7 (5.2–6.4) days]. Our study highlights for the first time that the Mn²⁺-cofactored, cytosolic BbSod2 contributes significantly to the virulence and stress tolerance of B. bassiana and reveals possible means to improving field persistence and efficacy of a fungal formulation by manipulating the antioxidant enzymes of a candidate strain.     

Modulation of Bacillus pasteurii cytochrome c 553 reduction potential by structural and solution parameters

 Direct cyclic voltammetry and ¹H NMR spectroscopy have been combined to investigate the electrochemical and spectroscopic properties of cytochrome c ₅₅₃ isolated from the alkaliphilic soil bacterium Bacillus pasteurii. A quasi-reversible diffusion-controlled redox process is exhibited by cytochrome c ₅₅₃ at a pyrolitic graphite edge microelectrode. The temperature dependence of the reduction potential, measured using a non-isothermal electrochemical cell, revealed a discontinuity at 308 K. The thermodynamic parameters determined in the low-temperature range (275–308 K;ΔS°′=–162.7±1.2 J mol^–1 K^–1, ΔH°′=–53.0±0.5 kJ mol^–1, ΔG°′=–4.5±0.1 kJ mol^–1, E°′=+47.0±0.6 mV) indicate the presence of large enthalpic and entropic effects, leading, respectively, to stabilization and destabilization of the reduced form of cytochrome c ₅₅₃. Both effects are more accentuated in the high-temperature range (308–323 K;ΔS°′=–294.1±8.4 J mol^–1 K^–1, ΔH°′=–93.4±3.1 kJ mol^–1, ΔG°′=–5.8±0.6 kJ mol^–1, E°′=+60.3±5.8 mV), with the net result being a slight increase of the standard reduction potential. These thermodynamic parameters are interpreted using the compensation theory of hydration of biopolymers as indicating the extrusion, upon reduction, of water molecules from the hydration sphere of the cytochrome. The low-T and high-T conformers differ by the number of water molecules in the solvation sphere: in the high-T conformer, the number of water molecules extruded upon reduction increases, as compared to the low-T conformer. The ionic strength dependence of the reduction potential at 298 K, treated within the frame of extended Debye-Hückel theory, yields values of E ^°′ _(I=0) =–25.4±1.4 mV, z _red=–11.3, and z _ox=–10.3. The pH dependence of the reduction potential at 298 K shows a plateau in the pH range 7–10 and an increase at more acidic pH, allowing the calculation of pK _O=5.5 and pK _R=5.7, together with the estimate of the reduction potentials of completely protonated (+71 mV) and deprotonated (+58 mV) forms of cytochrome c ₅₅₃. ¹H NMR spectra of the oxidized paramagnetic cytochrome c ₅₅₃ indicate the presence of a His-Met axial coordination of the low-spin (S=1/2) heme iron, which is maintained in the temperature interval 288–340 K at pH 7 and in the pH range 4.8–10.0 at 298 K. The temperature dependence of the hyperfine-shifted signals shows both Curie-type and anti-Curie-type behavior, with marked deviations from linearity, interpreted as indicating the presence of a fast equilibrium between the low-T and high-T conformers, having slightly different heme electronic structures resulting from the T-induced conformational change. Increasing the NaCl concentration in the range 0–0.2 M causes a slight change of the ¹H NMR chemical shifts of the hyperfine-shifted signals, with no influence on their linewidth. The calculated lower limit value of the apparent affinity constant for specific ion binding is estimated as 5.2±1.1 M^–1. The pH dependence of the isotropically shifted ¹H NMR signals of the oxidized cytochrome displays at least one ionization step with pK _O=5.7. The thermodynamic and spectroscopic data indicate a large solvent-derived entropic effect as the main cause for the observed low reduction potential of B. pasteurii cytochrome c ₅₅₃. Received: 9 January 1998 / Accepted: 8 April 1998     

Particle size effects in bioleaching of pyrite by acidophilic thermophile Sulfolobus metallicus (BC)

The effect of mineral particle size on the bioleaching of pyrite by the acidophilic thermophile Sulfolobus metallicus was investigated in a batch bioreactor. Decreasing the particle size from a mean diameter of 202 micron (size fraction: 150–180 micron) to a mean diameter of 42.5 micron (size fraction: 25–45 micron) enhanced the bioleaching rate from 0.05 kg m⁻³ h⁻¹ to 0.098 kg m⁻³ h⁻¹. The particle size distribution of the mineral in this range did not influence the morphology and growth kinetics of the cells. The values of specific growth rate (μ) and yield factor (Y) were 0.018–0.025 h⁻¹ and 0.67 × 10¹¹–1.45 × 10¹¹ cells (g iron)⁻¹, respectively. Decreasing the particle size of the mineral to a mean diameter of 6.40 micron (size fraction <25 micron) adversely influenced the activity of the cells. The presence of fine particles apparently damaged the structure of the cells, resulting in their inability to oxidise pyrite. Received: 11 December 1998 / Accepted: 9 April 1999