Continuous monitoring of cellulase action on microcrystalline cellulose

Summary Cellobiose oxidase from Phanerochaete chrysosporium was used for continuous monitoring of cellulase action on microcrystalline cellulose (Avicel). Two protocols are described, the parameter monitored being either the decline in electrode potential as ferricyanide is reduced or consumption of dioxygen. Most experiments used a commercial cellulase preparation from Trichoderma reesei and ferricyanide as acceptor. Within 1 min of an addition of cellulase, ferricyanide reduction reached a steady rate. This was converted into a rate of production of substrate for celobiose oxidase, in mol·min^–1. Experiments were conducted either with a constant concentration of cellulase and increasing Avicel, or with constant Avicel and increasing cellulase. Kinetic analysis of the experiments with constant cellulase indicated a K _mof 4.8 ± 1.0 (g cellulose)·1^–1, which was close to the value predicted from binding studies. The specific activity of the cellulase was measured as 375±25 mol·(g cellulase)^–1·min^–1 in experiments with a high cellulose concentration, but was less than half this value when the cellulose was saturated with cellulase. The maximal rate of cellulose degradation was 9.6±1.3 mol·(g cellulose)^–1·min^–1.     

Ca2+ transport by opercular epithelium of the fresh water adapted euryhaline teleost,Fundulus heteroclitus

We examined transepithelial transport of Ca²⁺ across the isolated opercular epithelium of the euryhaline killifish adapted to fresh water. The opercular epithelium, mounted in vitro with saline on the serosal side and fresh water (0.1 mmol·l^–1 Ca²⁺) bathing the mucosal side, actively transported Ca²⁺ in the uptake direction; net flux averaged 20–30 nmol·cm^–2·h^–1. The rate of Ca²⁺ uptake varied linearly with the density of mitochondria-rich cells in the preparations. Ca²⁺ uptake was saturable, apparent K _1/2 of 0.348 mmol·l^–1, indicative of a multistep transcellular pathway. Ca²⁺ uptake was inhibited partially by apically added 0.1 mmol·l^–1 La³⁺ and 1.0 mmol·l^–1 Mg²⁺. Addition of dibutyryl-cyclic adenosine monophosphate (0.5 mmol·l^–1)+0.1 mmol·l^–1 3-isobutyl-l-methylxanthine inhibited Ca²⁺ uptake by 54%, but epinephrine, clonidine and isoproterenol were without effect. Agents that increase intracellular Ca²⁺, thapsigargin (1.0 mol·l^–1, serosal side), ionomycin (1.0 mol·l^–1, serosal side) and the calmodulin blocker trifluoperazine (50 mol·l^–1, mucosal side) all partially inhibited Ca²⁺ uptake. In contrast, apically added ionomycin increased mucosal to serosal unidirectional Ca²⁺ flux, indicating Ca²⁺ entry across the apical membrane is rate limiting in the transport. Verapamil (10–100 mol·l^–1, mucosal side), a Ca²⁺ channel blocker, had no effect. Results are consistent with a model of Ca²⁺ uptake by mitochondria rich cells that involves passive Ca²⁺ entry across the apical membrane via verapamil-insensitive Ca²⁺ channels, intracellular complexing of Ca²⁺ by calmodulin and basolateral exit via an active transport process. Increases in intracellular Ca²⁺ invoke a downregulation of transcellular Ca²⁺ transport, implicating Ca²⁺ as a homeostatic mediator of its own transport.Abbreviations DASPEI 2-(4-dimethylaminostyryl)-N-ethylpyridinium iodide - db-cAMP dibutyryl-cyclic adenosine monophosphate - FW fresh water - G _t transepithelial conductance - I _sc short-circuit current - IBMX 3-isobutyl-1-methylxanthine - SW sea water - TFP trifluoperazine - V _t transepithelial potential     

Light response of D1 turnover and photosystem II efficiency in the seagrassZostera capricorni

Biochemical and biophysical parameters, including D1-protein turnover, chlorophyll fluorescence, oxygen evolution activity and zeaxanthin formation were measured in the marine seagrassZostera capricorni (Aschers) in response to limiting (100 mol·m^–2·^–1), saturating (350 mol·m^–2·s^–1) or photoinhibitory (1100 mol·m^–2·s^–1) irradiances. Synthesis of D1 was maximal at 350 mol·m^–2·s^–1 which was also the irradiance at which the rate of photosynthetic O₂ evolution was maximal. Degradation of D1 was saturated at 350 mol·m^–2·s^–1. The rate of D1 synthesis at 1100 mol·m^–2·s^–1 was very similar to that at 350 mol·m^–2·s^–1 for the first 90 min but then declined. At limiting or saturating irradiance little change was observed in the ratio of variable to maximal fluorescence (Fv/Fm) measured after dark adaptation of the leaves, while significant photoinhibition occurred at 1100 mol·m^–2·s^–1. The proportion of zeaxanthin in the total xanthophyll pool increased with increasing irradiance, indicative of the presence of a photoprotective xanthophyll cycle in this seagrass. These results are consistent with a high level of regulatory D1 turnover inZostera under non-photoinhibitory irradiance conditions, as has been found previously for terrestrial plants.We would like to thank Professor Peter Böger (Department of Plant Biochemistry, University of Konstanz, Germany) for the kind gift of D1 antibodies. This work was partly supported by a University of Queensland Enabling Grant to CC.     

The dose of 1-naphthaleneacetic acid determines flower-bud regeneration in tobacco explants at a large range of concentrations

Short-term applications of very high concentrations of 1-naphthaleneacetic acid (NAA) to expiants from flower stalks of tobacco (Nicotiana tabacum L. cv. Samsun) induced flower-bud regeneration to the same extent as longer or continuous incubation on lower concentrations. The maximum number of flower buds per explant after 15 d of culture was obtained not only by continuous culturing at 1 mol·l^–1 NAA but also by 12 h of culturing at 22 mol·l^–1 or 0.5 h at 220 mol· l^–1, followed by incubation on medium without auxin for the remaining period. Continuous application of such high concentrations resulted in callus formation or caused the death of the explanted tissue. In all experiments in which auxin concentration and time of application were independently varied, the product of concentration and time determined the number of buds formed. Most, but not all, of the NAA taken up by the tissues was converted into conjugates. In expiants which had received a dose which was optimal for regeneration, the internal concentration of free NAA remaining beyond the pulse period was between 1.7 and 6.2 mol·l^–1. Suboptimal applications led to lower values, supraoptimal treatments to much higher internal concentrations. The physiological effect, which depends on the internal hormone concentration, thus manifested itself as dose-dependent with regard to applied hormone.Abbreviations BAP N⁶-benzylaminopurine - NAA 1-naphthaleneacetic acid     

Photosynthetic production of hydrogen peroxide by Ulva rigida C. Ag. (Chlorophyta)

Production of hydrogen peroxide has been found in Ulva rigida (Chlorophyta). The formation of H₂O₂ was light dependent with a production of 1.2 mol·g FW^–1·h^–1 in sea water (pH 8.2) at an irradiance of 700 mol photons m^–2·s^–1. The excretion was also pH dependent: in pH 6.5 the production was not detectable (< 5 nmol·g FW^–1·h^–1) but at pH 9.0 the production was 5.0 mol·g FW^–1·h^–1. The production of H₂O₂ was totally inhibited by 3-(3,4-dichlorophenyl)-1,1 dimethylurea (DCMU). The ability of U. rigida growing in tanks (7501) under a natural light regime to excrete H₂O₂ was checked and found to be seven times higher at 08.00 hours than other times of the day. The H₂O₂ concentration in the cultivation tank (density: 2 g FW·l^–1) reached the highest value (3 M) at 11.00 hours. Photosynthesis was not influenced by H₂O₂ formation. The H₂O₂ is suggested to come from the Mehler reaction (pseudocyclic photophosphorylation). With an oxygen evolution of 120 mmol·g FW^–1·h^–1 at pH 8.2 and 90 mmol·g FW^–1·h^–1 at pH 9.0, 0.5% and 2.7% of the electrons were used for extracellular H₂O₂ production. The H₂O₂ production is sufficiently high to be of physiological and ecological significance, and is suggested to be a part of the defence against epi and endophytes.Abbreviations ACL artificial, continuous light - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - GNL greenhouse - LDC Luminol-dependent chemiluminescence - SOD Superoxide dismutase This investigation was supported by SAREC (Swedish Agency for Research Cooperation with Developing Countries), Hierta-Retzius Foundation, Marianne and Marcus Wallenberg Foundation, the Swedish Environmental Protection Board, and CICYT Spain.     

Thermal behavior of round and wagtail dancing honeybees

The thermal behavior of round and wagtail dancing honeybees (Apis mellifera carnica) gathering sucrose solutions of concentrations between 0.5 and 2 mol·l^-1 was investigated under field conditions by infrared thermography (30–506 m flight distance). During the stay inside the hive thoracic surface temperature ranged from 31.4 to 43.9 °C. In both round and wagtail dancing honeybees the concentration of sucrose in the food influenced dancing temperature in a non-linear way. Average dancing temperature was 37.9 °C in foragers gathering a 0.5 mol·l^-1 sucrose solution, 40.1°C with a 1 mol·l^-1, 40.6°C with a 1.5 mol·l^-1 and 40.7°C with a 2 mol·l^-1 solution. The variability of thoracic temperature was highest with the 0.5 mol·l^-1 and lowest with the 1.5 and 2 mol·l^-1 concentrations. Thoracic temperatures during trophallactic contact with hive bees were similar to dancing temperature at 1.5 mol·l^-1 but lower at the other concentrations. During periods of distribution of food to hive bees (trophallactic contact >2.5s) the dancers' thorax cooled down by more than 0.5°C considerably more frequently with the 0.5 mol·l^-1 solution (65% of cases) than with the 1.5 mol·l^-1 solution (26%). By contrast, heating the thorax up by more than 0.5°C was infrequent with the 0.5 mol·l^-1 solution (2%) but occurred at a maximum rate of 26% with the 1.5 mol·l^-1 solution. Bees gathering the 1 or 2 mol·l^-1 solutions showed intermediate behavior. Linear model analysis showed that at higher concentrations the dancers compensated better for variations of hive air temperature: per 1 °C increase of hive temperature dancing temperature increased by 0.34, 0.22, 0.12, and 0.13 °C with 0.5, 1, 1.5, and 2 mol·l^-1 sucrose solutions, respectively. The results furnish evidence that dancing honeybees follow a strategy of selective heterothermy by tuning their thermal behavior to the needs of the behavior performed at the moment. Thoracic temperature is regulated to a high level and more accurately when fast exploitation of profitable food sources is recommended. Thoracic temperature is lowered when the ratio of gain to costs of foraging becomes more unfavorable.Abbreviations SD standard deviation - SD _reg SD around regression line - H _rel relative humidity at feeding station - T _a air temperature at feeding station - T _i air temperature near the dancers - T _d Thoracic surface temperatures - T _d dancing - T _tr trophallactic contact (distribution of food) - T _w walking - T _stay mean temperature of total stay in the hive     

Nitrate induction in spruce: an approach using compartmental analysis

Using ¹³NO ₃ ^– -efflux analysis, the induction of nitrate uptake by externally supplied nitrate was monitored in roots of intact Picea glauca (Moench) Voss. seedlings over a 5-d period. In agreement with our earlier studies, efflux analysis revealed three compartments, which have been identified as surface adsorption, apparent free space, and cytoplasm. While induction of nitrate uptake was pronounced, NO ₃ ^– fluxes in induced plants were decidedly lower and the induction response was slower than in other species. Influx rose from 0.1 mol·g^–1·h^–1 (measured at 100 M [NO ₃ ^– _o) in uninduced plants to a maximum of 0.5 mol·g^–1h^–1 after 3 d of exposure to 100 M [NO ₃ ^– _o and declined to 0.3–0.4 mol·g^–1h^–1 at the end of the 5-d period. Efflux remained relatively constant around 0.02-0.04 mol·g^–1h^–1, but its percentage with respect to influx declined from initially high values (around 30%) to steady-state values of 4–7%. Cytoplasmic [NO ₃ ^– ] ranged from the low micromolar in uninduced plants to a maximum of 2 mM in plants fully induced at 100 M [NO ₃ ^– ]_o. In-vivo root nitrate reductase activity (NRA) was measured over the same time period, and was found to follow a similar pattern of induction as influx. The maximum response in NRA slightly preceded that of influx. It increased from 25 nmol·g^–1·h^–1 without prior exposure to NO ₃ ^– to peak values around 150 nmol· g^–1h^–1 after 2 d of exposure to 100 M [NO ₃ ^– ]_o. Subsequently, NRA declined by about 50%. The dynamics of flux partitioning to reduction, to the vacuole, the xylem, and to efflux during the induction process are discussed.The research was supported by an Natural Sciences and Engineering Research Council, Canada, grant to Dr. A.D.M. Glass and by a University of British Columbia Graduate Fellowship to Herbert J. Kronzucker. Our thanks go to Dr. M. Adam and Mr. P. Culbert at the particle accelerator facility TRIUMF on the University of British Columbia campus for providing ¹³N, to Drs. R.D. Guy and S. Silim for providing plant material, and to Dr. M.Y. Wang, Mr. J. Bailey, Mr. J. Mehroke and Mr. J. Vidmar for essential assistance in experiments.     

Inversion of gravitropism by symmetric blue light on the clinostat

Gravitropic stimulation of maize (Zea mays L.) seedlings resulted in a continuous curvature of the coleoptiles in a direction opposing the vector of gravity when the seedlings were rotated on a horizontal clinostat. The orientation of this response, however, was reversed when the gravitropic stimulation was preceeded by symmetric preirradiation with blue light (12.7 mol photons·m^–2). The fluence-response curve of this blue light exhibited a lower threshold at 0.5 mol·m^–2, and could be separated into two parts: fluences exceeding 5 mol·m^–2 reversed the direction of the gravitropic response, whereas for a range between the threshold and 4 mol·m^–2 a split population was obtained. In all cases a very strong curvature resulted either in the direction of gravity or in the opposite orientation. A minor fraction of seedlings, however, curved towards the caryopsis. Furthermore, the capacity of blue light to reverse the direction of the gravitropic response disappeared with the duration of gravitropic stimulation and it depended on the delay time between both stimulations. Thistonic blue-light influence appears to be transient, which is in contrast to the stability observed fortropistic blue-light effects.This work was supported by the Deutsche Forschungsgemeinschaft.     

Sensitivity to far-red radiation in stomata of Phaseolus vulgaris L.: Rhythmic effects on conductance and photosynthesis

The influence of far-red (FR; 700–800 nm) radiation on steady-state stomatal conductance and net photosynthesis in P. vulgaris has been studied. Whereas FR radiation alone was relatively ineffective, addition of FR to a background of white light (WL; predominantly 400–700 nm) resulted in increased stomatal conductance. Stomata exhibited a marked diurnal sensitivity to FR. The action maximum for enhancing stomatal conductance was near 714 nm. A combination of FR and infra-red (IR; >800 nm) enhanced net photosynthesis when added to a background of WL. When IR alone was added to WL, there was a net decrease in photosynthesis, indicating that it is the FR waveband which is responsible for the observed photosynthetic effects. Naturally occurring levels of FR radiation (235 mol·m^-2·s^-1) in vegetation-canopy shade enhanced net photosynthetic CO₂ gain by 28% when added to a background of 55 mol·m^-2·s^-1 WL.Abbreviations BL blue - FR far-red - IR infra-red - PAR photosynthetically active radiation - R red - WL white light     

Mucosal acidification and an acid microclimate in the hen colon in vitro

Experiments were performed on isolated, stripped colonic epithelia of low-salt-adapted hens (Gallus domesticus) in order to characterize acid secretion by this tissue. With symmetric, weak buffer solutions, colonic epithelia acidified both mucosal and serosal sides. Titration measurements of the mucosal acidification rate (pH-stat technique) averaged 1.63±0.25 Eq·cm^-2·h^-1. Mucosal acidification was also evident in colons from high-salt-adapted birds and in low-salt-adapted coprodeum, but was completely abolished in the high-salt coprodeum. Mucosal acidification by low-salt-adapted colonic epithelium was unaffected by sodium replacement, mucosal amiloride (10^-3 mol·l^-1), and serosal ouabain (5x10^-4 mol·l^-1), although all three treatments significantly reduced or reversed the short-circuit current. Acetazolamide (10^-3 mol·l^-1, serosal) reduced mucosal acidification by 15% and simultaneously increased short-circuit current by a similar amount. Colonic epithelia incubated in glucose-free solutions had significantly lower acidification rates (0.59±0.13 Eq·cm^-2·h^-1, P<0.002 versus controls) and addition of glucose (15 mmol·l^-1), but not galactose, partially restored acidification to control levels. Anoxia (N₂ gassing) completely inhibited short-circuit current, but reduced acidification by only 30%. A surface microclimate pH, nearly 2 pH units more acidic than the bath pH of 7.1–7.4 was measured in low-salt-adapted colon and coprodeum. The acid microclimate of both tissues was partially attenuated by adaptation to a high-salt diet. Colonic microclimate pH was dependent on the presence of glucose and sensitive to the bath pH. Histochemical staining for carbonic anhydrase localized this enzyme to cytoplasm and lateral margins of one subfraction of colonic cells, and to cytoplasm in a second subpopulation Intense staining was also evident in subepithelial capillaries. These results suggest that a large part of mucosal acidification and maintenance of the acid microclimate in hen colon may be dependent on glycolysis and metabolic acid production, although a smaller, electrogenic and acetazolamidesensitive component also appears to exist. This latter component may become more prominent under conditions of cellular acidification.Abbreviations CA carbonic anhydrase - I _SC short circuit current - NFM N-ethylmaleimide - PD transepithelial potential - SCFA short chain fatty acids     

Decreased ribulose-1,5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with “antisense” rbcS

Tobacco (Nicotiana tabacum L.) plants transformed with antisense rbcS to decrease the expression of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) have been used to investigate the contribution of Rubisco to the control of photosynthesis in plants growing at different irradiances. Tobacco plants were grown in controlled-climate chambers under ambient CO₂ at 20°C at 100, 300 and 750 mol·m^–2·s^–1 irradiance, and at 28°C at 100, 300 and 1000 mol·m^–2·s^–1 irradiance. (i) Measurement of photosynthesis under ambient conditions showed that the flux control coefficient of Rubisco (C _infRubisco ^supA ) was very low (0.01–0.03) at low growth irradiance, and still fairly low (0.24–0.27) at higher irradiance. (ii) Short-term changes in the irradiance used to measure photosynthesis showed that C _infRubisco ^supA increases as incident irradiance rises, (iii) When low-light (100 mol·m^–2·s^–1)-grown plants are exposed to high (750–1000 mol·m^–2·s^–1) irradiance, Rubisco is almost totally limiting for photosynthesis in wild types. However, when high-light-grown leaves (750–1000 mol·m^–2·s^–1) are suddenly exposed to high and saturating irradiance (1500–2000 mol·m^–2·s^–1), C _infRubisco ^supA remained relatively low (0.23–0.33), showing that in saturating light Rubisco only exerts partial control over the light-saturated rate of photosynthesis in sun leaves; apparently additional factors are co-limiting photosynthetic performance, (iv) Growth of plants at high irradiance led to a small decrease in the percentage of total protein found in the insoluble (thylakoid fraction), and a decrease of chlorophyll, relative to protein or structural leaf dry weight. As a consequence of this change, high-irradiance-grown leaves illuminated at growth irradiance avoided an inbalance between the light reactions and Rubisco; this was shown by the low value of C _infRubisco ^supA (see above) and by measurements showing that non-photochemical quenching was low, photochemical quenching high, and NADP-malate dehydrogenase activation was low at the growth irradiance. In contrast, when a leaf adapted to low irradiance was illuminated at a higher irradiance, Rubisco exerted more control, non-photochemical quenching was higher, photochemical quenching was lower, and NADP-malate dehydrogenase activation was higher than in a leaf which had grown at that irradiance. We conclude that changes in leaf composition allow the leaf to avoid a one-sided limitation by Rubisco and, hence, overexcitation and overreduction of the thylakoids in high-irradiance growth conditions, (v) Antisense plants with less Rubisco contained a higher content of insoluble (thylakoid) protein and chlorophyll, compared to total protein or structural leaf dry weight. They also showed a higher rate of photosynthesis than the wild type, when measured at an irradiance below that at which the plant had grown. We propose that N-allocation in low light is not optimal in tobacco and that genetic manipulation to decrease Rubisco may, in some circumstances, increase photosynthetic performance in low light.Abbreviations A rate of photosynthesis - C _infRubisco ^supA flux control coefficient of Rubisco for photosynthesis - c_i internal CO₂ concentration - qE energy-dependent quenching of chlorophyll fluorescense - qQ photochemical quenching of chlorophyll fluorescence - NADP-MDH NADP-dependent malate dehydrogenase - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - RuBP ribulose-1,5-bisphosphate This work was supported by the Deutsche Forschungsgemeinschaft (SFB 137).     

Transport of monoamine and amino acid neurotransmitters by primary astroglial cultures

The transport of [³H]l-glutamate, [³H]l-aspartate, [³H]-aminobutyric acid ([³H]GABA), [³H]dopamine, [³H]norepinephrine and [³H]5-hydroxytryptamine (³H-5-HT) was measured in primary astroglial cultures from newborn rat cerebral hemispheres. There was a high-affinity uptake with aK _m of 69.0 M for L-glutamate, 12.3 M forl-aspartate and 3.1 M for GABA. The uptake showed properties of high capacity with aV _max of 17.0 nmol·mg prot^–1·min^–1 forl-glutamate, 1.1 nmol·mg prot^–1·min^–1 forl-aspartate and 0.04 nmol·mg prot^–1·min^–1 for GABA. No high-affinity high capacity transport system was found for the monoamines studies. Autoradiographic examination demonstrated a heavy deposit of grains suggesting a prominent accumulation of [³H]l-glutamate and [³H]l-aspartate in the astroglial-like cells of the cultures, while the [³H]GABA accumulation was less intense. On the other hand, there was only a weak accumulation of grains after incubating the cultures with [³H]dopamine, [³H]norepinephrine or [³H]5-HT. Thus, astroglial cells in culture accumulate amino acid neurotransmitters and monoamines in different ways with a high-affinity high-capacity uptake of glutamate, aspartate and GABA and a diffusion-uptake of dopamine, norepinephrine and 5-HT.     

Noradrenalin antagonizes effects of serotonin and acetylcholine in the seawater eel intestine

After inhibiting ion and water transport with 10^-6 mol·l^-1 serotonin and 10^-6 mol·l^-1 methacholine, a muscarinic agonist of acetylcholine, 10^-5 mol·l^-1 (±)noradrenaline restored the serosa-negative transepithelial potential difference and short-circuit current in a step-like manner, accompanied by an increase in water absorption across the seawater eel intestine. Such recovery by noradrenalin was not obtained after pretreatment with 10^-7 mol·l^-1 eel atrial natriuretic peptide. This means that the inhibitory mechanisms of serotonin and acetylcholine are different from those of atrial natriuretic peptide. Similarly, 10^-7 mol·l^-1 clonidine and guanabenz (₂-agonists) also reversed the inhibitory action of serotonin and methacholine, but 10^-7 mol·l^-1 phenylephrine (₁-agonists) and 10^-7 mol·l^-1 isoproterenol (-agonist) did not antagonize serotonin and methacholine actions. Further, the enhancement by 10^-5 mol·l^-1 noradrenalin was blocked by 10^-4 mol·l^-1 yohimbine (₂-agonists) and 10^-4 mol·l^-1 prazosin (₁-agonists), but not by 10^-4 mol·l^-1 propranolol (-antagonist). Although relatively high dosage is required to obtain a significant effect, and discrimination between ₁- and ₂- is not successful in the present study, these results suggest that noradrenalin acts on an -type receptor. The -type receptor may exist on the enterocytes, since the effects of noradrenalin are observed even in the presence of 10^-6 mol·l^-1 tetrodotoxin. Interestingly, the tissue resistance also increased in parallel with increase in the short-circuit current after treatment with noradrenalin in the posterior part of the seawater eel intestine.Abbreviations ACh acetylcholine - 5-HT serotonin - eANP eel atrial natriuretic peptide - I _sc short-circuit current - MCh methacholine - NA noradrenalin - PD transepithelial potential difference - R _t tissue resistance - TTX tetrodotoxin - VIP vasoactive intestinal peptide     

Ion transport across leech integument

The dorsal skin of the leech Hirudo medicinalis was used for electrophysiological measurements performed in Ussing chambers. The leech skin is a tight epithelium (transepithelial resistance = 10.5±0.5 k· cm^-2) with an initial short-circuit current of 29.0±2.9 A·cm^-2. Removal of Na⁺ from the apical bath medium reduced short-circuit current about 55%. Ouabain (50mol·l^-1) added to the basolateral solution, depressed the short-circuit current completely. The Na⁺ current saturated at a concentration of 90 mmol Na⁺·l^-1 in the apical solution (K _M=11.2±1.8 mmol·l^-1). Amiloride (100 mol·l^-1) on the apical side inhibited ca. 40% of the Na⁺ current and indicated the presence of Na⁺ channels. The dependence of Na⁺ current on the amiloride concentration followed Michaclis-Menten kinetics (K _i=2.9±0.4 mol·l^-1). The amiloride analogue benzamil had a higher affinity to the Na⁺ channel (K _i=0.7±0.2 mol·l^-1). Thus, Na⁺ channels in leech integument are less sensitive to amiloride than channels known from vertebrate epithelia. With 20 mmol Na⁺·l^-1 in the mucosal solution the tissue showed an optimum amiloride-inhibitable current, and the amiloride-sensitive current under this condition was 86.8±2.3% of total short-circuit current. Higher Na⁺ concentrations lead to a decrease in amiloride-blockade short-circuit current. Sitmulation of the tissue with cyclic adenosine monophosphate (100 mol·l^-1) and isobutylmethylxanthine (1 mmol·l^-1) nearly doubled short-circuit current and increased amiloride-sensitive Na⁺ currents by 50%. By current fluctuation analysis we estimated single Na⁺ channel current (2.7±0.9 pA) and Na⁺ channel density (3.6±0.6 channels·m^-2) under control conditions. After cyclic adenosine monophosphate stimulation Na⁺ channel density increased to 5.4±1.1 channels·m^-2, whereas single Na⁺ channel current showed no significant change (1.9±0.2 pA). These data present a detailed investigation of an invertebrate epithelial Na⁺ channel, and show the similarities and differences to vertebrate Na⁺ channels. Whereas the channel properties are different from the classical vertebrate Na⁺ channel, the regulation by cyclic adenosine monophosphate seems similar. Stimulation of Na⁺ uptake by cyclic adenosine monophosphate is mediated by an increasing number of Na⁺ channels.Abbreviations slope of the background noise component - ADH antidiuretic hormone - cAMP cyclic adenosine monophosphate - f frequency - f _c coner frequency of the Lorentzian noise component - Hepes N-hydroxyethylpiperazine-N-ethanesulphonic acid - BMX isobutyl-methylxanthine - i _Na single Na⁺ channel current - I _Na max, maximal inhibitable Na⁺ current - I _SC short circuit current - K _i half maximal blocker concentration - K _M Michaelis constandard error of the mean - S _(f) power density of the Lorentzian noise component - S ₀ plateau value of the Lorentzian noise component - TMA tetramethylammonium - Trizma TRIS-hydroxymethyl-amino-methane - V _max maximal reaction velocity - V _T transepithelial potential - K half maximal blocker concentration     

Thiophene accumulation in relation to morphology in roots of Tagetes patula

Roots of marigold (Tagetes patula L.) accumulate thiophenes, heterocyclic sulfurous compounds with strong biocidal activity. In detached roots cultured in vitro, the thiophene content was 5 mol·(g fresh weight)^-1 which is 25-times higher than in roots attached to the plant. In roots derived from tissues transformed by Agrobacterium tumefaciens and A. rhizogenes, the morphology and thiophene content varied with the bacterial strain used. Transformation stimulated the elongation of the root tips and the formation of lateral roots but lowered the thiophene level to 20–50% relative to the concentration in untransformed detached roots. A negative correlation was found between the number of laterals in a root system and the thiophene content. Extensive branching and a decrease in thiophene accumulation was evoked in untransformed roots by indole-3-acetic acid (1–10 mol·l^-1) added to the medium. Within the roots, the highest thiophene concentrations were found in the tips. The results indicate that auxin directly or indirectly plays a role in the regulation of the thiophene level in root tips.Abbreviations B5 Gamborg's B5 medium - IAA Indole-3-acetic acid     

Temperature-dependent feedback inhibition of photosynthesis in peanut

Arachis hypogaea L. is a tropical crop that is slow-growing at temperatures below 25°C. Unadapted CO₂-assimilation rate (A) showed insufficient variation between 15 and 30°C in the short term (hours) to explain this marked reduction in growth. However, at longer periods (12 d), A was depressed as were growth rate and leafproduction rate. To examine the possible relationship between growth, A and sink demand plants were transferred from 30°C, which is near the optimum for growth, to a suboptimal temperature (19°C). In the first 2 d of cooling, A decreased by 50–70%, the stomata stayed open, and the intercellular CO₂ concentration (c_i) rose, i.e. the decrease in A of the cooled plants was the result of non-stomatal factors. Changes in dark respiration did not account for the decline in A.Clear evidence was obtained of sink control of A by independently manipulating the temperature of different leaves on the plant. Cooling (to 19°C) most of the plant (the sink) led to a 70% decline in A of the remaining leaves at 30°C after 3 d, whereas the converse treatments (30°C sink, 19°C source) resulted in small changes (17%). In plants at 19°C which were exposed to low CO₂ concentration to prevent photosynthesis, A was not reduced when measured at normal CO₂ concentrations, indicating that carbohydrate accumulation was responsible for the decline in A. Dry-matter build-up at suboptimal temperature was also consistent with end-product inhibition of photosynthesis.Abbreviations and symbols A (mol·m^-2·s^-1) rate of net CO₂ assimilation - C_i (l·l^-1) substomatal CO₂ concentration - DW (g) dry weight - g (mol·m^-2·s^-1) stomatal conductance to diffusion of water vapour - PFD (mol·m^-2·s^-1) photon flux density     

Thiamin transport by human erythrocytes and ghosts

Summary Thiamin transport in human erythrocytes and resealed pink ghosts was evaluated by incubating both preparations at 37 or 20°C in the presence of [³H]-thiamin of high specific activity. The rate of uptake was consistently higher in erythrocytes than in ghosts. In both preparations, the time course of uptake was independent from the presence of Na⁺ and did not reach equilibrium after 60 min incubation. At concentrations below 0.5 m and at 37°C, thiamin was taken up predominantly by a saturable mechanism in both erythrocytes and ghosts. Apparent kinetic constants were: for erythrocytes,K _m =0.12, 0.11 and 0.10 m andJ _max=0.01, 0.02 and 0.03 pmol·l^–1 intracellular water after 3, 15, and 30 min incubation times, respectively; for ghosts,K _m =0.16 and 0.51 m andJ _max=0.01 and 0.04 pmol·l^–1 intracellular water after 15 and 30 min incubation times, respectively. At 20°C, the saturable component disappeared in both preparations. Erythrocyte thiamin transport was not influenced by the presence ofd-glucose or metabolic inhibitors. In both preparations, thiamin transport was inhibited competitively by unlabeled thiamin, pyrithiamin, amprolium and, to a lesser extent, oxythiamin, the inhibiting effect being always more marked in erythrocytes than in ghosts. Only approximately 20% of the thiamin taken up by erythrocytes was protein-(probably membrane-) bound. A similar proportion was esterified to thiamin pyrophosphate. Separate experiments using valinomycin and SCN^– showed that the transport of thiamin, which is a cation at pH 7.4, is unaffected by changes in membrane potential in both preparations.     

A rapid method for naphtalene dioxygenase assay in whole cells of naphtalene cis-dihydrodiol dehydrogenase blocked Pseudomonas fluoresecens: Screening of potential inducers of dioxygenase activity

A rapid and sensitive photometric method was devised to assay naphthalene dioxygenase in whole cells of Pseudomonas fluorescens NCIMB 40531, a strain derived from a naphthalene-metabolizing isolate by means of N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The naphthalene-assimilating pathway of NCIMB 40531 is functionally blocked at the level of cis-1,2-dihydroxy-1,2-dihydronaphthalene dehydrogenase and therefore cis-1,2-dihydroxy-1,2-dihydronaphthalene (napthalene dihydrodiol) is accumulated when cultures are supplied with naphthalene.This modified metabolism allowed dioxygenase to be assayed by monitoring product formation. Optimal conditions were selected to give linear optical density vs time curves and reaction rates proportional to dry cell weight (DCW): specific activities of 0.125(+-0.008) mol·min^–1·mgDCW^–1 were consistently obtained in cultures grown on succinate in the presence of naphthalene as inducer. By means of the developed assay, 62 compounds (mainly mono- and bicyclic aromatics) were screened as potential inducers of the dioxygenase activity, when added to the growth medium at the concentration of 100 mg·1^–1: besides naphthalene, the highest activities were induced by 3-methylsalicyclic acid (2-hydroxy-3-methylbenzoic acid), O-acetylsalicylic acid and 5-chlorosalicyclic acid with 0.198, 0.167 and 0.076 mol·min^–1mg DCW^–1, respectively. Under the conditions used, no detectable dioxygenase activity was induced by salicylic acid, which is recognized as the natural inducer of the enzyme in Pseudomonas.     

Na/H exchange in cultured epithelial cells from fish gills

Using primary cultures of gill pavement cells from freshwater rainbow trout, a method is described for achieving confluent monolayers of the cells on glass coverslips. A continuous record of intracellular pH was obtained by loading the cells with the pH-sensitive flourescent dye 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein and mounting the coverslips in the flowthrough cuvette of a spectrofluorimeter. Experiments were performed in HEPES-buffered media nominally free of HCO₃. Resting intracellular pH (7.43 at extracellular pH=7.70) was insensitive to the removal of Cl^– or the application of 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (0.1 mmol·l^–1), but fell by about 0.3 units when Na⁺ was removed or in the presence of amiloride (0.2 mmol·l^–1). Exposure to elevated ammonia (ammonia prepulse; 30 mmol·l^–1 as NH₄Cl for 6–9 min) produced an increase in intracellular pH (to about 8.1) followed by a slow decay, and washout of the pulse caused intracellular pH to fall to about 6.5. Intracellular non-HCO ₃ ^– buffer capacity was about 13.4 slykes. Rapid recovery of intracellular pH from intracellular acidosis induced by ammonia prepulse was inhibited more than 80% in Na⁺-free conditions or in the presence of amiloride (0.2 mmol·l^–1). Neither bafilomycin A₁ (3 mol·l^–1) nor Cl removal altered the intracellular pH recovery rate. The K _m for Na⁺ of the intracellular pH recovery mechanism was 8.3 mmol·l^–1, and the rate constant at V _max was 0.008·s^–1 (equivalent to 5.60 mmol H⁺·l^–1 cell water·min^–1), which was achieved at external Na⁺ levels from 25 to 140 mmol·l^–1. We conclude that intracellular pH in cultured gill pavement cells in HEPES-buffered, HCO ₃ ^– -free media, both at rest and during acidosis, is regulated by a Na⁺/H⁺ antiport and not by anion-dependent mechanisms or a vacuolar H⁺-ATPase.Abbreviations BCECF 2,7-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein - BCECF/AM 2,7-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein, acetoxymethylester - Cholin-Cl choline chloride - DMSO dimethyl sulfoxide - EDTA ethylene diamine tetra-acetic acid - FBS foetal bovine serum - H ⁺ -ATPase Proton-dependent adenosine triphosphatase - HEPES N-[2-hydroxyethyl]piperazine-N[2-ethanesulfonic acid] - pH _i intracellular pH - pH _e extracellular pH - PBS phosphate-buffered saline - SITS 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid     

Organic reduction of tapioca wastewaters using modified RBC

A modified Rotating Biological Contactor (RBC) was used for the treatability studies of synthetic tapioca wastewaters. The RBC used was a four stage laboratory model and the discs were modified by attaching porous nechlon sheets to enhance biofilm area. Synthetic tapioca wastewaters were prepared with influent concentrations from 927 to 3600 mg/l of COD. Three hydraulic loads were used in the range of 0.03 to 0.09 m³·m^–2·d^–1 and the organic loads used were in the range of 28 to 306 g COD· m^–2·d^–1. The percentage COD removal were in the range from 97.4 to 68. RBC was operated at a rotating speed of 18 rpm which was found to be the optimal rotating speed. Biokinetic coefficients based on Kornegay and Hudson models were obtained using linear analysis. Also, a mathematical model was proposed using regression analysis.List of Symbols A m² total surface area of discs - d m active depth of microbial film onany rotating disc - K _s mg ·l^–1 saturation constant - P mg·m^–2·^–1 area capacity - Q l·d^–1 hydraulic flow rate - q m³·m^–2·d^–1 hydraulic loading rate - S ₀ mg·l^–1 influent substrate concentration - S _e mg·l^–1 effluent substrate concentration - w rpm rotational speed - V m³ volume of the reactor - X _f mg·l^–1 active biomass per unit volume ofattached growth - X _s mg·l^–1 active biomass per unit volume ofsuspended growth - X mg·l^–1 active biomass per unit volume - Y _s yield coefficient for attachedgrowth - Y _A yield coefficient for suspendedgrowth - Y yield coefficient, mass of biomass/mass of substrate removed Greek Symbols hr mean hydraulic detention time - (_max)_A d^–1 maximum specific growth rate forattached growth - (_max)_s d^–1 maximum specific growth rate forsuspended growth - _max d^–1 maximum specific growth rate - d^–1 specific growth rate - _v mg·l^–1·hr^–1 maximum volumetric substrateutilization rate coefficient