Evidence for K+ channel control in Vicia guard cells coupled by G-proteins to a 7TMS receptor mimetic

To explore the involvement of a class of seven-trans-membrane-span (7TMS) receptors in cellular signalling, a synthetic analogue (mas7) of the amphipathic tetradecapeptide mastoparan was used to mimic hormonal stimulus in guard cells of Vicia faba. The ability for mas7 to substitute for an activated receptor complex was assayed by the effect on guard cell ion channel activities in the absence of any hormonal stimulus. Currents carried by inward-(I_K,in) and outward-(I_K,out) rectifying potassium channels were determined under voltage clamp conditions before, during, and after exposure to mas7. The dominant effect of mas7 was to inactivate I_K,in within 30 sec of application. By contrast, I_K,out was largely unaffected under these conditions. The effect of mas7 on I_K,in was both concentration- and voltage-dependent. At any one clamp voltage, mas7 inactivation showed Michaelian behaviour, with a mean K_i of 0.05 ± 0.02 µM at ?240 mV. Increasing mas7 concentration also shifted the voltage for half-maximal activation of the current negative, with 0.5 µM mas7 effecting a ?13 ± 2 mV displacement and lengthening the halftime for activation of the current by up to threefold. By contrast, the non-amphipathic analogue of mas7, masCP, had no appreciable effect on the steady-state current or its activation kinetics; nor was the poly-cation polylysine able to substitute for mas7 in its action on the K⁺ channels. Application of the non-hydrolysable analogue of GDP, GDP-β-S, either by iontophoresis or by diffusion from the microelectrode, effectively blocked mas7-induced inactivation of I_K,in. These, and additional results provide in vivo evidence for the involvement of G-protein-linked 7TMS receptors in the regulation of membrane transport in a higher plant cell.     

Influence of enhanced CO2 on growth and photosynthesis of the red algaeGracilaria sp. andG. chilensis

The influence of elevated CO₂ concentrations on growth and photosynthesis ofGracilaria sp. andG. chilensis was investigated in order to procure information on the effective utilization of CO₂. Growth of both was enhanced by CO₂ enrichment (air + 650 ppm CO₂, air + 1250 ppm CO₂, the enhancement being greater inGracilaria sp. Both species increased uptake of NO₃ ^– with CO₂ enrichment. Photosynthetic inorganic carbon uptake was depressed inG. chilensis by pre-culture (15 days) with CO₂ enrichment, but little affected inGracilaria sp. Mass spectrometric analysis showed that O₂ uptake was higher in the light than in the dark for both species and in both cases was higher inGracilaria sp. The higher growth enhancement inGracilaria sp. was attributed to greater depression of photorespiration by the enrichment of CO₂ in culture.     

Differences in cellular fatty acids and lipid composition inClostridium pasteurianum under nitrogen-and non-nitrogen-fixing conditions

Clostridium pasteurianum total cellular saturated fatty acids increased through its growth cycle from 81% to 91% but varied significantly in the composition under nitrogen- and non-nitrogen-fixing conditions. During ammonia-assimilating growth, palmitic acid decreased from 67.7% to 43.5% by late log while marked increases in shorter chain saturated fatty acids (C_15:0 and below) and a long chain saturated C_22:0 occured. In contrast, under N₂-fixing growth conditions, palmitic acid increased from 45.5% to 84.3% by late log, representing nearly the total amound of saturated fatty acids found inC. pasteurianum. The total cellular lipid concentration decreased as the culture aged. irrespective of the nitrogen sources; however, the phospholipid concentration increased significantly during N₂-fixing growth as compared with a 50% decrease during ammonia-assimilating conditions. The implication of these differences and possible role of palmitic acid and phospholipids inC. pasteurianum nitrogen fixation process are discussed.     

Fluctuations in circulating cell numbers following chemotherapy or bone marrow transplant

We study the effect of noise on the behaviour of a dynamic cell population model in which cell replication and maturation take place simultaneously. We assume that the maximum proliferative potential fluctuates uniformly about a mean value of v, and show that a decrease in v and/or the input flux u _in into the population can lead to an increase in the variance in the cellular efflux u _f. We draw a qualitative correspondence between this behaviour and the commonly observed increase in the variance of circulating blood cell numbers following chemotherapy and radiotherapy, both of which lead to a decrease in v and u _in , and bone marrow transplant which probably corresponds to a decrease in u _in .     

Regulation of the basolateral potassium conductance of theNecturus proximal tubule

Summary Two methods, the measurement of the response of the basolateral membrane potential (V _bl) of proximal tubule cells ofNecturus to step changes in basolateral K⁺ concentration, and cellular cable analysis, were used to assess the changes in basolateral potassium conductance (G _K) caused by a variety of maneuvers. The effects of some of these maneuvers on intracellular K⁺ activity (a _K ⁱ ) were also evaluated using double-barreled ion-selective electrodes. Perfusion with 0mm K⁺ basolateral solution for 15 min followed by 45 min of 1mm K⁺ solution resulted in a fall in basolateral potassium (apparent) transference number (t _K),V _bl anda _K ⁱ . Results of cable analysis showed that total basolateral resistance,R _b , rose. The electrophysiological effects of additional manipulations, known to inhibit net sodium reabsorption across the proximal tubular epithelium ofNecturus, were also investigated. Ouabain caused a fall int _K accompanied by large decreases ina _K ⁱ andV _bl. Lowering luminal sodium caused a fall int _K and a small reduction inV _bl. Selective reduction of peritubular sodium, a maneuver that has been shown to block sodium transport from lumen to peritubular fluid, also resulted in a significant decrease int _K. These results suggest thatG _K varies directly with rate of transport of the sodium pump, irrespective of the mechanism of change in pump turnover.Part of this material has been presented at the 10th International Conference on Biological Membranes (Cohen & Giebisch, 1984).     

The internal rotenone‐insensitivae NADPH dehydrogenase contributes to malate oxidation by potato tuber and pea leaf mitochondria

Inside-out submitochondrial particles from both potato (Solanum tuberosum L. cv. Bintje) tubers and pea (Pisum sativum L. cv. Oregon) leaves possess three distinct dehydrogenase activities: Complex I catalyzes the rotenone-sensitive oxidation of deamino-NADH, ND_in(NADPH) catalyzes the rotenone-insensitive and Ca²⁺-dependent oxidation of NADPH and ND_in(NADH) catalyzes the rotenone-insensitive and Ca²⁺-independent oxidation of NADH. Diphenylene iodonium (DPI) inhibits complex I, ND_in(NADPH) and ND_in (NADH) activity with a K_i of 3.7, 0.17 and 63 µM, respectively, and the 400-fold difference in K_i between the two ND_in made possible the use of DPI inhibition to estimate ND_in (NADPH) contribution to malate oxidation by intact mitochondria. The oxidation of malate in the presence of rotenone by intact mitochondria from both species was inhibited by 5 µM DPI. The maximum decrease in rate was 10–20 nmol O₂ mg^?1 min^?1. The reduction level of NAD(P) was manipulated by measuring malate oxidation in state 3 at pH 7.2 and 6.8 and in the presence and absence of an oxaloacetate-removing system. The inhibition by DPI was largest under conditions of high NAD(P) reduction. Control experiments showed that 125 µM DPI had no effect on the activities of malate dehydrogenase (with NADH or NADPH) or malic enzyme (with NAD⁺ or NADP⁺) in a matrix extract from either species. Malate dehydrogenase was unable to use NADP⁺ in the forward reaction. DPI at 125 µM did not have any effect on succinate oxidation by intact mitochondria of either species. We conclude that the inhibition caused by DPI in the presence of rotenone in plant mitochondria oxidizing malate is due to inhibition of ND_in(NADPH) oxidizing NADPH. Thus, NADP turnover contributes to malate oxidation by plant mitochondria.     

Studies on the phospholipase A of dermatophytes

The presence of phospholipase A activity was detected in three dermatophytes:Microsporum cookei, Trichophyton mentagrophytes, andEpidermophyton floccosum. The activity was always higher inT. mentagrophytes than inM. cookei andE. floccosum. All exhibited phospholipase A₁ and A₂ activities, but the activity was largely A₂ inM. cookei and A₁ inE. floccosum. T. mentagrophytes possessed almost equal activities of phospholipase A₁ and A₂.     

ALGORITHM FOR APPLICATION OF FOURIER ANALYSIS FOR BIORHYTHMIC BASELINES OF PHARMACODYNAMIC INDIRECT RESPONSE MODELS

The change of an indirect pharmacological response R(t) can be described by a periodic time-dependent production rate k_in (t) and a first-order loss constant k_out. If k_in(t) follows some biological rhythm (e.g., circadian), then the response R(t) also displays a periodic behavior. A new approach for describing the input function in indirect response models with biorhythmic baselines of physiologic substances is introduced. The present approach uses the baseline (placebo) response R_b(t) to recover the equation for k_in(t). Fourier analysis provides an approximate equation for R_b(t) that consists of terms (usually two or three) of the Fourier series (harmonics) that contribute most to the overall sum. The model differential equation is solved backward for k_in(t), yielding the equation involving R_b(t). A computer program was developed to perform the square L²-norm approximation technique. Fourier analysis was also performed based on nonlinear regression. Cortisol suppression after inhalation of fluticasone propionate (FP) was modeled based on the inhibition of the secretion rate k_in(t) using ADAPT II. The pharmacodynamic parameters k_out and IC₅₀ were estimated from the model equation with k_in(t) derived by the new approach. The proposed method of describing the input function needs no assumption about the behavior of k_in(t), is as efficient as methods used previously, and is more flexible in describing the baseline data than the nonlinear regression method. (Chronobiology International, 17(1), 77–93, 2000)     

Glucose catabolism during alkaloid production in a strain ofClaviceps purpurea

Badiorespirometric experiments with glucose labelled in positions 1; 2; 3,4 and 6 were carried out inClaviceps purpurea strain pepty 695. The pattern of¹⁴CO₂ evolution from 5 – 50 mM glucose indicated the operation of the pentose phosphate cycle during alkaloid production. The authors thank Mrs. H. Pechfelder for her skilled technical assistance and Prof. Dr. H. Reinbothe and Dr. C. Wasternack for helpful discussions.     

Effects of concentrated carbon dioxide on the fruiting of several cultivated basidiomycetes (II)

Edible basidiomycetesFlammulia velutipes andPleurotus ostreatus were cultivated in the usual manner on media based on sawdust and rice bran, and the cultures were exposed to slowly flowing CO₂-enriched air (550 (control), 3,000, 6,000, and 9,000µl/l) for seven days at different stages of cultivation. When the cultures were exposed at the primordium stage (less than 10 mm in length), length and yield of fruit-bodies increased and pileus expansion was slightly inhibited inF. velutipes, while inP. ostreatus length increased, yield decreased, and pileus expansion was greatly inhibited. When the cultures with fruit-bodies larger than 10 mm were exposed, length and yield were insensitive and pileus expansion was greatly inhibited inF. velutipes, while inP. ostreatus length was insensitive, but pileus expansion was heavily damaged by trumpet-like deformation and yield decreased. The different action of CO₂ on the two species appeared to be due to the different anatomical structures of their fruit-bodies.     

The Status and the Role of Glutathione under Disturbed Ionic Balance and pH Homeostasis in Escherichia coli

The study of glutathione status in aerobically grown Escherichia coli cultures showed that the total intracellular glutathione (GSH_in + GSSG_in) level falls by 63% in response to a rapid downshift in the extracellular pH from 6.5 to 5.5. The incubation of E. coli cells in the presence of 50 mM acetate or 10 g/ml gramicidin S decreased the total intracellular glutathione level by 50 and 25%, respectively. The fall in the total intracellular glutathione level was accompanied by a significant decrease in the (GSH_in : GSSG_in) ratio. The most profound effect on the extracellular glutathione level was exerted by gramicidin S, which augmented the total glutathione level by 1.8 times and the (GSH_out : GSSG_out) ratio by 2.1 times. The gramicidin S treatment and acetate stress inhibited the growth of mutant E. coli cells defective in glutathione synthesis 5 and 2 times more severely than the growth of the parent cells. The pH downshift and the exposure of E. coli cells to gramicidin S and 50 mM acetate enhanced the expression of the sodA gene coding for superoxide dismutase SodA.     

The xylem pressure potential of shrub species in an oak-hornbeam forest

Diurnal and seasonal changes of the xylem pressure potential (Ψ_xylem) were investigated in five species during three years. Intraspecific comparison was made on the basis of the mathematically expressed relationship Ψ_xylem of the individual species to Ψ_xylem inCrataegus oxyacantha, which exhibited the highest drought resistance. With increasing water stress the value for Ψ_xylem of the individual species decreases linearly in comparison with that ofC. oxyacantha, namely to −1.02 MPa inLigustrum vulgare, to −1.33 MPa inCornus mas, and to −2.09 MPa inEuonymus verrucosa. At a higher water deficit the value for Ψ_xylem of these species decreases more rapidly than inC. oxyacantha. On the basis of these findings, the relative drought resistance of the species may by evaluated, and from the value of Ψ_xylem forC. oxyacantha Ψ_xylem of the individual species may be derived. By measuring the difference between Ψ_xylem of free- and polyethylene-covered individuals the existence of water redistribution within the shrub individual was confirmed.     

The effects of extremely alkaline water (pH 9·5) on rainbow trout gill function and morphology

Rainbow trout that were held under control conditions, at pH8·0, in moderately hard Hamilton tap water, had Cl^? and Na⁺ influx rates (J^CLin and J^Na, respectively) of 270 and 300 μmol kg^?1 h^?1, respectively. Exposure to pH 9·5 water led to an immediate 67% decline in J^CL_in and a 45% reduction in J^Na_in at 0–1 h. Influx rates declined further and by 4–5 h the net decreases in both J^CL_in and J^Na_in approximated 80%. By 24 h J^CL_in had recovered to rates not significantly different from those at pH 8·0; while J^Na_in only partially recovered and remained about 50% lower than control measurements through 72 h. The complete recovery of J^CL_in and partial recovery of J^Na_in may have been related to a fourfold greater branchial chloride cell (CC) fractional surface area observed in rainbow trout exposed to pH 9·5 for 72 h. Ammonia excretion (J_Amm) was about 170 μmol N kg^?1 h^?1 at pH 8·0 but was initially reduced by 90% over the first hour of high pH exposure. J_Amm rapidly recovered and by 24 h it had returned to pre-exposure levels. This recovery tended to parallel the partial recovery of J^Na_in. However, subsequent addition of amiloride (10^?4M) to the water at 75 h led to no change in J_Amm, despite a 50% reduction in J^Na_in. Thus, it does not appear that there is a linkage between Na⁺ influx and the recovery of ammonia excretion under highly alkaline conditions.     

Ultrastructure of leaves in C4 Cyperus iria and C3 Carex siderosticta

The ultrastructural aspects ofCyperus iria leaves showing the C₄ syndrome and the typical C₃ species,Carex siderosticta, in the Cyperaceae family were examined.C. iria exhibited the chlorocyperoid type, showing an unusual Kranz structure with vascular bundles completely surrounded by two bundle sheaths. The cellular components of the inner Kranz bundle sheath cells were similar to those found in the NADP-ME C₄ subtype, having centrifugally arranged chloroplasts with greatly reduced grana and numerous starch grains. Their chloroplasts contained convoluted thyla-koids and a weakly-developed peripheral reticulum, although it was extensive mostly in mesophyll cell chloroplasts. The outer mestome bundle sheath layer was sclerenchymatous and generally devoid of organelles, but had unevenly thickened walls. Suberized lamellae were present on its cell walls, and they became polylamellate when traversed by plasmodesmata. Mesophyll cell chloroplasts showed well-stacked grana with small starch grains. InC. siderosticta, vascular bundles were surrounded by the inner mestome sheath and the outer parenchymatous bundle sheath with intercellular spaces. The mestome sheath cells degraded in their early development and remained in a collapsed state, although the suberized lamellae retained polylamellate features. Plastids with a crystalline structure, sometimes membrane-bounded, were found in the epidermal cells. The close interveinal distance was 35–50 μm inC. iria, whereas it was 157–218 μm inC. siderosticta. These ultrastructural characteristics were discussed in relation to their photosynthetic functions.     

Nystatin affects zinc uptake in human fibroblasts

The mechanism(s) by which zinc is transported into cells has not been identified. Since zinc uptake is inhibited by reducing the temperature, zinc uptake may depend on the movement of plasma membrane micoenvironments, such as endocytosis or potocytosis. We investigated the potential role of potocytosis in cellular zinc uptake by incubating normal and acrodermatitis enteropathica fibroblasts with nystatin, a sterol-binding drug previously shown to inhibit potocytosis. Zinc uptake was determined during initial rates of uptake (10 min) following incubation of the fibroblasts in 50 μg nystatin/mL or 0.1% dimethyl-sulfoxide for 10 min at 37°C. The cells were then incubated with 1 to 30 μM ⁶⁵zinc. Michaelis-Menten kinetics were observed for zinc uptake. Nystatin inhibited zinc uptake in both the normal and AE fibroblasts. Reduced cellular uptake of zinc was associated with its internalization, not its external binding. In normal fibroblasts, nystatin significantly reduced theK _m 56% and theV _max 69%. In the AE fibroblasts, nystatin treatment significantly reduced theV _max 59%, but did not significantly affect theK _m. The AE mutation alone affected theV _max for cellular zinc uptake. The control AE fibroblasts exhibited a 40% reduction inV _max compared to control normal fibroblasts. We conclude that nystatin exerts its effect on zinc uptake by reducing the velocity at which zinc traverses the cell membrane, possibly through potocytosis. Furthermore, the AE mutation also effects zinc transport by reducing zinc transport.     

Insertion mutations in the control region of the galactose operon of E. coli. II. Physical characterization of the mutations

Summary Four gal negative mutations, which affect the expression of the gal operon severely as described in the preceding paper (Saedler et al.), are characterized as insertions of DNA by CsCl density gradient centrifugation of transducing phages carrying the mutations and by electron microscopy of hybrid DNA molecules in which the insertion forms a singlestranded loop.Mutation galOP _in 308 is shown by both procedures to be about twice as large as the three other insertions, which are similar in size. The length of the insertions as determined by electron microscopy corresponds to about 1500 nucleotide pairs galOP _in 308 and 800, 700, and 700 nucleotide pairs for galOP _in 128, 141, and 306 respectively. Single-stranded regions are seen in hybrid molecules prepared between DNAs from galOP _in 306 and 128, 141 or 308 as well as from galOP _in 308 and 128. No such single-stranded regions are observed in hybrid molecules between DNAs from galOP _in 128 and 141.Thus, at least three of the four insertions are not identical.     

Mechanism of action of aflatoxin B1

The inhibitory effects of aflatoxin B₁ were found to be related to the gram character in procaryotes, used in this study. Ethylene diamine tetra chloroacetic acid (0.05% w/v) or Tween-80 (0.05 % v/v) addition accentuated the aflatoxin B₁ growth inhibition inSalmonella typhi andEscherichia coli at different pH values. The inhibition of lipase production was only 5–20 % inPseudomonas fluorescence ca. 25–48% inStaphylococcus aureus andBacillus cereus at different aflatoxin B₁ concentrations (4–16μg/ml).However, inhibition of α-amylase induction was complete in1Bacillus megaterium whereas the inhibition was partial inPseudomonas fluorescence (27–40%) at 32μg aflatoxin B₁ concentration. An increase in leakage of cell contents and decreased inulin uptake were observed in toxin incubated sheep red blood cell suspension (1 %) with increased aflatoxin B₁ concentration     

Voltage- and time dependence of apical membrane conductance during current clamp inNecturus gallbladder epithelium

Summary The effects of short (1 sec) and long (1 min) transepithelial current clamps on membrane voltages and resistances ofNecturus gallbladder were investigated. Transepithelial and cell membrane current-voltage relationships determined from 1-sec clamps revealed that: a) depolarization of the apical membrane voltage (V _mc) results in a marked decrease in apical membrane fractional resistance (fR _a), whereas hyperpolarization ofV _mc results in either no change infR _a or a small increase, and b) the voltage-dependent changes infR _a are essentially complete within 500 msec. Exposure of the tissue to 5mm TEA⁺ on the mucosal side caused no significant change in baselineV _mc (–69±2 mV) and yet virtually abolished the voltage dependence offR _a. A possible interpretation of these results is that two types of K⁺ channels exist in the apical membrane, with different voltage dependencies and TEA⁺ sensitivities. Acidification or Ba²⁺ addition to the mucosal solution also reduced the voltage-dependent changes infR _a. The time courses of the changes infR _a and in the cable properties of the epithelium were assessed during 1-min transepithelial current clamps (±200 A/cm²). No secondary change infR _a was observed with mucosa-to-serosa currents, but a slow TEA⁺-sensitive decrease infR _a (half-time of seconds) was evident with serosa-to-mucosa currents. Cable analysis experiments demonstrated that the initial (<500 msec) voltage-dependent decrease infR _a is due to a fall in apical membrane resistance. The later decrease infR _a is due to changes in both cell membrane resistances attributable to the increase in transcellular current flow resulting from a fall in paracellular conductance. The voltage dependence of the apical membrane conductance is a more significant problem in estimatingfR _a than the current-induced effects on the lateral intercellular spaces. In principle, TEA⁺ can be used to prevent the nonlinear behavior ofR _a during measurements of the voltage divider or membrane resistance ratio.     

Kinetics of Catalase Inactivation Induced by Ultrasonic Cavitation

Kinetic patterns of sonication-induced inactivation of bovine liver catalase (CAT) were studied in buffer solutions (pH 4.0–11.0) within the temperature range from 36 to 55^o. Solutions of CAT were exposed to LF (20.8 kHz) ultrasound (specific power, 48–62 W/cm²). The kinetics of CAT inactivation was characterized by effective first-order rate constants (s^–1) of total inactivation (k _in), thermal inactivation (*k _in), and ultrasonic inactivation (k _in(us)). In all cases, the following inequality was valid: k _in > *k _in. The value of k _in(us) increased with the ultrasound power (range, 48–62 W/cm²) and exhibited a strong dependence on the pH of the medium. On increasing initial concentration of CAT (0.4–4.0 nM), k _in(us) decreased. The three rate constants were minimum within the range pH 6.5–8.0; their values increased considerably at pH < 6.0 and pH > 9.0. At 36–55^o, the temperature dependence of k _in(us) was characterized by an activation energy (E _act) of 19.7 kcal/mol, whereas the value of E _act for CAT thermoinactivation was equal to 44.2 kcal/mol. Bovine and human serum albumins (BSA and HSA, respectively) inhibited sonication-induced CAT inactivation; complete prevention was observed at concentrations above 2.5 g/ml. Dimethyl formamide (DMFA), a scavenger of hydroxyl radicals (O ), prevented sonication-induced CAT inactivation at 10% (k _in and *k _in increased with the content of DMFA at concentrations in excess of 3%). The results obtained indicate that free radicals generated in the field of ultrasonic cavitation play a decisive role in the inactivation of CAT, which takes place when its solutions are exposed to low-frequency ultrasound. However, the efficiency of CAT inactivation by the radicals is determined by (1) the degree of association between the enzyme molecules in the reaction medium and (2) the composition thereof.