Production of a high activity of an extremely thermostable β-mannanase by the thermophilic eubacterium Rhodothermus marinus, grown on locust bean gum

The production of a highly thermostable mannanase by Rhodothermus marinus was increased 16.5-fold by optimising the concentrations of locust bean gum and yeast extract using central composite designs. The optimised medium and culture conditions yielded mannanase activity at 495 nkat ml–1 (248 nkat mg–1 protein). In addition, -L-arabinofuranosidase, -xylanase, -xylosidase, -glucosidase, -mannosidase, -galactosidase, -galactosidase and endoglucanase activities were detected at 32 nkat ml–1, 30 nkat ml–1, 16 nkat ml–1, 15 nkat ml–1, 0.1 nkat ml–1, 1 nkat ml–1, 0.5 nkat ml–1 and 8 nkat ml–1, respectively. No filter paper cellulase activity could be detected. The optimum pH of the mannanase was 5.0–6.5 and it showed high stability from pH 5 to 10 after 16 h incubation at 50 °C. The enzyme activity was maximum at 85 °C, with half lives of 45.3 h at 85 °C and 4.2 h at 90 °C. This is the first report on the production of such a high activity of extremely thermostable mannanase by an extreme thermophilic bacterium. © Rapid Science Ltd. 1998     

Pulmonary diffusing capacity in reptiles (Relations to temperature and O2-uptake)

Summary Pulmonary CO-diffusing capacity (D l _CO), lung volume, pulmonary perfusion and O₂-uptake were measured by non-invasive techniques in the lizardsVaranus exanthematicus andTupinambis teguixin (mean body weight 2.2 kg for both species).The CO-diffusing capacity was at 25–27°C 0.059 mlstpd·kg^–1·min^–1·Torr^–1 inVaranus, which is 47% greater than the value of 0.040 mlstpd·kg^–1·min^–1·Torr^–1 inTupinambis. The lung volume ofVaranus was 36 ml·kg^–1 and that ofTupinambis 20 ml·kg^–1. At 35–37°C the diffusing capacity of lizard lungs are about 25% of those for mammals of comparable size.InVaranus pulmonary CO-diffusing capacity increased with temperature from 0.027 mlstpd·kg^–1·min^–1·Torr^–1 at 17–19 °C to 0.075 mlstpd·kg^–1·min^–1·Torr^–1 at 35–37 °C. This change closely matched a concomitant increase of O₂-uptake. Pulmonary perfusion increased from 27 ml·kg^–1·min^–1 to 55 ml·kg^–1·min^–1 within this temperature range.The study emphasizes that pulmonary diffusing capacity cannot be fully evaluated without information on pulmonary perfusion and O₂-uptake. In reptiles and other ectotherms diffusing capacity must be reported at specified body temperature.     

Hydrogen photoproduction by Rhodobacter sphaeroides immobilised on polyurethane foam

H2 photoproduction by Rhodobacter sphaeroides GL-1 immobilised on polyurethane foam in a continuous flow photobioreactor was shown to occur for prolonged periods. Under optimal conditions (300 W m–2; dilution rate 0.023 h–1) the rate of H2 production was 0.21 ml h–1 ml–1 foam matrix with an efficiency for lactic acid to H2 conversion of 86%. The duration of the process (35 days of operation) showed no major limitations. © Rapid Science Ltd. 1998     

Effect of tunicamycin on the activity and immunoreactivity of ascorbate oxidase (Cucurbita pepo medullosa) expressed in cultured green zucchini cells

Ascorbate oxidase activity and immunoreactivity were evaluated in crude tissue extracts obtained from callus cell cultures induced by green zucchini sarcocarp and grown in the presence of tunicamycin, a powerful N-glycosylation inhibitor. Tunicamycin at 2 or 4 g ml–1 blocked cell growth within a couple of weeks, although a sustained cell viability was observed in the same period. A significant inhibition of total protein synthesis was observed at 10 and 15 days of culture time, with a decrease of 30% and 43% respectively when cells were grown in the presence of 2 g ml–1 tunicamycin, and of 48% and 57% respectively when the tunicamycin concentration was 4 g ml–1. After the same culture times ascorbate oxidase specific activity assayed in crude tissue extracts showed increases of about 1.9-fold and 3.5-fold (10 days) and 1.7-fold and 3.1-fold (15 days) at 2 and 4 g ml–1 tunicamycin, respectively. Ascorbate oxidase mRNA levels, however, did not appreciably differ between control and treated samples, measured at the same growing times. Lectin-blot, based on the use of concanavalin A, indicated a marked decrease of glycosylated proteins in tunicamycin-treated cultures. As judged by immunoblot, anti-native ascorbate oxidase antibodies scarcely recognized the enzyme expressed in tunicamycin-treated cells; on the contrary, anti-deglycosylated ascorbate oxidase antibodies were more reactive to the enzyme expressed in tunicamycin-treated cultures.     

Maximal oxygen uptake, anaerobic threshold and running economy in women and men with similar performances level in marathons

Sex differences in running economy (gross oxygen cost of running, C_R), maximal oxygen uptake (VO_2max), anaerobic threshold (Th_an), percentage utilization of aerobic power (% VO_2max), and Th_an during running were investigated. There were six men and six women aged 20–30 years with a performance time of 2 h 40 min over the marathon distance. The VO_2max, Th_an, and CR were measured during controlled running on a treadmill at 1° and 3° gradient. From each subject's recorded time of running in the marathon, the average speed (v _M) was calculated and maintained during the treadmill running for 11 min. The VO_{2 max} was inversely related to body mass (m _b), there were no sex differences, and the mean values of the reduced exponent were 0.65 for women and 0.81 for men. These results indicate that for running the unit ml·kg^–0.75·min^–1 is convenient when comparing individuals with different m _b. The VO_2max was about 10% (23 ml·kg^–0.75·min^–1) higher in the men than in the women. The women had on the average 10–12 ml·kg^–0.75·min^–1 lower VO₂ than the men when running at comparable velocities. Disregarding sex, the mean value of C_R was 0.211 (SEM 0.005) ml·kg^–1·m^–1 (resting included), and was independent of treadmill speed. No sex differences in Th_an expressed as % VO_2max or percentage maximal heart rate were found, but Than expressed as VO₂ in ml·kg^–0.75·min^–1 was significantly higher in the men compared to the women. The percentage utilization of f _emax and concentration of blood lactate at v _M was higher for the female runners. The women ran 2 days more each week than the men over the first 4 months during the half year preceding the marathon race. It was concluded that the higher VO_2max and Th_an in the men was compensated for by more running, superior C_R, and a higher exercise intensity during the race in the performance-matched female marathon runners.     

Induction of astaxanthin accumulation by nitrogen and magnesium deficiencies in Haematococcus pluvialis

Haematococcus pluvialis was cultured under N– and Mg+2-deficient conditions with two light intensities: 40 and 230 mol m² s–1. Highest astaxanthin concentration, 49.5 g·ml–1, was obtained when high light was applied under N-deficient conditions. N-deficiency has a greater effect than high light intensity on astaxanthin synthesis by exerting a stronger blocking effect on cell division. The effect of high light was synergetic with the other stress conditions in stimulating the synthesis of astaxanthin. Mg+2 deficiency also stimulated the synthesis of astaxanthin but produced lower concentrations: 7 and 26 g·ml–1 for low and high light intensities respectively. When both N and Mg+2 were absent from the culture media the concentration of astaxanthin was lower than with N-deficiency alone but higher than with Mg+2-deficiency. © Rapid Science Ltd. 1998     

Blood-brain glucose transfer in the mouse

The intracarotid injection method has been utilized to examine blood-brain barrier (BBB) glucose transport in normal mice, and after a 2-day fast. In anesthetized mice, cerebral blood flow (CBF) rates were reduced from 0.86 ml·min^–1·gm^–1 in control to 0.80 ml·min^–1·gm^–1 in fasted animals (p>0.05). Brain Uptake Indices were significantly (p<0.05) higher in fasted (plasma glucose =4.7 mM) than control (plasma glucose = 6.5 mM) mice, while plasma glucose was significantly lower. The maximal velocity (V_max) for glucose transport was 1562±303 nmoles·min^–1·g^–1, and the half-saturation constant (Km =) 6.67±1.46 mM in normally fed mice. In fasted mice the Vmax was 2053±393 nmoles·min^–1·g^–1 (p>0.05), and the half-saturation constant (Km =) 7.30±1.60 mM (not significant, P>0.05). A rabbit polyclonal antiserum to a synthetic peptide encoding the 13 C-terminal amino acids of the human erythrocyte glucose transporter (GLUT-1) immunocytochemically confirmed that the mouse brain capillary endothelial glucose transporter is a GLUT-1 transporter, and immunoreactivity was similar in brain endothelia from fed and fasted animals. In conclusion, after a 2-day fast in the mouse, we saw significant reductions in forebrain weight (7%), and plasma glucose levels (27%). Increased brain glucose extraction (25%, p<0.05), and a 22% increase in the unsaturatedpermeability-surface area product (p<0.05) was also observed.     

Water and energy metabolism in the rock hyrax (Procavia habessinica)

Summary The influence of ambient temperature and water supply on water metabolism and O₂-consumption was measured in rock hyraxes (Procavia habessinica).With ad libitum food and water (control), water turnover rates of hyraxes were significantly lower than the general eutherian mean; water turnover rates were 61.4, 44.1 and 55.1 ml·kg^–0.82·24 h^–1 at 20, 27 and 35°C respectively. When greens were fed ad libitum but no drinking water was given, water turnover rate at 20°C was twofold higher, but at 27 and 35°C it was similar to that in control experiments.Water turnover rates were significantly reduced when no drinking water and only 25 g greens per day were offered (25.8, 22.0 and 29.3 ml·kg^–0.82·24 h^–1 at 20, 27 and 35°C respectively). Highest urine osmolality (3,200 mosm·kg^–1) was recorded at 20°C.Oxygen consumption under control conditions was 43% below that predicted on the basis of body weight for most eutherian mammals. The thermoneutral zone ranged from 27 to 35°C, and the basal metabolic rate was 165 kJ·kg^–0.75·h^–1.     

Conformation of the high molecular weight form ofDunaliella salina phosphofructokinase in solution by means of small-angle X-ray diffraction and viscosity measurements

Summary The intrinsic viscosity of phosphofructokinase fromDunaliella salina in different states of aggregation was determined. The instrinsic viscosity [], of the biologically active tetramer, with a molecular weight of 320,000, was found to be 6.5 ml·g^–1 at 4°C. Moreover, for the inactive dimer, with a molecular weight of 160,000, a value of []=8.0 ml·g^–1 was determined. The high molecular weight aggregate of phosphofructokinase fromDunaliella salina, that shows little activity, has an intrinsic viscosity of 23.2 ml·g^–1, which is significantly higher than that found for the active tetramer and the inactive dimer.Small angle X-ray scattering experiments in solution of this high molecular from of phosphofructokinase fromDunaliella salina reveal a radius of gyration of the cross section ofR _c=49.0 Å at an ionic strength of 0.15 M and_pH 7.2. Furthermore, a comparison of the values obtained for the tetramer and the radius of gyration (R _g=52.9 Å) with those of typical spherical proteins (3–4 ml·g^–1) shows that the values of [] andR _g are significantly larger for the high molecular weight form of phosphofructokinase than for the spherical proteins. The high intrinsic viscosity of the polymeric form of phosphofructokinase suggests an end-to-end aggregation consisting of monomeric units with heights,h=80–90 Å, and a cylindrical diameter of approximately 140.0 Å, resulting in a long rod of a total length of 1,800 Å and a molecular weight of two million. On the basis of the experimentally observedR _c and [] values, using a prolate ellipsoid of revolution as a model, the hydrodynamic volume and the hydration, the axial ratio could be determined to be 12. The native tetrameric form contains 0.4 g H₂O/g protein, whereas the higher aggregate structure corresponds to a hydration of 0.60 g H₂O/g protein.     

Secretion by the mandibular gland of the red kangaroo (Macropus rufus) during isoprenaline infusion

Summary Intracarotid infusion of isoprenaline, either alone or in combination with acetylcholine infusion was used to stimulate salivation by the mandibular glands of anaesthetized red kangaroos. Isoprenaline alone (0.20–1.25 mol·kg^–1·min^–1) elicited flow rates ranging from 0.014 to 0.239 ml·min^–1 (1.21–28.1 l·g gland^–1·min^–1). Salivary concentrations of sodium, chloride, phosphate and urea were negatively correlated with flow, whereas potassium, calcium, magnesium, hydrogen ion, bicarbonate, protein, and osmolality were poorly correlated with flow. Relative to cholinergic saliva produced at equivalent flow rates, isoprenaline-evoked saliva had higher osmolality, saliva/plasma urea ratios and concentrations of protein, potassium, magnesium, bicarbonate, and phosphate, but lower sodium, chloride and hydrogen ion levels. At a steady salivary flow (0.5 ml·min^–1), superimposition of isoprenaline infusion (0.15 mol·kg^–1·min^–1) on a pre-existing acetylcholine infusion reduced the rate of acetylcholine administration necessary to maintain flow, increased osmolality and the concentrations of protein, urea, potassium, calcium, magnesium, bicarbonate and phosphate and decreased sodium, chloride and hydrogen ion in the saliva. Salivary amylase activity was low and highly variable and the amylase activity/protein ratio fell substantially during isoprenaline stimulation. These results support the conclusion that the enzyme is of extrinsic origin. The response of the kangaroo mandibular gland to isoprenaline stimulation was very similar to that reported for rat mandibular gland, suggesting that the same ion transport phenomena underlie mandibular secretion in both species and probably in therian mammals generally.     

Nutrient composition of spent wash and its impact on sugarcane growth and biochemical attributes

Nutrient composition of crude and digested spent wash and effect of their application on sugarcane growth and biochemical attributes were studied. Higher concentrations of essential nutrients (P, S, Fe, Mn, Zn, Cu) and heavy metals (Cd, Cr, Ni and Pb) were present in crude spent wash (CSW) as compared to the digested spent wash (DSW); sulphur content was the highest (765 μg ml⁻¹ in DSW and 1,609 μg ml⁻¹ in CSW) among all nutrients analyzed. Sugarcane (Saccharum spp. hybrid cultivar CoLk 8102) setts grown in soil pot culture conditions with different rates of crude spent wash (5, 10, 20 and 100 ml kg⁻¹ soil) along with digested spent wash (100 ml kg⁻¹ soil) showed improvement in bud sprouting (10.5 %), settling height (40 %), root number (9.4 %), root length (13.2 %), chlorophyll a (52.9 %) and b (55.3 %) contents and activity of catalase (98 %) enzyme over control at low rate of crude spent wash (5 ml kg⁻¹ soil). Whereas, higher doses of spent wash (20 and 100 ml kg⁻¹ soil) decreased these parameters markedly except peroxidase which was found higher at all the levels of both CSW and DSW. Findings indicated stimulatory effect of low rate of crude spent wash (5 ml kg⁻¹ soil) on root and shoot growth and inhibitory effect of higher dose (100 ml kg⁻¹ soil) of both crude and digested spent wash, therefore, judicious application of spent wash will improve crop productivity and alleviate environmental pollution problems.     

Renal response to head-out water immersion in Korean women divers

Head-out water immersion (HOI) induces a profound diuresis and natriuresis, which may endanger the body fluid balance of breath-hold divers during prolonged diving work. To investigate if adaptation is acquired by professional breath-hold divers, we have evaluated renal responses to 3-h HOI in 5 Korean women divers (Amas) and 11 nondiving housewives (controls). In both control and diver groups, the average urine flow during 3-h immersion was four times greater and Na⁺ excretion was 70%–80% greater than the pre-immersion value [urine flow: 3.7 (SD 1.0) ml·min^–1 vs 0.9 (SD 0.4), P<0.001, in controls; 4.3 (SD 0.9) vs 1.1 (SD 0.4), P<0.001, in divers; Na⁺ excretion: 270 (SD 176) mol· min^–1 vs 161 (SD 84), P<0.025, in controls; 303 (SD 31) vs 164 (SD 62), P<0.005, in divers]. In all cases, the values for a given period were not significantly different between the two groups. The plasma concentrations of Na⁺ and osmolality, and renal clearance of creatinine did not change significantly. However, the osmolal clearance increased [from 2.0 (SD 0.8) ml·min^–1 to 2.8 (SD 0.7), P<0.05, in the controls; from 2.2 (SD 0.4) to 2.6 (SD 0.4), P<0.05, in the divers] and free water clearance changed from negative to positive values [from -1.1 (SD 0.5) ml·min^–1 to 1.2 (SD 0.3), P<0.005, in the controls; from -1.2 (SD 0.4) to 1.6 (SD 1.1), P<0.01, in the divers] during immersion, again the pattern of change being similar in the two groups. It was, therefore, concluded from our study that the renal response to HOI was unchanged in the Korean women professional breath-hold divers compared to the nondiving women.     

Adaptation of renal function to hypotonic medium in the winter flounder (Pseudopleuronectes americanus)

Summary The kidneys of winter flounders transferred to hypotonic medium were investigated for glomerular and tubular handling of fluid and electrolytes and for the urinary excretion of proteins. Media were sea water (925 mosm·kg^–1) and brackish water (70 mosm·kg^–1).In sea water, the urine was hypertonic to the plasma in 7 fish of this study. Urine flow rate was correlated with the GFR. After adaptation to brackish water a delay of 1 to 3 days was observed until the kidneys switched from fluid retention to the excretion of large amounts of dilute urine. GFR and urine flow rate were increased from 0.61±0.08 to 1.58±0.29 ml·h^–1·kg^–1 and from 0.14±0.02 to 0.68±0.08 ml·h^–1·kg^–1, respectively . With increased filtered load the tubular reabsorption of fluid decreased from 74±2.4% to 45±11.2%. The excretion rates of sodium and potassium were increased due to decreased fractional sodium and potassium reabsorption. The urinary excretion of divalent cations, however, was reduced because the net tubular reabsorption of calcium was increased and the net secretion of magnesium was diminished.Both the urinary total protein concentration and the protein pattern showed no significant change, but the rate of protein excretion was increased from 0.21±0.04 to 0.60±0.05 mg·h^–1·kg^–1. The comparison of protein patterns obtained from urine and serum samples revealed that high molecular weight (HMW) proteins prevail in the serum whereas low molecular weight (LMW) proteins dominate in the urine. The diminished quantity of the HMW-protein fraction in the urine thus may reflect size selectivity of the glomerular filtration barrier for serum proteins also in the winter flounder.Abbreviations BW brackish water - SW sea water - GFR glomerular filtration rate - HMW heigh molecular weight - LMW low molecular weight     

An aid to the determination of the ventilatory threshold

Detection of the ventilatory threshold during an incremental load exercise test by eye can be difficult. Although various alternative methods employing information other than the ventilation can be used to assist in determining the ventilatory threshold, they rely on underlying assumptions about the physiological basis for the ventilatory threshold. The method presented here (CUSUM) uses only the ventilation data, and therefore avoids such assumptions. Twelve subjects performed a total of 47 incremental exercise tests to exhaustion. Determinations of the ventilatory thresholds made by eye from the ventilation data (mean of three independent observers) were used as a standard for comparison with determinations using the modified V-slope method and the CUSUM method. A mean (SD) difference of 0.6 (2.84) ml·min^–1·kg^–1 was found between the standard ventilatory thresholds and those determined using the modified V-slope method. A similar comparison between the standard ventilatory thresholds and those determined using the CUSUM method yielded a difference of –0.11 (2.35) ml min^–1·kg^–1. It was concluded that the CUSUM method was a useful aid for the detection of the ventilatory threshold using the ventilation data alone.     

Bovine αs1-casein gene sequences direct high level expression of human granulocyte-macrophage colony-stimulating factor in the milk of transgenic mice

The generation is reported of transgenic mice expressing human granulocyte-macrophage colony-stimulating factor (GM-CSF) or human erythropoietin (EPO) under the control of bovine s1- casein regulatory sequences. GM-CSF expression was specific to the mammary gland, and levels of human GM-CSF in transgenic mouse milk were in the range of mg ml–1. The specific activity of the milk GM-CSF was similar to that of the recombinant protein produced in Escherichia coli, and the glycosylation-derived size heterogeneity corresponded to that of the native human protein. In spite of the identical bovine regulatory sequences of the fusion genes, the levels of human EPO in transgenic mouse milk were 103--106 times lower than those of GM-CSF, ranging from 0.003 to 3 g ml–1. There appeared to be a positive correlation between the amount of EPO in the milk of lactating females and blood haematocrit values. In view of this, other type of constructs should be used to achieve more efficient EPO expression and to circumvent concomitantly-occurring adverse effects. In contrast, the high-level production of recombinant GM-CSF, its resemblance to the native mammalian protein, and mild adverse consequences of transgene expression imply that the current construct could be used for generation of larger GM-CSF transgenic anim als to produce this protein in quantities sufficient for therapeutic purposes     

Physiological and subjective responses to maximal repetitive lifting employing stoop and squat technique

To establish safe levels for physical strain in occupational repetitive lifting, it is of interest to know the specific maximal working capacity. Power output, O₂ consumption, heart rate and ventilation were measured in ten experienced forestry workers during maximal squat and stoop repetitive lifting. The two modes of repetitive lifting were also compared with maximal treadmill running. In addition, electromyogram (EMG) activity in four muscles was recorded and perceived central, local low-back and thigh exertion were assessed during the lifting modes. No significant difference was found in power output between the two lifting techniques. Despite this the mean O₂ consumption was significantly greater during maximal squat lifting [38.7 (SD 5.8) ml·kg^–1-·min^–1] than maximal stoop lifting [32.9 (SD 5.7) ml·kg^–1·min^–1] (P<0.001). No significant correlation was found between O₂ consumption (in millilitres per kilogram per minute) during maximal treadmill running and maximal stoop lifting, while O₂ consumption during maximal squat lifting correlated highly with that of maximal treadmill running (r=0.928, P<0.001) and maximal stoop lifting (r=0.808, P<0.01). While maximal heart rates were significantly different among the three types of exercise, no such differences were found in the central rated perceived exertions. Perceived low-back exertion was rated significantly lower during squat lifting than during stoop lifting. The EMG recordings showed a higher activity for the vastus lateralis muscle and lower activity for the biceps femoris muscle during squat lifting than during stoop lifting. Related to the maximal voluntary contraction, the erector spinae muscle showed the highest activity irrespective of lifting technique.     

Expression of Pseudomonas amyloderamosa isoamylase gene in Saccharomyces cerevisiae

Isoamylase gene (iso) of Pseudomonas amyloderamosa was amplified by polymerase chain reaction and cloned into Saccharomyces cerevisiae vectors under the control of alcohol dehydrogenase gene and glyceraldehyde-3-phosphate dehydrogenase gene promoters. The signal sequence of iso gene was also replaced with that of Schwanniomyces occidentalis -amylase gene. The extracellular isoamylase activity of transformed Sacc. cerevisiae could reach 86 U ml–1 after a 4-days cultivation. © Rapid Science Ltd. 1998     

The influence of a 1-year programme of brisk walking on endurance fitness and body composition in previously sedentary men aged 42–59 years

This study examined the influence of a 1-year brisk walking programme on endurance fitness and the amount and distribution of body fat in a group of formerly sedentary men. Seventy-two males, aged 42–59 years, body mass index 25.2 (0.3) kg·m^–2 [mean (SEM)], were randomly allocated to either a walking group (n = 48) or control group (n = 24). Brisk walking speed was evaluated using a 1.6-km track walk. Changes in endurance fitness were assessed by measuring blood lactate concentration and heart rate during submaximal treadmill walking. Body composition was determined by hydrostatic weighing and anthropometry; energy intake was assessed from 7-day weighed food inventories. Differences in the response of walkers and controls were examined using two-way analyses of variance. Forty-two walkers (87.5%) completed the study and averaged 27.9 (1.4) min·day^–1 of brisk walking (range 11–46). Brisk walking speed averaged 1.95 (0.03) m·s^–1 and elicited approximately 68 (1) % of maximum heart rate. Heart rate and blood lactate concentration during submaximal treadmill walking were significantly reduced in the walkers after 3, 6 and 12 months and the oxygen uptake at a reference blood lactate concentration of 2 mmol·l^–1 was increased by 3.2 ml·kg^–1. min^–1 (14.9%) in the walkers at 6 months (P< 0.01). Although skinfold thicknesses at anterior thigh and medial calf sites decreased significantly for the walkers, the response of the two groups did not differ significantly for other body composition variables or for energy intake.