Bioremediation of Fungicides by Spent Mushroom Substrate and Its Associated Microflora

Experiments were conducted both under in vitro and in situ conditions to determine the biodegradation potential of button mushroom spent substrate (SMS) and its dominating microbes (fungi and bacteria) for carbendazim and mancozeb, the commonly used agricultural fungicides. During 6 days of incubation at 30 ± 2°C under broth culture conditions, highest degradation of carbendazim (17.45%) was recorded with B-1 bacterial isolate, while highest degradation of mancozeb (18.05%) was recorded with Trichoderma sp. In fungicide pre-mixed sterilized SMS, highest degradation of carbendazim (100.00–66.50 μg g⁻¹) was recorded with mixed inoculum of Trichoderma sp. and Aspergillus sp., whereas highest degradation of mancozeb (100.00–50.50 μg g⁻¹) was with mixed inoculum of Trichoderma sp., Aspergillus sp. and B–I bacterial isolate in 15 days of incubation at 30 ± 2°C. All these microbes both individually as well as in different combinations grew well and produced extracellular lignolytic enzymes on SMS, which helped in fungicides degradation. Under in situ conditions, among three different proportions of SMS (10, 20 and 30%, w/w) mixed with fungicide pre-mixed soil (100 μg g⁻¹ of soil), the degradation of carbendazim was highest in 30% SMS treatment, while for mancozeb it was in 20% SMS treatment. The residue levels of both fungicides decreased to half of their initial concentration after 1 month of SMS mixing.     

Isolation and characterization of <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. CBW capable of degrading carbendazim

With the intensive application of carbendazim in greenhouse production of vegetables and the production of medicinal herbs, there is an increasing need to find a way to remediate carbendazim-contaminated soil. A bacterial stain capable of utilizing carbendazim as the sole source of carbon and energy was isolated from soil. The isolate was designated CBW and identified as a member of Pseudomonas sp. based on its colony morphology, 16S rRNA gene sequencing and Biolog analysis. About 87.1 and 99.1% of carbendazim at concentrations of 1.0 and 10.0 mg l⁻¹ in mineral salts medium were removed by the isolate CBW after incubation for 3 days, respectively. The optimal pH value for the isolate CBW to degrade carbendazim was 7.0. The degradation rate of carbendazim by the isolate CBW was found to increase slightly with temperature. According to the metabolites detected and identified in the present study, it was proposed that carbendazim was first converted to 2-aminobenzimidazole, which was then transformed to 2-hydroxybenzimidazole, 1,2-diaminobenzene, catechol, and finally to carbon dioxide. The results indicate that the isolate CBW is a new bacterial resource for biodegrading carbendazim and might be used for bioremediation of sites heavily contaminated by carbendazim and its derivatives.     

Biodegradation kinetics of the benzimidazole fungicide thiophanate-methyl by bacteria isolated from loamy sand soil

Degradation of the fungicide thiophanate-methyl (TM) by Enterobacter sp. TDS-1 and Bacillus sp. TDS-2 isolated from sandy soil previously treated with TM was studied in mineral salt medium (MSM) and soil. Both strains were able to grow in MSM supplemented with TM (50 mg l⁻¹) as the sole carbon source. Over a 16 days incubation period, 60 and 77% of the initial dose of TM were degraded by strains TDS-1 and TDS-2, respectively, and disappearance of TM was described by first-order kinetics. Medium supplementation with glucose markedly stimulated bacterial growth; while the final rate of TM degradation was reduced by 21 and 27% for strains TDS-1 and TDS-2, respectively as compared to medium with TM only. Moreover, this additional carbon source changed the TM degradation kinetics, which proceeded according to a zero-order model. This effect was linked to substrate competition and/or a strong decrease of medium pH. Isolates degraded TM (100 mg kg⁻¹) in soil with rate constants of 0.186 and 0.210 day⁻¹, following first-order rate kinetics, and the time in which the initial TM concentration was reduced by 50% (DT50) in soils inoculated with strains TDS-1 and TDS-2 were 6.3 and 5.1 days, respectively. Analysis of TM degradation products in soil showed that the tested strains may have the potential to transform carbendazim (MBC) to 2-aminobenzimidazole (2-AB), and may be useful for a bioremediation of MBC-polluted soils.     

Effect of culture conditions on growth of green alga — <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis> and astaxanthin production

Influence of culture conditions such as light, temperature and C/N ratio was studied on growth of Haematococcus pluvialis and astaxanthin production. Light had significant effect on astaxanthin production and it varied with its intensity and direction of illumination and effective culture ratio (ECR, volume of culture medium/volume of flask). A 6-fold increase in astaxanthin production (37 mg/L) was achieved with 5.1468·10⁷ erg·m⁻²·s⁻¹ light intensity (high light, HL) at effective culture ratio of 0.13 compared to that at 0.52 ECR, while the difference in the astaxanthin production was less than 2 — fold between the effective culture ratios at 1.6175·10⁷ erg·m⁻²·s⁻¹ light intensity (low light, LL). Multidirectional (three-directional) light illumination considerably enhanced the astaxanthin production (4-fold) compared to unidirectional illumination. Cell count was high at low temperature (25 °C) while astaxanthin content was high at 35 °C in both autotrophic and heterotrophic media. In a heterotrophic medium at low C/N ratio H. pluvialis growth was higher with prolonged vegetative phase, while high C/N ratio favoured early encystment and higher astaxanthin formation.     

Indigestible dextrin is an excellent inducer for α-amylase, α-glucosidase and glucoamylase production in a submerged culture of Aspergillus oryzae

α-Amylase activities of Aspergillus oryzae grown on dextrin or indigestible dextrin were 7·8 and 27·7 U ml⁻¹, respectively. Glucoamylase activities of the cultures grown on dextrin or indigestible dextrin were 5·4 and 301 mU ml⁻¹, respectively. The specific glucoamylase production rate in indigestible dextrin batch culture reached 1·35 U g DW⁻¹ h⁻¹. In contrast, biomass concentration of A. oryzae in indigestible dextrin culture was 35% of that in dextrin culture. Thus, the culture method using indigestible dextrin has the potential to improve amylolytic enzyme production and fungal fermentation broth rheology.     

Isolation and Characterization of Carbendazim-degrading Rhodococcus erythropolis djl-11

Carbendazim (methyl 1H-benzimidazol-2-yl carbamate) is one of the most widely used fungicides in agriculture worldwide, but has been reported to have adverse effects on animal health and ecosystem function. A highly efficient carbendazim-degrading bacterium (strain dj1-11) was isolated from carbendazim-contaminated soil samples via enrichment culture. Strain dj1-11 was identified as Rhodococcus erythropolis based on morphological, physiological and biochemical characters, including sequence analysis of the 16S rRNA gene. In vitro degradation of carbendazim (1000 mg·L⁻¹) by dj1-11 in minimal salts medium (MSM) was highly efficient, and with an average degradation rate of 333.33 mg·L⁻¹·d⁻¹ at 28°C. The optimal temperature range for carbendazim degradation by dj1-11 in MSM was 25–30°C. Whilst strain dj1-11 was capable of metabolizing cabendazim as the sole source of carbon and nitrogen, degradation was significantly (P<0.05) increased by addition of 12.5 mM NH₄NO₃. Changes in MSM pH (4–9), substitution of NH₄NO₃ with organic substrates as N and C sources or replacing Mg²⁺ with Mn²⁺, Zn²⁺ or Fe²⁺ did not significantly affect carbendazim degradation by dj1-11. During the degradation process, liquid chromatography-mass spectrometry (LC-MS) detected the metabolites 2-aminobenzimidazole and 2-hydroxybenzimidazole. A putative carbendazim-hydrolyzing esterase gene was cloned from chromosomal DNA of djl-11 and showed 99% sequence homology to the mheI carbendazim-hydrolyzing esterase gene from Nocardioides sp. SG-4G.     

Culture of human hepatocytes from small surgical liver biopsies. Biochemical characterization and comparison with in vivo

Summary High yields of human hepatocytes (up to 23×10⁶ viable cells/g) were obtained from small surgical liver biopsies (1 to 3 g) by a two-step collagenase microperfusion method. Cell viability was about 95%, attachment efficiency of hepatocytes seeded on fibronectin-coated plates was 80% within 1 h after plating, and cells survived for about 2 wk in serum-free Ham’s F12 containing 0.2% bovine serum albumin, 10⁻⁸ M insulin, and 10⁻⁸ M dexamethasone. To evaluate the metabolism of human hepatocytes in serum-free conditions, we measured their most characteristic biochemical functions and compared them to those reported for human liver. After 24 h in culture, glycogen content was 1250±177 nmol glucose/mg cell protein and remained stable for several days. Gluconeogenesis from lactate in hormone-free media was (3.50±0.17 nmol glucose·mg⁻¹·min⁻¹) similar to that reported for human liver. Insulin at 10⁻⁸ M activated glycolysis (×1.40) and glycogenesis (×1.34), and glucagon at 10⁻⁹ M stimulated gluconeogenesis (×1.35) and glycogenolysis (×2.18). Human hepatocytes synthesized albumin, transferrin, fibrinogen, α₁-antitrypsin, α₁-antichymotrypsin, α₁-acid glycoprotein, haptoglobin, α₂-macroglobulin, and plasma fibronectin and excreted them to the culture medium. Maximum protein synthesis was stimulated by 10⁻⁹ M dexamethasone. Basal urea synthesis oscillated between 2.5 and 3.5 nmol·mg⁻¹ cell protein·min⁻¹, about 5 times the value estimated for human liver. Cytochrome P-450 decreased in culture but it was still 20% of freshly isolated hepatocytes by Day 5 in culture. In addition, ethoxycumarin-O-deethylase and aryl hydrocarbon hydroxylase could be induced in vitro by treatment with methyl cholanthrene. Glutathione levels were similar to those reported for human liver (35 nmol·mg⁻¹). The results of our work show that adult human hepatocytes obtained from small surgical biopsies and cultured in chemically defined conditions express their most important metabolic functions to an extent that is similar to that reported for adult human liver.     

Plant regeneration from shoot apical meristems of Melia azedarach L. (Meliaceae)

A protocol was developed for plant regeneration of Melia azedarach L. by in vitro culture of apical meristem (0.5 mm in length). The influence of six clones was investigated. The culture procedure comprised two sequential steps: 1) Induction of shoots by in vitro culture of axillary buds from adult trees (10–15 years old) by culture on Murashige and Skoog (1962) medium (MS) supplemented with 0.5 mg·dm⁻³ BAP (6-benzylaminopurine), 0.1 mg·dm⁻³ IBA (indolebutyric acid), and 0.1 mg·dm⁻³ GA₃ (gibberellic acid). The Multiplication of the regenerated shoots was achieved in MS + 0.5 mg·dm⁻³ BAP + 0.1 mg·dm⁻³ GA₃. 2) In vitro culture of the apical meristems from the regenerated shoots in MS medium (0.7 %) supplemented with various combinations of BAP and IBA. Maximum shoot proliferation was obtained on MS medium supplemented with 0.5 mg·dm⁻³ BAP and 0.1 mg·dm⁻³ IBA. Regenerated shoots were rooted on MS + 3.5 mg·dm⁻³ IBA (4 days) followed by subculture on MS lacking growth regulators (30 days). Complete plants were transferred to soil.     

Isolation and characterization of Rhodococcus rhodochrous for the degradation of the wastewater component 2-hydroxybenzothiazole

2-Hydroxybenzothiazole (OBT) is present in wastewaters from the industrial production of the rubber vulcanization accelerator 2-mercaptobenzothiazole (MBT). We have achieved the first isolation of axenic bacterial cultures capable of the degradation of OBT and growth on this substrate as the sole source of carbon, nitrogen and energy. All isolates had similar characteristics corresponding to one particular isolate, which was studied in more detail and identified as Rhodococcus rhodochrous. The strains were also capable of degrading benzothiazole (BT) but not MBT or benzothiazole-2-sulphonate (BTSO₃). OBT was degraded at a concentration of up to 600 mg · l⁻¹. BT was toxic above 300 mg · l⁻¹. MBT inhibited OBT degradation. Growth on OBT was not significantly different at pH values of between 6.3 and 7.9 or salt concentrations between 1 % and 3 %. In shake flasks the cells clumped together, which resulted in a lower rate of oxygen transfer and slower degradation as compared to cells grown on OBT in a stirred reactor. Received: 22 August 1996 / Received revision: 29 November 1996 / Accepted: 29 November 1996     

Cellular signalling of PCH-induced pigment aggregation in the crustacean Macrobrachium potiuna erythrophores

The cellular system responsible for the transduction of the pigment-concentrating hormone (PCH) signal was investigated in erythrophores of the freshwater shrimp, Macrobrachium potiuna. Dose-response curves to the hormone were determined in the absence and in the presence of several drugs that affect sequential steps of the Ca²⁺-dependent signalling pathway. Additionally, the ability of forskolin to induce pigment dispersion was evaluated. Neomycin sulphate (10⁻⁴ and 10⁻³ mol · l⁻¹), trifluoperazine (10⁻⁵ and 10⁻⁴ mol · l⁻¹), 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (10⁻⁷ and 10⁻⁵ mol · l⁻¹) and okadaic acid (10⁻⁷ mol · l⁻¹) significantly (P<0.05) decreased the responses to PCH. However, okadaic acid at low concentration (10⁻⁹ mol · l⁻¹) and cyclosporin A (10⁻⁶ and 10⁻⁵ mol · l⁻¹) did not significantly (P>0.05) affect PCH activity. Forskolin (10⁻⁴ mol · l⁻¹) was able to half-maximally reverse the hormone-induced aggregation. Our results suggest that the pigment-concentrating hormone induces pigment aggregation through a Ca²⁺-dependent pathway with a posteriori phosphatase activation, probably the serine/threonine phosphatase 1. Accepted: 30 June 1997     

Optimization of culture medium for the continuous cultivation of the microalga Haematococcus pluvialis

The freshwater microalga Haematococcus pluvialis is one of the best microbial sources of the carotenoid astaxanthin, but this microalga shows low growth rates and low final cell densities when cultured with traditional media. A single-variable optimization strategy was applied to 18 components of the culture media in order to maximize the productivity of vegetative cells of H. pluvialis in semicontinuous culture. The steady-state cell density obtained with the optimized culture medium at a daily volume exchange of 20% was 3.77 · 10⁵ cells ml⁻¹, three times higher than the cell density obtained with Bold basal medium and with the initial formulation. The formulation of the optimal Haematococcus medium (OHM) is (in g l⁻¹) KNO₃ 0.41, Na₂HPO₄ 0.03, MgSO₄ · 7H₂O 0.246, CaCl₂ · 2H₂O 0.11, (in mg l⁻¹) Fe(III)citrate · H₂O 2.62, CoCl₂ · 6H₂O 0.011, CuSO₄ · 5H₂O 0.012, Cr₂O₃ 0.075, MnCl₂ · 4H₂O 0.98, Na₂MoO₄ · 2H₂O 0.12, SeO₂ 0.005 and (in μg l⁻¹]) biotin 25, thiamine 17.5 and B₁₂ 15. Vanadium, iodine, boron and zinc were demonstrated to be non-essential for the growth of H. pluvialis. Higher steady-state cell densities were obtained by a three-fold increase of all nutrient concentrations but a high nitrate concentration remained in the culture medium under such conditions. The high cell productivities obtained with the new optimized medium can serve as a basis for the development of a two-stage technology for the production of astaxanthin from H. pluvialis. Received: 10 September 1999 / Received revision: 2 December 1999 / Accepted: 3 December 1999     

Effect of capsaicin on thermosensitive receptors of the rat skinin vitro

The spike activity of cold- and heat-sensitive receptors was recorded in experiments on skin-nerve preparations (scrotum skin-n. pudendum) of male ratsin vitro. The effects of capsaicin in different concentrations (dilutions of 1·10⁻⁵, 1·10⁻⁶, and 1·10⁻⁷) on the background activity of these receptors and their sensitivity to thermal stimulation were studied. An excitatory effect of capsaicin in the concentrations of 1·10⁻⁵ and 1·10⁻⁶ on skin cold receptors, expressed as intensification of their background activity, was accompanied by complete suppression of their responses to cooling. Against the background of capsaicin in a lower concentration (1·10⁻⁷), some receptors preserved their responses to cooling, but the response intensity dropped, and the thresholds shifted toward lower temperatures. In heat receptors influenced by capsaicin, initial intensification of their responses to increased temperature and their subsequent desensitization to warming were observed.     

Relations between carbon dioxide fluxes and environmental factors of Kobresia humilis meadows and Potentilla fruticosa meadows

Carbon dioxide fluxes of Kobresia humilis and Potentilla fruticosa shrub meadows, two typical ecosystems in the Qinghai-Tibet Plateau, were measured by eddy covariance technology and the data collected in August 2003 were employed to analyze the relations between carbon dioxide fluxes and environmental factors of the ecosystems. August is the time when the two ecosystems reach their peak leaf area indexes and stay stable, and also the period when the net carbon absorptions of Kobresia humilis and Potentilla fruticosa shrub meadows reach 56.2 g C·m⁻² and 32.6 g C·m⁻², with their highest daily carbon dioxide absorptions standing at 12.7 μmol·m⁻²·s⁻¹ and 9.3 μmol·m⁻²·s⁻¹, and their highest carbon discharges at 5.1 μmol·m⁻²·s⁻¹ and 5.7 μmol·m⁻²·s⁻¹, respectively. At the same photosynthetic photo flux densities (PPFD), the carbon dioxide-uptake rate of the Kobresia humilis meadow is higher than that of the Potentilla fruticosa shrub meadow; where the PPFD are higher than 1,200 μmol·m⁻²·s⁻¹. The carbon dioxide uptake rates of the two ecosystems declined as air temperature increased, but the carbon dioxide uptake rate of the Kobresia humilis meadow decreased more quickly (−0.086) than that of the Potentilla fruticosa shrub meadow (−0.016). Soil moistures exert influence on the soil respirations and this varies with the vegetation type. The daily carbon dioxide absorptions of the ecosystems increase with increased diurnal temperature differences and higher diurnal temperature differences result in higher carbon dioxide exchanges. There exists a negative correlation between the vegetation albedos and the carbon dioxide fluxes. Translated from Acta Bot Boreal—Occident Sin, 2006, 26(1): 133–142 [译自: 西北植物学报]     

Comparative analysis of the digestive efficiency and nitrogen and energy requirements of the phyllostomid fruit-bat (Artibeus jamaicensis) and the pteropodid fruit-bat (Rousettus aegyptiacus)

Nitrogen (N) and energy (E) requirements of the phyllostomid fruit bat, Artibeus jamaicensis, and the pteropodid fruit bat Rousettus aegyptiacus, were measured in adults that were fed on four experimental diets. Mean daily food intake by A. jamaicensis and R. aegyptiacus ranged from 1.1–1.6 times body mass and 0.8–1.0 times body mass, respectively. Dry matter digestibility and metabolizable E coefficient were high (81.1% and 82.4%, respectively) for A. jamaicensis and (77.5% and 78.0%, respectively) for R. aegyptiacus. Across the four diets, bats maintained constant body mass with mean metabolizable E intakes ranging from 1357.3 kJ · kg^−0.75 · day⁻¹ to 1767.3 kJ · kg^−0.75 · day⁻¹ for A. jamaicensis and 1282.6–1545.2 kJ · kg^−0.75 · day⁻¹ for R. aegyptiacus. Maintenance E costs were high, in the order of 3.6–5.4 times the basal metabolic rate (BMR). It is unlikely that the E intakes that we observed represent a true measure of maintenance E requirements. All evidence seems to indicate that fruit bats are E maximizers, ingesting more E than required and regulating storage by adjusting metabolic output. We suggest that true maintenance E requirements are substantially lower than what we observed. If it follows the eutherian norm of two times the BMR, fruit bats must necessarily over-ingest E on low-N fruit diet. Dietary E content did affect N metabolism of A. jamaicensis. On respective low- and high-E diets, metabolic fecal N were 0.492 mg N · g⁻¹ and 0.756 mg N · g⁻¹ dry matter intake and endogenous urinary N losses were 163.31 mg N · kg^−0.75 · day⁻¹ and 71.54 mg N · kg^−0.75 · day⁻¹. A. jamaicensis required 332.3 mg · kg^−0.75 · day⁻¹ and 885.3 mg · kg^−0.75 · day⁻¹ of total N on high- and low-E diets, respectively, and 213.7 mg · kg^−0.75 · day⁻¹ of truly digestible N to achieve N balance. True N digestibilities were low (29% and 49%) for low- and high-E diets, respectively. For R. aegyptiacus, metabolic fecal N and endogenous urinary N losses were 1.27 mg N · g⁻¹ dry matter intake and 96.0 mg N · kg^−0.75 · day⁻¹, respectively, and bats required 529.8 mg · kg^−0.75 · day⁻¹ (total N) or 284.0 mg · kg^−0.75 · day⁻¹ (truly digestible N). True N digestibility was relatively low (50%). Based on direct comparison, we found no evidence that R. aegyptiacus exhibits a greater degree of specialization in digestive function and N retention than A. jamaicensis. When combined with results from previous studies, our results indicate that all fruit bats appear to be specialized in their ability to retain N when faced with low N diet. Accepted: 24 November 1998     

Water and sodium balances and metabolic physiology of house mice (Mus domesticus) and short-tailed mice (Leggadina lakedownensis) under laboratory conditions

A laboratory study investigated the metabolic physiology, and response to variable periods of water and sodium supply, of two arid-zone rodents, the house mouse (Mus domesticus) and the Lakeland Downs short-tailed mouse (Leggadina lakedownensis) under controlled conditions. Fractional water fluxes for M. domesticus (24 ± 0.8%) were significantly higher than those of L. lakedownensis (17 ± 0.7%) when provided with food ad libitum. In addition, the amount of water produced by M. domesticus and by L. lakedownensis from metabolic processes (1.3 ± 0.4 ml · day⁻¹ and 1.2 ± 0.4 ml · day⁻¹, respectively) was insufficient to provide them with their minimum water requirement (1.4 ± 0.2 ml · day⁻¹ and 2.0 ± 0.3 ml · day⁻¹, respectively). For both species of rodent, evaporative water loss was lowest at 25 °C, but remained significantly higher in M. domesticus (1.1 ± 0.1 mg H₂O · g^−0.122 · h⁻¹) than in L. lakedownensis (0.6 ± 0.1 mg H₂O · g^−0.122 · h⁻¹). When deprived of drinking water, mice of both species initially lost body mass, but regained it within 18 days following an increase in the amount of seed consumed. Both species were capable of drinking water of variable saline concentrations up to 1 mol · l⁻¹, and compensated for the increased sodium in the water by excreting more urine to remove the sodium. Basal metabolic rate was significantly higher in M. domesticus (3.3 ± 0.2 mg O₂ · g^−0.75 · h⁻¹) than in L. lakedownensis (2.5 ± 0.1 mg O₂ · g^−0.75 · h⁻¹). The study provides good evidence that water flux differences between M. domesticus and L. lakedownensis in the field are due to a requirement for more water in M. domesticus to meet their physiological and metabolic demands. Sodium fluxes were lower than those observed in free-ranging mice, whose relatively high sodium fluxes may reflect sodium associated with available food. Accepted: 16 August 1999     

Biomass, carbonate standing stock and production of the mediterranean coralCladocora caespitosa (L.)

Summary The Mediterranean coralCladocora caespitosa often occurs in large beds, i.e. populations of hemispherical clonies with stock densities varying between 1.9 and 4 coloneis ·m⁻². Laboratory measurements of volume, skeleton weight, surface and number of corallites per colony, coupled with mean annual growth rates evaluated through sclerochronology, allowed for the estimation of biomass, skeleton bulk density, calcimass (carbonate standing stock) and secondary production (both organic and inorganic) of twoC. caespitosa beds at 4 and 9 m depth. The mean colony biomass varied between 0.73 and 0.99 kg dw ·m⁻², corresponding to a calcimass between 2 and 5 kg CaCO₃·m⁻². Organic secondary production was 215.5–305.4 g dw of polyps ·m⁻²·y⁻¹, while the potential (mineral) production was 1.1–1.7 kg CaCO₃·m⁻²·y⁻¹, for the year 1996–1997. These values show thatC. caespitosa is one of the major carbonate producers within the Mediterranean and one of the major epibenthic species originating stable carbonate frameworks both in recent and past times.     

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     

NADPH oxidase as the source of ROS produced under waterlogging in roots of mung bean

The objective of this study was to examine the role of NADPH oxidase on superoxide radical production under waterlogging in mung bean (Vigna radiata) cvs. T 44 (tolerant) and Pusa Baisakhi (PB) (susceptible), and wild species Vigna luteola. Two days of waterlogging caused decline in superoxide radical (O₂ ^·−) contents in all the genotypes, however, further waterlogging up to 8 d caused significant increase in O₂ ^·− contents. In control and revived plants O₂ ^·− contents were higher in PB, while under waterlogging stress T 44 and V. luteola showed greater increases in the O₂ ^·− contents. During waterlogging the increase in O₂ ^·− content was found to be due to the diphenylene iodonium chloridesensitive NADPH oxidase (NOX). This was further confirmed by the waterlogging induced increase in NOX activity, which was higher in tolerant genotypes T 44 and V. luteola compared with PB. Gene expression studies showed enhanced expression of NOX in the roots of waterlogged V. luteola and T 44, while little expression was observed in control or treated plants of PB. PCR band products were cloned and sequenced, and partial cDNAs of NOX was obtained. Results suggest that increase in O₂ ^·− content during waterlogging could be due to the induction of membrane linked NOX.     

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     

The development of an isokinetic squat device: reliability and relationship to functional performance

The aims of the present study were: (1) to assess aerobic metabolism in paraplegic (P) athletes (spinal lesion level, T4–L3) by means of peak oxygen uptake (V˙O_2peak) and ventilatory threshold (VT), and (2) to determine the nature of exercise limitation in these athletes by means of cardioventilatory responses at peak exercise. Eight P athletes underwent conventional spirographic measurements and then performed an incremental wheelchair exercise on an adapted treadmill. Ventilatory data were collected every minute using an automated metabolic system: ventilation (l · min⁻¹), oxygen uptake (V˙O₂, l · min⁻¹, ml · min⁻¹ · kg⁻¹), carbon dioxide production (V˙CO₂, ml · min⁻¹), respiratory exchange ratio, breathing frequency and tidal volume. Heart rate (HR, beats · min⁻¹) was collected with the aid of a standard electrocardiogram. V˙O_2peak was determined using conventional criteria. VT was determined by the breakpoint in the V˙CO₂−V˙O₂ relationship, and is expressed as the absolute VT (V˙O₂, ml · min⁻¹ · kg⁻¹) and relative VT (percentage of V˙O_2peak). Spirometric values and cardioventilatory responses at rest and at peak exercise allowed the measurement of ventilatory reserve (VR), heart rate reserve (HRr), heart rate response (HRR), and O₂ pulse (O₂ P). Results showed a V˙O_2peak value of 40.6 (2.5) ml · min⁻¹ · kg⁻¹, an absolute VT detected at 23.1 (1.5) ml · min⁻¹ · kg⁻¹ V˙O₂ and a relative VT at 56.4 (2.2)% V˙O_2peak. HRr [15.8 (3.2) beats · min⁻¹], HRR [48.6 (4.3) beat · l⁻¹], and O₂ P [0.23 (0.02) ml · kg⁻¹ · beat⁻¹] were normal, whereas VR at peak exercise [42.7 (2.4)%] was increased. As wheelchair exercise excluded the use of an able-bodied (AB) control group, we compared our V˙O_2peak and VT results with those for other P subjects and AB controls reported in the literature, and we compared our cardioventilatory responses with those for respiratory and cardiac patients. The low V˙O_2peak values obtained compared with subject values obtained during an arm-crank exercise may be due to a reduced active muscle mass. Absolute VT was somewhat comparable to that of AB subjects, mainly due to the similar muscle mass involved in wheelchair and arm-crank exercise by P and AB subjects, respectively. The increased VR, as reported in patients with chronic heart failure, suggested that P athletes exhibited cardiac limitation at peak exercise, and this contributed to the lower V˙O_2peak measured in these subjects. Accepted: 22 April 1997