One-step purification and characterization of a β-1,4-glucosidase from a newly isolated strain of Stereum hirsutum

A highly efficient β-1,4-glucosidase (BGL) secreting strain, Stereum hirsutum SKU512, was isolated and identified based on morphological features and sequence analysis of internal transcribed spacer rDNA. A BGL containing a carbohydrate moiety was purified to homogeneity from S. hirsutum culture supernatants using only a single chromatography step on a gel filtration column. The relative molecular weight of S. hirsutum BGL was determined as 98 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis or 780 kDa by size exclusion chromatography, indicating that the enzyme is an octamer. S. hirsutum BGL showed the highest activity toward p-nitrophenyl-β-D-glucopyranoside (V _max = 3,028 U mg-protein⁻¹, k _cat = 4,945 s⁻¹) ever reported. The enzyme also showed good stability at an acidic pH ranging from 3.0 to 5.5. The BGL was able to promote transglycosylation with an activity of 42.9 U mg-protein⁻¹ using methanol as an acceptor and glucose as a donor. The internal amino acid sequences of the isolated enzyme showed significant homology with hydrolases from glycoside hydrolase family 1 (GH1), indicating that the S. hirsutum BGL is a member of GH1 family. The characteristics of S. hirsutum BGL could prove to be of interest in several potential applications, especially in enhancing flavor release during the wine fermentation process.     

Ecological Characterisation of the Colonic Microbiota in Arctic and Sub-Arctic Seals

Dominant colonic bacteria in wild hooded (n = 9), harbour (n = 1) and grey (n = 1) seals were identified using 16S rRNA gene clone libraries (313 clones), revealing 52.7% Bacteroidetes, 41.5% Firmicutes, 4.5% Proteobacteria and 1.0% Fusobacteria. Thirty (77%) of the 39 phylotypes identified were novel, showing <97% sequence similarity to their nearest cultivated relatives. Mean colonic bacterial cell density, determined by real-time PCR, was high (12.8 log₁₀ cells/g wet wt) for the hooded seals, while the number of methanogenic Archea was low (4.0 log₁₀ cells/g wet wt). The level of ampicillin (amp^r) and tetracycline-resistant (tet^r) isolates was investigated by cultivation. Aerobic amp^r isolates were only detected in colon contents from four hooded seals, whereas aerobic tet^r isolates were found in seven of the nine hooded seals. These data provide novel insight to the gut microbiota of Arctic and sub-Arctic seals living in the wild.     

Localization of ion-regulatory epithelia in embryos and hatchlings of two cephalopods

The tissue distribution and ontogeny of Na⁺/K⁺-ATPase has been examined as an indicator for ion-regulatory epithelia in whole animal sections of embryos and hatchlings of two cephalopod species: the squid Loligo vulgaris and the cuttlefish Sepia officinalis. This is the first report of the immunohistochemical localization of cephalopod Na⁺/K⁺-ATPase with the polyclonal antibody α (H-300) raised against the human α1-subunit of Na⁺/K⁺-ATPase. Na⁺/K⁺-ATPase immunoreactivity was observed in several tissues (gills, pancreatic appendages, nerves), exclusively located in baso-lateral membranes lining blood sinuses. Furthermore, large single cells in the gill of adult L. vulgaris specimens closely resembled Na⁺/K⁺-ATPase-rich cells described in fish. Immunohistochemical observations indicated that the amount and distribution of Na⁺/K⁺-ATPase in late cuttlefish embryos was similar to that found in juvenile and adult stages. The ion-regulatory epithelia (e.g., gills, excretory organs) of the squid embryos and paralarvae exhibited less differentiation than adults. Na⁺/K⁺-ATPase activities for whole animals were higher in hatchlings of S. officinalis (157.0 ± 32.4 μmol g_FM⁻¹ h⁻¹) than in those of L. vulgaris (31.8 ± 3.3 μmol g_FM⁻¹ h⁻¹). S. officinalis gills and pancreatic appendages achieved activities of 94.8 ± 18.5 and 421.8 ± 102.3 μmol_ATP g_FM⁻¹ h⁻¹, respectively. High concentrations of Na⁺/K⁺-ATPase in late cephalopod embryos might be important in coping with the challenging abiotic conditions (low pH, high pCO₂) that these organisms encounter inside their eggs. Our results also suggest a higher sensitivity of squid vs. cuttlefish embryos to environmental acid-base disturbances.     

Production of hexanoic acid from d-galactitol by a newly isolated Clostridium sp. BS-1

In a study screening anaerobic microbes utilizing d-galactitol as a fermentable carbon source, four bacterial strains were isolated from an enrichment culture producing H₂, ethanol, butanol, acetic acid, butyric acid, and hexanoic acid. Among these isolates, strain BS-1 produced hexanoic acid as a major metabolic product of anaerobic fermentation with d-galactitol. Strain BS-1 belonged to the genus Clostridium based on phylogenetic analysis using 16S rRNA gene sequences, and the most closely related strain was Clostridium sporosphaeroides DSM 1294^T, with 94.4% 16S rRNA gene similarity. In batch cultures, Clostridium sp. BS-1 produced 550 ± 31 mL L⁻¹ of H₂, 0.36 ± 0.01 g L⁻¹ of acetic acid, 0.44 ± 0.01 g L⁻¹ of butyric acid, and 0.98 ± 0.03 g L⁻¹ of hexanoic acid in a 4-day cultivation. The production of hexanoic acid increased to 1.22 and 1.73 g L⁻¹ with the addition of 1.5 g L⁻¹ of sodium acetate and 100 mM 2-(N-morpholino)ethanesulfonic acid (MES), respectively. Especially when 1.5 g L⁻¹ of sodium acetate and 100 mM MES were added simultaneously, the production of hexanoic acid increased up to 2.99 g L⁻¹. Without adding sodium acetate, 2.75 g L⁻¹ of hexanoic acid production from d-galactitol was achieved using a coculture of Clostridium sp. BS-1 and one of the isolates, Clostridium sp. BS-7, in the presence of 100 mM MES. In addition, volatile fatty acid (VFA) production by Clostridium sp. BS-1 from d-galactitol and d-glucose was enhanced when a more reduced culture redox potential (CRP) was applied via addition of Na₂S·9H₂O.     

Extraction and characterisation of alginate from brown seaweeds (Fucales,Phaeophyceae) collected from Port Dickson,Peninsular Malaysia

Four species of brown seaweeds, namely Sargassum baccularia, Sargassum binderi, Sargassum siliquosum and Turbinaria conoides, harvested from Port Dickson, Negeri Sembilan, Malaysia were analysed for ash content, alginate yield and alginate properties. Seaweeds calcined at 450°C were found to have low amount of non-combustible residue as these were not contaminated by calcareous animals. Alginate was extracted from these seaweeds by two methods: hot and cold. In the hot method, the storing time was 3 h and the processing temperature was 50°C, whilst in the cold method, the sample was stored overnight at room temperature. Higher yield of alginate was obtained by the hot method compared to the cold method, but alginate extracted by the cold method gave higher molecular weight. In the hot method, 49.9% of alginate was extracted from S. siliquosum, followed by T. conoides (41.4%), S. binderi (38.9%) and S. baccularia (26.7%). Alginate extracted from T. conoides has an average molecular weight, M _w, of 8.06 × 10⁵ g mol⁻¹, whereas alginate from S. siliquosum was the lowest in M _w (4.81 × 10⁵ g mol⁻¹) when the extraction was done at room temperature. Alginate extracted from S. baccularia was found to be very heat-sensitive. Its M _w has dropped more than 83%, from 7.52 × 10⁵ to 1.23 × 10⁵ g mol⁻¹, when the extraction temperature was raised. The effect of heat on the extent of depolymerisation of the alginate molecule of the other three brown seaweed species was less significant, with decrease in molecular weight ranging between 13% and 16%.     

Overexpression and biochemical characterization of DagA from <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> A3(2): an endo-type β-agarase producing neoagarotetraose and neoagarohexaose

The DagA product of Streptomyces coelicolor is an agarase with a primary translation product (35 kDa) of 309 amino acids, including a 30-amino acid signal peptide. Although dagA expression in Streptomyces lividans under the control of its own set of promoters was previously reported, its enzymatic properties have never been elucidated. To develop an improved expression system for dagA, three types of strong promoters for the Streptomyces host were linked to dagA, and their efficiencies in DagA production were compared in S. lividans TK24. All of the transformants with dagA grew at improved rates and produced larger amounts of DagA in the modified R2YE medium containing 0.5% agar as the sole carbon source. Of the three transformants, the S. lividans TK24/pUWL201-DagA (ermE promoter) produced the highest agarase activity (A ₅₄₀ = 4.24), and even the S. lividans TK24/pHSEV1-DagA (tipA promoter) and S. lividans TK24/pWHM3-DagA (sprT promoter) produced higher agarase activity (A ₅₄₀ = 0.24 and 0.12, respectively) than the control (A ₅₄₀ = 0.01) in the modified R2YE medium. The mature form of DagA protein (32 kDa) was successfully purified by one-step affinity column chromatography by using agarose beads with excellent yield. The purified DagA was found to exhibit maximal agarase activity at 40°C and pH 7.0. The K _m, V _max, and K _cat values for agarose were 2.18 mg/ml (approximately 1.82 × 10⁻⁵ M), 39.06 U/mg of protein, and 9.5 × 10³/s, respectively. Thin layer chromatography (TLC) analysis, matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry, and Fourier transform nuclear magnetic resonance (FT-NMR) spectrometry of the hydrolyzed products of agarose by DagA revealed that DagA is an endo-type β-agarase that degrades agarose into neoagarotetraose and neoagarohexaose.     

An altered camelid-like single domain anti-idiotypic antibody fragment of HM-1 killer toxin: acts as an effective antifungal agent

Phage-display and competitive panning elution leads to the identification of minimum-sized antigen binders together with conventional antibodies from a mouse cDNA library constructed from HM-1 killer toxin neutralizing monoclonal antibody (nmAb-KT). Antigen-specific altered camelid-like single-domain heavy chain antibody (scFv K2) and a conventional antibody (scFv K1) have been isolated against the idiotypic antigen nmAb-KT. The objectives of the study were to examine (1) their properties as compared to conventional antibodies and also (2) their antifungal activity against different pathogenic and non-pathogenic fungal species. The alternative small antigen-binder, i.e., the single-domain heavy chain antibody, was originated from a conventional mouse scFv phage library through somatic hyper-mutation while selection against antigen. This single-domain antibody fragment was well expressed in bacteria and specifically bound with the idiotypic antigen nmAb-KT and had a high stability and solubility. Experimental data showed that the binding affinity for this single-domain antibody was 272-fold higher (K _d = 1.07 × 10⁻¹⁰ M) and antifungal activity was three- to fivefold more efficient (IC₅₀ = 0.46 × 10⁻⁶ to 1.17 × 10⁻⁶ M) than that for the conventional antibody (K _d = 2.91 × 10⁻⁸ M and IC₅₀ = 2.14 × 10⁻⁶ to 3.78 × 10⁻⁶ M). The derived single-domain antibody might be an ideal scaffold for anti-idiotypic antibody therapy and the development of smaller peptides or peptide mimetic drugs due to their less complex antigen-binding site. We expect that such single-domain synthetic antibodies will find their way into a number of biotechnological or medical applications.     

Repeated batch cultivation of the microalga Spirulina platensis

Summary The cultivation of photosynthetic microorganisms such as the microalga Spirulina platensis can provide an alternative source of food. The water in Mangueira Lagoon (Rio Grande do Sul state, southern Brazil) has several required nutrients for the growth of Spirulina and could be added to culture medium to reduce the cost of producing S. platensis. Although little studied, repeated batch cultivation is a very useful technique because it has a better cost–benefit ratio than other cultivation methods. In a series of runs, we studied the influence of cell concentration, renewal rate and strain on the specific growth rate and biomass productivity of S. platensis during repeated batch cultivation, the runs taking place in 2-l Erlenmeyer flasks for 2160 h at 30 °C and a light intensity of 2500 lux under a 12 h photoperiod. The three factors studied had a significant (P < 0.05) effect on the results (specific growth rate and productivity). Using Zarrouk’s medium, the highest specific growth rate (μ_X) was 0.111 day⁻¹ while the biomass productivity (P _X) was 0.0423 g l⁻¹ day⁻¹, while Mangueira Lagoon water supplemented with 10% Zarrouk’s medium gave μ_X = 0.113 day⁻¹ and a productivity P _X = 0.0467 g l⁻¹ day⁻¹. These values were two to three times higher than the results obtained in batch cultivation, indicating that the repeated batch cultivation of S. platensis is attractive and convenient.     

Production and regeneration of protoplasts from <Emphasis Type="Italic">Grateloupia turuturu</Emphasis> Yamada (Rhodophyta)

Protoplasts were isolated enzymatically from the carrageenophyte red alga Grateloupia turuturu (Halymeniales, Rhodophyta) that occurs along the coast of the French Channel in Normandy. Effects of the main factors on the protoplast yield were identified to improve the isolation protocol. The optimal enzyme composition for cell wall digestion and protoplast viability consisted of 2% cellulase Onozuka R-10, 0.5% macerozyme R-10, 2% crude extract from viscera of Haliotis tuberculata, 0.8 M mannitol, 20 mM sodium citrate, 0.3% bovine serum albumin at 25°C, and 4-h incubation period. The protoplasts were approximately 5–15 μm in diameter, liberated mainly from the surface cell layers. Maximum yield was 1.5 × 10⁷ protoplasts g^-1 fresh tissue. The protoplasts underwent initial division after 14 days with a high density level of 1 × 10⁶ cells mL^-1 in culture medium and developed into microthalli of a line of two to six cells.     

The Relative Rates of Thiol–Thioester Exchange and Hydrolysis for Alkyl and Aryl Thioalkanoates in Water

This article reports rate constants for thiol–thioester exchange (k _ex), and for acid-mediated (k _a), base-mediated (k _b), and pH-independent (k _w) hydrolysis of S-methyl thioacetate and S-phenyl 5-dimethylamino-5-oxo-thiopentanoate—model alkyl and aryl thioalkanoates, respectively—in water. Reactions such as thiol–thioester exchange or aminolysis could have generated molecular complexity on early Earth, but for thioesters to have played important roles in the origin of life, constructive reactions would have needed to compete effectively with hydrolysis under prebiotic conditions. Knowledge of the kinetics of competition between exchange and hydrolysis is also useful in the optimization of systems where exchange is used in applications such as self-assembly or reversible binding. For the alkyl thioester S-methyl thioacetate, which has been synthesized in simulated prebiotic hydrothermal vents, k _a = 1.5 × 10⁻⁵ M⁻¹ s⁻¹, k _b = 1.6 × 10⁻¹ M⁻¹ s⁻¹, and k _w = 3.6 × 10⁻⁸ s⁻¹. At pH 7 and 23°C, the half-life for hydrolysis is 155 days. The second-order rate constant for thiol–thioester exchange between S-methyl thioacetate and 2-sulfonatoethanethiolate is k _ex = 1.7 M⁻¹ s⁻¹. At pH 7 and 23°C, with [R″S(H)] = 1 mM, the half-life of the exchange reaction is 38 h. These results confirm that conditions (pH, temperature, pK _a of the thiol) exist where prebiotically relevant thioesters can survive hydrolysis in water for long periods of time and rates of thiol–thioester exchange exceed those of hydrolysis by several orders of magnitude.     

Field metabolic rates and water uptake in the blossom-bat Syconycteris australis (Megachiroptera)

Blossom-bats, Syconycteris australis (18 g) are known to be highly active throughout the night. Since this species frequently enters torpor, we postulated that their use of heterothermy may be related to a high energy expenditure in the field. To test this hypothesis we measured field metabolic rates (FMR) of S. australis at a subtropical site using the doubly labelled water (DLW) method. We also measured DLW turnover in captive animals held at constant ambient temperature (T _a) with ad libitum food to estimate whether T _a and food availability affect energy expenditure under natural conditions. The FMR of S. australis was 8.55 ml CO₂ g⁻¹h⁻¹ or 76.87 kJ day⁻¹ which is 7.04 times the basal metabolic rate (BMR) and one of the highest values reported for endotherms to date. Mass-specific energy expenditure by bats in the laboratory was about two-thirds of that of bats in the field, but some of this difference was explained by the greater body mass in captive bats. This suggests that foraging times in the field and laboratory were similar, and daily energy expenditure was not strongly affected by T _a or ad libitum food. Water uptake in the field was significantly higher than in the laboratory, most likely because nectar contained more water than the laboratory diet. Our study shows that S. australis has a FMR that is about double that predicted for its size although its BMR is lower than predicted. This supports the view that caution must be used in making assumptions from measurements of BMR in the laboratory about energy and other biological requirements in free-ranging animals. Accepted: 4 January 1999     

High-quality RNA preparation for cDNA library construction of the Antarctic sea–ice alga <Emphasis Type="Italic">Chlamydomonas</Emphasis> sp. ICE-L

To study the molecular mechanism of the Antarctic sea–ice alga in adaptation to polar sea–ice environments, the RNA was prepared for cDNA library construction of Chlamydomonas sp. ICE-L. Three different methods were tested to prepare total RNA from this psychrophilic, unicellular green alga rich in protein and polysaccharide. Lauryl sodium sulfate- based method allowed a most effective extraction of high-quality total RNA compared to the other methods. Total RNA extracted with this protocol was used for cDNA library construction. The recombination rate of constructed cDNA library was 98.60%, the primary titer was 7.15 × 10⁶ pfu, and an average sequence length was 1.2 kb. These results show that with a high-quality RNA preparation, a cDNA library can be constructed successfully for Chlamydomonas sp. ICE-L.     

Comparison of metabolite levels in callus of <Emphasis Type="Italic">Tecoma stans</Emphasis> (L.) Juss. ex Kunth. cultured in photoperiod and darkness

Tecoma stans is a tropical plant from the Americas. Antioxidant activity and both phenolic compound and flavonoid total content were determined for callus tissue of T. stans cultured in either a set photoperiod or in darkness. Callus lines from three explant types (hypocotyls, stem, and leaf) were established on B5 culture medium supplemented with 0.5 μM 2,4-D and 5.0 μM kinetin. While leaf-derived callus grew slower under a 16-h photoperiod (specific growth rate, μ = 0.179 d⁻¹, t _D = 3.9 d) than in darkness (μ = 0.236 d⁻¹, t _D = 2.9 d), it accumulated the highest amount (p < 0.05) of both phenolics (86.6 ± 0.01 mg gallic acid equivalents/g) and flavonoids (339.6 ± 0.06 mg catechin equivalents/g). Similarly, antioxidant activity was significantly higher (p < 0.05) when callus was cultured in period light than when grown in extended darkness. Antioxidant activity measured with a 2,20-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS)-based assay was 350.5 ± 15.8 mmol Trolox/g extract for callus cultured under a defined photoperiod compared to 129.1 ± 7.5 mmol Trolox/g extract from callus cultured in darkness. Content of phenolic compounds and flavonoids was in agreement with a better antioxidant power (EC₅₀ = 450 μg extract/mg 1,1-diphenyl-2-picrylhydrazyl) and antiradical efficiency. Results of the present study show that calli of T. stans are a source of compounds with antioxidant activity that is favored by culture under a set photoperiod.     

Age and growth of Schizopygopsis younghusbandi younghusbandi in the Yarlung Zangbo River in Tibet, China

Schizopygopsis younghusbandi younghusbandi is an endemic species whose distribution is restricted to the middle reaches of the Yarlung Zangbo River, being one of the most important commercial fishes in this area. Age and growth of 606 specimens captured between October 2002 and April 2005 were studied. The range in standard length (L) was 65.7–387.3 mm and total weight (W) was 3.3–772.0 g. The relationship between L and W was W = 0.000909L^2.2493 for males and W = 0.000259L^2.4781 for females. Age, determined from anal scales and lapillus otoliths, ranged from 3 to 18 years. The parameters of von Bertalanffy growth functions, estimated by back-calculated length, were L_￥ = 442.7mm  LL_\infty = 442.7mm\;L, k = 0.0738 year⁻¹ and t ₀  = −1.4 year for males, and L_￥ = 471.4mm  LL_\infty = 471.4mm\;L, k = 0.0789 year⁻¹ and t ₀ = 0.2 year for females. Males and females exhibited statistically significant differences in growth. χ ²-test indicated that von Bertalanffy growth functions could well describe the growth of S. y. younghusbandi. The longevities were 39.2 and 38.2 years for males and females, respectively. Growth inflexion points were 10.2 and 12.0 years for males and females, respectively, but 84.8% of the captures were at the smaller ages. So conservation and management schemes for this population should be considered urgently. In addition, we found that populations from the upstream of the Lhasa River, the downstream of the Lhasa River and the middle reaches of the Yarlung Zangbo River showed statistically significant differences in growth patterns.     

A novel bifunctional endo-/exo-type cellulase from an anaerobic ruminal bacterium

An anaerobic microorganism termed AN-C16-KBRB was isolated from the bovine rumen and demonstrated cellulolytic activity on a NB agar plate containing azo-carboxymethyl cellulose. The 16S rRNA gene of the strain was 98% similar to that of Clostridiaceae bacterium SK082 (AB298754) as the highest homology. A novel celEdx16 gene encoding a bifunctional endo-/exocellulase (CelEdx16) was cloned by the shotgun method from AN-C16-KBRB, and the enzyme was characterized. The celEdx16 gene had an open reading frame of 1,104-base pairs, which encoded 367 amino acids to yield a protein of molecular mass 40.4 kDa. The amino acid sequence was 53% identical to that of an endoglucanase from Clostridium thermocellum. CelEdx16 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography. The specific endocellulase and exocellulase activities of CelEdx16 were 15.9 and 3.6 × 10⁻² U mg⁻¹, respectively. The Michaelis–Menten constant (K _m values) and the maximal reaction velocities (V _max values) of CelEdx16 were 47.1 μM and 9.6 × 10⁻³ μmole min⁻¹ when endocellulase activity was measured and 106.3 μM and 2.1 × 10⁻⁵ μmole min⁻¹ when exocellulase activity was assessed. CelEdx16 was optimally active at pH 5.0 and 40°C.     

Nitrifying granules cultivation in a sequencing batch reactor at a low organics-to-total nitrogen ratio in wastewater

It is possible to cultivate aerobic granular sludge at a low organic loading rate and organics-to-total nitrogen (COD/N) ratio in wastewater in the reactor with typical geometry (height/diameter = 2.1, superficial air velocity = 6 mm/s). The noted nitrification efficiency was very high (99%). At the highest applied ammonia load (0.3 ± 0.002 mg NH₄⁺–N g total suspended solids (TSS)⁻¹ day⁻¹, COD/N = 1), the dominating oxidized form of nitrogen was nitrite. Despite a constant aeration in the reactor, denitrification occurred in the structure of granules. Applied molecular techniques allowed the changes in the ammonia-oxidizing bacteria (AOB) community in granular sludge to be tracked. The major factor influencing AOB number and species composition was ammonia load. At the ammonia load of 0.3 ± 0.002 mg NH₄⁺–N g TSS⁻¹ day⁻¹, a highly diverse AOB community covering bacteria belonging to both the Nitrosospira and Nitrosomonas genera accounted for ca. 40% of the total bacteria in the biomass.     

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.     

Kinetics of styrene biodegradation by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. E-93486

The research into kinetics of styrene biodegradation by bacterial strain Pseudomonas sp. E-93486 coming from VTT Culture Collection (Finland) was presented in this work. Microbial growth tests in the presence of styrene as the sole carbon and energy source were performed both in batch and continuous cultures. Batch experiments were conducted for initial concentration of styrene in the liquid phase changed in the range of 5–90 g m⁻³. The Haldane model was found to be the best to fit the kinetic data, and the estimated constants of the equation were: μ _m = 0.1188 h⁻¹, K _S = 5.984 mg l⁻¹, and K _i = 156.6 mg l⁻¹. The yield coefficient mean value Y_\textxs^\textapp Y_{\text{xs}}^{\text{app}} for the batch culture was 0.72 g_{dry cells weight} (g_substrate)⁻¹. The experiments conducted in a chemostat at various dilution rates (D = 0.035–0.1 h⁻¹) made it possible to determine the value of the coefficient for maintenance metabolism m _d = 0.0165 h⁻¹ and the maximum yield coefficient value Y_\textxs^\textM = 0.913 Y_{\text{xs}}^{\text{M}} = 0.913 . Chemostat experiments confirmed the high value of yield coefficient Y_\textxs^\textapp Y_{\text{xs}}^{\text{app}} observed in the batch culture. The conducted experiments showed high activity of the examined strain in the styrene biodegradation process and a relatively low sensitivity to inhibition of its growth at higher concentrations of styrene in the solution. Such exceptional features of Pseudomonas sp. E-93486 make this bacterial strain the perfect candidate for technical applications.     

The influence of maximal aerobic power on recovery of skeletal muscle following anaerobic exercise

Phosphorus magnetic resonance spectroscopy (³¹P-MRS) was used to investigate the influence of maximal aerobic power (˙VO _2max) on the recovery of human calf muscle from high-intensity exercise. The (˙VOO_2max) of 21 males was measured during treadmill exercise and subjects were assigned to either a low-aerobic-power (LAP) group (n = 10) or a high-aerobic-power (HAP) group (n = 11). Mean (SE) ˙VO _2max of the groups were 46.6 (1.1) and 64.4 (1.4) ml · kg⁻¹ · min⁻¹, respectively. A calf ergometry work capacity test was used to assign the same relative exercise intensity to each subject for the MRS protocol. At least 48 h later, subjects performed the rest (4 min), exercise (2 min) and recovery (10 min) protocol in a 1.5 T MRS scanner. The relative concentration of phosphocreatine (PCr) was measured throughout the protocol and intracellular pH (pH_i) was determined from the chemical shift between inorganic phospate (P_i) and PCr. End-exercise PCr levels were 27 (3.4) and 25 (3.5)% of resting levels for LAP and HAP respectively. Mean resting pH_i was 7.07 for both groups, and following exercise it fell to 6.45 (0.04) for HAP and 6.38 (0.04) for LAP. Analysis of data using non-linear regression models showed no differences in the rate of either PCr or pH_i recovery. The results suggest that ˙VO_2max is a poor predictor of metabolic recovery rate from high-intensity exercise. Differences in recovery rate observed between individuals with similar ˙VO_2max imply that other factors influence recovery. Accepted: 17 December 1996     

Bioenergetics and RNA/DNA ratios in the common carp (Cyprinus carpio ) under hypoxia

Hypoxia caused by eutrophication occurs over large areas in aquatic systems worldwide. Common carp (Cyprinus carpio) exposed to hypoxia (1 mg · O₂ · l⁻¹ and 2 mg · O₂ · l⁻¹) for 1 week showed a significant reduction in feeding rate, respiration rate, faecal production and nitrogenous excretion compared to those maintained at normoxia (7 mg · O₂ · l⁻¹). Fish exposed to hypoxia showed negative scope for growth (SfG), but no significant difference in the specific growth rate was revealed after 1 week in both hypoxic groups. A significant reduction in RNA/DNA ratio was, however, clearly evident in the white muscle of the 1 mg · O₂ · l⁻¹ treatment group, but not in the 2 mg · O₂ · l⁻¹ treatment group. Both specific growth rate and RNA/DNA ratio were significantly reduced when fish were exposed to severe hypoxia (0.5 mg · O₂ · l⁻¹) for 4 weeks. At all levels of hypoxia, growth reduction was accompanied by a significant decrease in RNA/DNA ratio in white muscle. Covariance analysis showed no significant difference between the slope of RNA/DNA ratio and growth rate under normoxic conditions and 0.5 mg · O₂ · l⁻¹ for 4 weeks (F=1.036, P > 0.326), as well as 1.0 mg · O₂ · l⁻¹ and 2.0 mg · O₂ · l⁻¹ for 1 week (F = 0.457, P > 0.5), indicating that the RNA/DNA ratio serves as a biomarker of growth under all oxygen levels, at least under controlled experimental conditions. SfG also appears to be more sensitive than the RNA/DNA ratio in responding to hypoxia in fish. Accepted: 15 September 2000