Effects Of Ionophores Research Articles

Na+-dependent accumulation of sulfate and thiosulfate in marine sulfate-reducing bacteria

Uptake of 35S-labelled sulfate and thiosulfate was studied in twenty sulfate-reducing bacteria. Micromolar additions of these substrates were highly accumulated by washed cells of freshwater and marine strains. In marine strains accumulation required Na+. Generally, the uptake capacity was increased after sulfate limitation during growth. With two marine species, Desulfovibrio salexigens and Desulfobacterium autotrophicum, the effects of various ionophores and inhibitors affecting the transmembrane pH or Na+ gradient or the membrane potential were studied. In both strains transport was reversible. There was no discrimination between sulfate and thiosulfate. With increasing additions the amount taken up increased, while the accumulation factor (Cin/Cout) decreased. Uptake was not directly correlated with the ATP level inside the cells. From these results and the action patterns of the inhibitors tested it is concluded that marine sulfate-reducing bacteria accumulate sulfate and thiosulfate electrogenically in symport with Na+ ions, while in freshwater strains protons are symported. The high-accumulating systems are induced only at low sulfate concentration, while low-accumulating systems are active at sulfate-sufficient conditions.

The influence of lasalocid and cationomycin on nitrogen digestion in sheep: Comparison of methods for estimating microbial nitrogen

An in vivo study was carried out to evaluate the effect of a new ionophore (cationomycin) on nitrogen digestion compared with lasalocid. Three diets based on forage were given (a control diet, a "cationomycin" diet, a "lasalocid" diet) to six sheep fitted with rumen duodenal and ileal cannulae over three different periods in a Latin square design experiment. The supply of lasalocid or cationomycin (33 mg kg−1) decreased (P < 0.05) the breakdown of dietary proteins in the rumen and lowered the microbial protein synthesis whatever the microbial marker used (diaminopimelic acid or purine bases). The duodenal and ileal flows of organic matter (OM), ammonia nitrogen (NH3-N), nonammonia nitrogen, and amino acids (AA) were not affected by the ionophore addition. We could not use the AA profile method to estimate the microbial contribution to the duodenal flow of nonammonia nitrogen because of the strong similarities in AA composition of the feed and the rumen microbial sample. The efficiency of microbial synthesis was significantly (P < 0.05) decreased by cationomycin, and tended to decrease with lasalocid. Only lasalocid increased the digestibility of dietary nitrogen in all the digestive tract. Key words: Ionophore, lasalocid, cationomycin, digestion, nitrogen, sheep, rumen

Synergistic Effects of a Calcium Ionophore and Activators of Protein Kinase C on Epithelial Paracellular Permeability

Oxidants reversibly increase the paracellular permeability of Madin Darby canine kidney (MDCK) epithelial cell monolayers, and the decrease in resistance occurs within 10 to 15 min of initiating oxidant exposure. Oxidants also initiate hydrolysis of phosphatidylinositol in MDCK cells, with resultant increases in diacylglycerol and inositol phosphates. Phorbol esters and synthetic diacylglycerols increase the paracellular permeability of MDCK monolayers with a time course similar to the oxidants. In contrast, calcium ionophores increase MDCK monolayer paracellular permeability only after 2 to 3 h of exposure. Because the products of the oxidant-initiated phospholipid hydrolysis would be likely to both activate protein kinase C and increase cell calcium, we asked if ionomycin, a calcium ionophore, and phorbol esters or diacylglycerols, activators of protein kinase C, might not act in concert to alter MDCK monolayer paracellular permeability. When ionomycin was added alone to MDCK monolayers, there was an increase in cell calcium, activation of a lumen negative current, a limited transitory decrease in transepithelial resistance, but no increase in mannitol flux across the monolayers. When phorbol dibutyrate (PDBU) or oleyl acetyl glycerol (OAG) were added to MDCK monolayers, there was no current activated, there was a progressive decrease in transepithelial resistance, and there was an increase in mannitol flux across the monolayers which was evident within 20 to 40 min of adding the agent. When 1 microM ionomycin was added to the monolayers along with PDBU or OAG, there was a synergistic increase in paracellular permeability of the monolayers when compared to addition of ionomycin, PDBU, or OAG alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of smooth muscle contraction in rabbit internal anal sphincter by protein kinase C and Ins(1,4,5)P3

We have examined the role of protein kinase C (PKC)-beta II and its functional relationship to inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and intracellular Ca2+ in the contraction of smooth muscle cells from the rabbit internal and sphincter (IAS). PKC-beta (0.1-100 U/ml) and Ins(1,4,5)P3 (10(-9) to 10(-6) M) caused concentration-dependent contraction of IAS smooth muscle cells permeabilized by saponin. The combination of threshold concentrations of Ins(1,4,5)P3 (10(-9) M) and PKC (0.1 U/ml) was more than additive, causing near maximal shortening (28.2 +/- 2.1% decrease in cell length from control). The response to high concentrations of Ins(1,4,5)P3 and PKC used in combination was not greater than the response to either agent alone. The calmodulin antagonist W-7 (10(-9) M) inhibited the maximal contraction induced by Ins(1,4,5)P3 but not contraction caused by PKC, whereas the PKC antagonist H-7 (10(-6) M) inhibited the maximal contraction induced by PKC but not contraction caused by Ins(1,4,5)P3. Threshold doses of the ionophores A23187 (10(-9) M) and ionomycin (0.2 ng/ml) caused little contraction by themselves, but they potentiated the response elicited by a threshold concentration of PKC (0.1 U/ml), inducing maximal contraction. Preincubation of IAS cells with 4 mM Sr2+, which inhibits the release of intracellular Ca2+, abolished the potentiating effect of Ins(1,4,5)P3 and calcium ionophores on PKC, but the calmodulin antagonist W-7 did not. These data suggest that the contractile effect of maximally effective doses of PKC is independent of the effects of Ins(1,4,5)P3. At submaximal concentrations, however, PKC-dependent contraction is potentiated by Ins(1,4,5)P3 or by ionophore-mediated release of intracellular Ca2+ without requiring calmodulin activation.

Delta pH-dependent accumulation of tetracycline in Escherichia coli

The effects of ionophores on tetracycline accumulation in Escherichia coli cells were investigated in the presence of polymyxin B nonapeptide. Accumulation was inhibited by nigericin but not by valinomycin. Tetracycline accumulation was stimulated by decreasing the pH of the medium and inhibited by the addition of magnesium ions. These results indicated that tetracycline enters cells through diffusion as a protonated form (TH2) and is accumulated as a membrane-impermeable magnesium-tetracycline chelate complex (THMg+). This noncarrier diffusion hypothesis was confirmed by the fact that tetracycline accumulated in protein-free liposomes through an artificially imposed pH difference.

Electrochemical studies of antibiotic 23187 (A23187) mediated permeability to divalent heavy-metal ions in phospholipid monolayers adsorbed on mercury electrodes

The effect of the carboxylic ionophore, A23187, on the permeability of adsorbed phospholipid monolayers to heavy-metal ions has been investigated using cyclic voltammetry. The influence of different cationic interactions and complexing agents in solution on the ionophore transport has been studied. A23187 selectively increases the permeability of the monolayer to CdII and CuII ions. The ionophore complexes the metal ion in the monolayer. This complex induces a conformational change in the monolayer at a critical potential, releasing the metal ion for reduction at the electrode surface. The oxidation of the metal and thus its transport from the electrode to the solution is always inhibited. The rate of CdII transfer through the ionophore system is faster than that of CuII, MgII, LaIII and protons interfere with the metal ion transport by binding active sites within the ionophore system.

Nitrogen utilization by ruminants during restricted intake of high-concentrate diets.

A series of experiments was conducted to determine the effects of limit-feeding high-concentrate (LFHC) diets on dietary CP requirements of steer calves. When steer calves were fed 80% concentrate diets at 78 g/kg of BW.75, increasing dietary CP resulted in increased ADG (P less than .001). Average daily gain was increased in steers as daily monensin dosage increased from 120 to 180 mg (P less than .05). Increasing the daily monensin dosage to 240 mg did not increase ADG further. There were no (P greater than .10) CP X monensin interactions, suggesting that the monensin response was caused by improved energy utilization and not be the possible protein-sparing effects of ionophores. Steer calves in the second feedyard experiment expressed similar ADG when provided equal NEg as limit-fed, high-moisture ear corn (HMEC) or when given ad libitum access to corn silage. The basal diet did not affect the steers' daily N requirement for growth. Gain per unit of protein intake declined quadratically (P less than .05) with increasing CP intake, indicating that CP requirements were near NRC estimates on both diets. The corn silage-based diet was less digestible (70.3 vs 77.4%; P less than .01) than the HMEC diet when fed to lambs. Fecal output differed (P less than .10) substantially (342 g/d of corn silage vs 205 g/d of HMEC), whereas fecal N output was only slightly higher (6.97 vs 6.34 g/d, respectively; P less than .10). Limited feeding of higher-concentrate diets to steer calves seemed to be an effective management procedure and did not cause acute digestion upset.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of ions, ion channel blockers, and ionophores on uptake of vitellogenin into cockroach follicles

Since calcium plays an important role in vitellogenin binding and uptake in Nauphoeta cinerea and because calcium channels have been described in follicles of this species, we investigated the effect of various ions, ionophores, and ion channel blockers on vitellogenin uptake in vitro. Calcium significantly stimulated vitellogenin uptake; this effect could be substituted best by barium and less well by strontium and magnesium. The stimulatory effect of calcium, and to a certain extent also that of barium, was dependent on the vitellogenin concentration, whereas the effect of strontium and magnesium was not. In the presence of calcium, vitellogenin uptake was inhibited by barium, strontium, and magnesium as well as by the transition elements nickel, cobalt, and zinc, but not by manganese which had a stimulatory effect. Valinomycin, verapamil, tetraethylammonium, and atropin reduced vitellogenin uptake, while amiloride and ouabain were ineffective. Our results indicate that calcium inward (and possibly potassium outward) fluxes play an important role in vitellogenin uptake.

PAF metabolism in resident and activated alveolar macrophages: Role of protein kinase C

Platelet-activating factor (PAF) metabolism was studied in resident and activated alveolar macrophages. Macrophages were obtained from normal Sprague-Dawley rats and from rats previously injected with complete Freund's adjuvant. Macrophages were attached and stimulated for 90 min. Then, cell PAF was extracted and quantitated by thin-layer chromatography. We found that in both resident and activated macrophages, calcium ionophore A23187 was a potent stimulus for PAF production while phorbol myristate acetate (PMA) was not. PMA and ionophore acted synergistically to increase PAF content in resident macrophages. This synergism was not observed in activated macrophages. To examine if this difference between resident and activated macrophages was due to a difference in PAF degradation, we assayed acetylhydrolase, the PAF-degrading enzyme. We found that ionophore stimulated acetylhydrolase activity in activated macrophages, but not in resident macrophages. Furthermore, PMA potentiated the ionophore effect in activated macrophages. This synergism was less obvious in resident cells. We conclude that PAF metabolism is different in activated and resident alveolar macrophages. Protein kinase C may play an important role in acetylhydrolase regulation in these cells.

CCCP activation of the reconstituted NaK-pump

In the NaK-ATPase proteoliposomes (PLs), the NaK-pump activity, Na+ uptake, and ATP hydrolysis were apparently enhanced by carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and other ionophores without ion gradients. These ionophore effects were not cation specific. Without ionophores, the PL's ATPase activity fell to its steady-state value within 3 sec at 15 degrees C. This decrease in activity disappeared in the presence of CCCP. Since CCCP is believed to enhance proton mobility across the lipid bilayer and dissipate membrane potential (Vm), we postulated that a Vm build-up partially inhibits the PLs by changing the conformation of the NaK-pump, and that CCCP eliminated this partial inhibition. Since this activation required extracellular K+ and high ATP concentration in the PLs, CCCP must affect the conversion between the phosphorylated forms of NaK-ATPase (EP); this step has been suggested by Goldschlegger et al. (1987) to be the voltage-sensitive step (J. Physiol. (London) 387:331-355). Although cytoplasmic K+ accelerated the change of ADP- and K(+)-sensitive EP (E*P) to K(+)-sensitive ADP-insensitive EP (E2P), CCCP did not complete with cytoplasmic K+ when cytoplasmic Na+ was saturated. When the PLs were phosphorylated with 20 microM ATP and 20 microM palmitoyl CoA instead of with high concentration of ATP, CCCP increased the E*P content and decreased the ADP-sensitive K(+)-insensitive EP (E1P). The results described above suggest that CCCP affects the E1P to E*P change in the E1P----E*P----E2P conversion and that this reaction step is inhibited by Vm.

Selective inhibition by lasalocid of hydrolysis of the ADP-insensitive phosphoenzyme in the catalytic cycle of sarcoplasmic reticulum Ca2(+)-ATPase.

The effect of a carboxylic ionophore (lasalocid) on the sarcoplasmic reticulum Ca2(+)-ATPase was investigated. The purified enzyme was preincubated with lasalocid in the presence of Ca2+ and the absence of K+ at pH 7.0 and 0 degrees C for 2 h. The Ca2(+)-dependent ATPase activity was strongly inhibited by this preincubation, whereas the activity of the contaminant Mg2(+)-ATPase was unaffected. The steady-state level of the phosphoenzyme (EP) intermediate remained constant over the wide range of lasalocid concentrations. The Ca2(+)-induced enzyme activation was unaffected. The kinetics of phosphorylation of the Ca2(+)-activated enzyme by ATP as well as the rate of conversion of ADP-sensitive EP to ADP-insensitive EP were also unaffected. Accumulation of ADP-insensitive EP was greatly enhanced, and almost all of the EP accumulating at steady state was ADP-insensitive. Hydrolysis of ADP-insensitive EP was strongly inhibited. A similar strong inhibition of the Ca2(+)-dependent ATPase activity by lasalocid was found with sarcoplasmic reticulum vesicles. To examine the effect of lasalocid on the conformational change in each reaction step, the Ca2(+)-ATPase of sarcoplasmic reticulum vesicles was labeled with a fluorescent probe (N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine) without a loss of catalytic activity and then preincubated with lasalocid as described above. The conformational changes involved in hydrolysis of ADP-insensitive EP and in the reversal of this hydrolysis were appreciably retarded by lasalocid. The conformational changes involved in other reaction steps were unaffected. These results demonstrate that hydrolysis of ADP-insensitive EP in the catalytic cycle of this enzyme is selectively inhibited by lasalocid.

PHYTOCHROME‐ and BLUE PIGMENT‐MEDIATED LEAF MOVEMENTS: EXPERIMENTAL APPROACH IN THE SIGNAL TRANSDUCTION

Abstract— Leaflet movements of Cassia fasciculata are induced by transferring leaves from light to darkness or from darkness to light. Phytochrome mediates the dark‐induced closure whereas a blue and far red light absorbing pigment (cryptochrome?) is the photoreceptor triggering the light‐induced opening. These movements are the result of reversible turgor variation driven by ionic migrations (H+, K+, Cl−) in cortical parenchyma cells of motor organs (“pulvini”) localized at the leaflet base. Calcium plays a predominant role in the regulation of the movements as shown by the inhibitory effects of chelators (EDTA, EGTA), intracellular antagonist TMB‐8 and by the promoting effect of ionophore A 23187. Compounds known as calcium channel blockers (LaCl3, verapamil and nifedipine) inhibited whereas Bay K 8644, a calcium channel activator, promoted the phytochrome‐mediated movement. In contrast, all these calcium channel modulators had no effect on the blue pigment‐mediated movement. From these results, it is suggested that calcium is not mobilized in the same manner in the two types of movements: possibly from external stores in the phytochrome‐mediated response and from internal stores in the blue pigment‐mediated response. Calcium acts possibly through calmodulin as suggested by a modification in the kinetics of the movements induced by inhibitors of calmodulin action (trifluoperazine, R 24571, W‐7). The unexpected promotion of the movements by these inhibitors shows that calmodulin action on the ion migrations is not simple and direct. Experimental observations suggested that regulation might be done through cAMP metabolism. db‐cAMP promoted the movements. Compounds known either to activate adenylate cyclase (prostaglandins, forskolin) or to inhibit phosphodiesterase (imidazolidinones, ICI 58301) induced the same modifications as db‐cAMP. By contrast, a phosphodiesterase activator (imidazole) inhibited the movements.

Ionophores and Nutrient Digestion and Absorption in Ruminants

This paper reviews the effects of feeding ionophores on nutrient digestion and absorption. In cattle, monensin and lasalocid increase apparent digestible energy by an average of 2.0 percentage units. In sheep, responses in digestible energy to ionophore feeding have been more variable, and neither monensin nor lasalocid have, on average, affected digestible energy. The effect of ionophores on fiber digestibility appears to depend on diet composition and source of fiber because both increases and decreases in fiber digestibility have been associated with ionophore feeding. Lasalocid and monensin reduce the percentage of starch digested in the rumen and increase the quantity of starch digested in the intestine. However, total gastrointestinal tract digestion of starch has generally not been affected by ionophores. Apparent nitrogen digestibility has been increased by ionophore feeding in a number of animal species. Apparent absorption of magnesium, phosphorus, zinc and selenium has been increased by ionophore supplementation. Absorption of calcium, potassium and sodium has been inconsistently affected by ionophores. Possible mechanisms whereby ionophores may affect nutrient digestion and absorption are discussed.

Effect of ionophores and pH on growth of Streptococcus bovis in batch and continuous culture

Batch cultures (pH 6.7) of Streptococcus bovis JB1 were severely inhibited by 1.25 and 5 microM lasalocid and monensin, respectively, even though large amounts of glucose remained in the medium. However, continuous cultures tolerated as much as 10 and 20 microM, respectively, and used virtually all of the glucose. Although continuous cultures grew with high concentrations of ionophore, the yield of bacterial protein decreased approximately 10-fold. When pH was decreased from 6.7 to 5.7, the potency of both ionophores increased, but lasalocid always caused a larger decrease in yield. The increased activity of lasalocid at pH 5.7 could largely be explained by an increased binding of the ionophore to the cell membrane. Because monensin did not show an increased binding at low pH, some other factor (e.g., ion turnover) must have been influencing its activity. There was a linear increase in lasalocid binding as the concentration increased, but monensin binding increased markedly at high concentrations. Based on the observations that (i) S. bovis cells bound significant amounts of ionophore (the ratio of ionophore to cell material was more important than the absolute concentration), (ii) batch cultures responded differently from continuous cultures, and (iii) pH can have a marked effect on ionophore activity, it appears that the term "minimum inhibitory concentration" may not provide an accurate assessment of microbial growth inhibition in vivo.

Energy transduction and amino acid transport in thermophilic aerobic and fermentative bacteria

Comparative studies on energy-transducing enzymes from thermophilic and mesophilic bacilli showed that functional analogues of enzymes from the thermophilic species are more thermostable and possess higher maximal turnover rates at the respective growth temperatures than those of the mesophilic species, Differences in membrane fatty-acid composition of thermophilic and mesophilic bacilli have been found which explain the similar apparent membrane-microviscosities of these bacteria at their respective growth temperatures. At elevated temperatures the efficiency of energy transduction is substantially declined in the thermophilic Bacillus stearothermophilus due to an extreme increase in H+-conductivity. In membrane vesicles of B. Stearothermophilus the acidic amino acids l-gultamate and l-aspartate are transported by a Na+-H+-solute symport mechanisms in a 1:1:1 stoichiometry. This is concluded from the observations that the membrane potential, pH-gradient and Δμ Na+ are equivalent as driving force for uptake. Uptake of neutral (brached chain) amino acids is also facilitated by Na+-solute mechanisms. This is confirmed by the stimulatory effect of monensin on the steady-state level of accumulation of these amino acids and the absence of uptake in the presence of nonactin in membrane vesicles. Furthermore, the initial rate of l-alanine or l-leucine uptake is specifically enchanced by the addition of sodium ions and to some extent by lithium ions. The affinities of both transport systems for sodium ions was rather low (mM-range). In contrast to this the Na+-H+-spl|-glutamate transport system in B. Stearothermophilus possesses an extremely high affinity fro sodium ionds (Kt<5 μM). l-Glutamate and l-alanine trasnport activities in B. stearothermophilus are allosterically influenced by the pH. Detailes mechanistic studies of amino acid transport have also been performed in a hybridmembrane system composed out of membrane vesicles of Clostridium fervidus and proteoliposomes containing the thermostable and thermoactive primary proton pump cytochrome c oxidase from B. stearothermophilus. The effects of ionophores, uptake driven by artificial gradients and the observed sodium-ion dependence of transport of neutral, acidic and aromatic amino acids indicates that these amino acids are transported in symport with one Na+- or Li+ in Cl. fervidus.

Energy transduction and amino acid transport in thermophilic aerobic and fermentative bacteria

Comparative studies on energy-transducing enzymes from thermophilic and mesophilic bacilli showed that functional analogues of enzymes from the thermophilic species are more thermostable and possess higher maximal turnover rates at the respective growth temperatures than those of the mesophilic species, Differences in membrane fatty-acid composition of thermophilic and mesophilic bacilli have been found which explain the similar apparent membrane-microviscosities of these bacteria at their respective growth temperatures. At elevated temperatures the efficiency of energy transduction is substantially declined in the thermophilic Bacillus stearothermophilus due to an extreme increase in H +-conductivity. In membrane vesicles of B. Stearothermophilus the acidic amino acids l-gultamate and l-aspartate are transported by a Na +-H +-solute symport mechanisms in a 1:1:1 stoichiometry. This is concluded from the observations that the membrane potential, pH-gradient and Δ μ Na + are equivalent as driving force for uptake. Uptake of neutral (brached chain) amino acids is also facilitated by Na +-solute mechanisms. This is confirmed by the stimulatory effect of monensin on the steady-state level of accumulation of these amino acids and the absence of uptake in the presence of nonactin in membrane vesicles. Furthermore, the initial rate of l-alanine or l-leucine uptake is specifically enchanced by the addition of sodium ions and to some extent by lithium ions. The affinities of both transport systems for sodium ions was rather low (mM-range). In contrast to this the Na +-H +-spl|-glutamate transport system in B. Stearothermophilus possesses an extremely high affinity fro sodium ionds ( K t<5 μM). l-Glutamate and l-alanine trasnport activities in B. stearothermophilus are allosterically influenced by the pH. Detailes mechanistic studies of amino acid transport have also been performed in a hybridmembrane system composed out of membrane vesicles of Clostridium fervidus and proteoliposomes containing the thermostable and thermoactive primary proton pump cytochrome c oxidase from B. stearothermophilus. The effects of ionophores, uptake driven by artificial gradients and the observed sodium-ion dependence of transport of neutral, acidic and aromatic amino acids indicates that these amino acids are transported in symport with one Na +- or Li + in Cl. fervidus.

Actin assembly in electropermeabilized neutrophils: role of intracellular calcium.

Assembly of microfilaments involves the conversion of actin from the monomeric (G) to the filamentous (F) form. The exact sequence of events responsible for this conversion is yet to be defined and, in particular, the role of calcium remains unclear. Intact and electropermeabilized human neutrophils were used to assess more directly the role of cytosolic calcium [( Ca2+]i) in actin assembly. Staining with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin and right angle light scattering were used to monitor the formation of F-actin. Though addition of Ca2+ ionophores can be known to induce actin assembly, the following observations suggest that an increased [Ca2+]i is not directly responsible for receptor-induced actin polymerization: (a) intact cells in Ca2(+)-free medium, depleted of internal Ca2+ by addition of ionophore, responded to the formyl peptide fMLP with actin assembly despite the absence of changes in [Ca2+]i, assessed with Indo-1; (b) fMLP induced a significant increase in F-actin content in permeabilized cells equilibrated with medium containing 0.1 microM free Ca2+, buffered with up to 10 mM EGTA; (c) increasing [Ca2+]i beyond the resting level by direct addition of CaCl2 to permeabilized cells resulted in actin disassembly. Conversely, lowering [Ca2+]i resulted in spontaneous actin assembly. To reconcile these findings with the actin-polymerizing effects of Ca2+ ionophores, we investigated whether A23187 and ionomycin induced actin assembly by a mechanism independent of, or secondary to the increase in [Ca2+]i. We found that the ionophore-induced actin assembly was completely inhibited by the leukotriene B4 (LTB4) antagonist LY-223982, implying that the ionophore effect was secondary to LTB4 formation, possibly by stimulation of phospholipase A2. We conclude that actin assembly is not mediated by an increase in [Ca2+]i, but rather that elevated [Ca2+]i facilitates actin disassembly, an effect possibly mediated by Ca2(+)-sensitive actin filament-severing proteins such as gelsolin. Sequential actin assembly and disassembly may be necessary for functions such as chemotaxis.

Regulation of the imbalance in light excitation between Photosystem II and Photosystem I by cations and by the energized state of the thylakoid membrane

The imbalance in photoactivity between the two photosystems in broken chloroplasts during steady-state electron transport was investigated using modulated chlorophyll a fluorimetry and oxygen evolution. No imbalance in favor of PS II (imbalance term equals zero) was found at low cation concentration (e.g., 10 mM NaCl) where the membranes are unstacked, while some imbalance in favor of PS II (imbalance term about 0.1–0.2) could be observed at ‘high’ cation medium (e.g., 100 mM for univalent, 5 mM for divalent and 100 μM for trivalent cations) where the membranes are stacked. At the high cation concentration the imbalance was particularly noticeable at a pH range 6–7.5 under conditions where the membranes were non-energized, e.g., in the presence of a range of uncouplers and ionophores (an imbalance term of between 0.44 and 1.1). In the absence of uncouplers or ionophores the imbalance term was initially high, decreasing to a low steady-state value during the light-induced energization of the membranes. The increase in the imbalance measured with the addition of gramicidin D was wavelength-dependent, implying changes in the allocation of excitation energy to the photosystems rather than any other mechanism. This effect was reversed at higher pH: At a pH higher than about 8, the imbalance in absence of uncouplers or ionophores was stronger than in their presence. The relation between the state of imbalance and the cross-membrane proton gradient (ΔpH) was not straightforward or simple, as follows: (i) Imbalance was induced by uncouplers and ionophores also at sufficiently low light intensities which produce only very small ΔpH. (ii) Valinomycin (+KCl) had the same effect as uncouplers like gramicidin D, nigericin, NH4Cl and others, although it presumably abolishes membrane potential but not ΔpH. (iii) The effect of gramicidin D and NH4Cl was close to saturation at concentrations which affect ΔpH still minutely. (iv) The same level of large imbalance could be achieved even without uncouplers or ionophores when a much higher cation concentration (approx. 10-fold) than that considered normal to achieve membrane stacking was used. It is therefore concluded that: (a) High cationic levels change the allocation of excitation energy, probably via the screening of negative charges on the thylakoid surface. (b) The effect of uncouplers and ionophores in the presence of high cationic levels most probably reflects, at least in part, the effect on external surface charges, their exposure by membrane energization and their screening by cations.

Differences between mechanisms of action of heat-stable and heat-labile enterotoxins of Escherichia coli

The mucosal-to-serosal and serosal-to-mucosal fluxes of Na + and Cl − were measured in control mice and mice treated with heat-stable (ST) and heat-labile (LT) enterotoxins in the presence or absence of: Ca 2+-ionophore A23187, an activator of Ca 2+-calmodulin; or phorbol-12-myristate-13-acetate (PMA), an activator of protein kinase C9PKC); or 1-(5-isoquinolinyl sulphonyl)-2-methyl piperazine (H-7), an inhibitor of PKC. There was net secretion of Na + and CL − in both experimental groups in contrast to net absorption in the control group. The addition of ionophore or PMA or ionophore + PMA resulted in net secretion of Na + and Cl − in the control group and the effect of ionophore and PMA was found to be additive. The addition of ionophore did not cause any change in electrolyte fluxes in the ST toxin treated group, however, it increased the net secretion of Na + and Cl − in the LT toxin treated group. PMA increased the net secretion of Na + and Cl − in the St toxin treated group, however, it did not cause any change in Na + and Cl − fluxes in the LT toxin treated group. H-7 did not reverse the effect of ST toxin, however, it reversed the effect of LT toxin. These studies suggest that ST toxin acts through the Ca 2+-calmodulin pathway and secretion of Na + and Cl − in ST toxin treated mice is due to an increase in the intestinal Ca 2+-calmodulin activity, whereas, LT toxin acts through the protein kinase C pathway and secretion of Na + and Cl − in LT toxin treated mice is due to an increase in the intestinal PKC activity.

Effect of fats and ionophores on in vitro fermentation of a high concentrate diet (1990)

Effect of fats and ionophores on in vitro fermentation of a high concentrate diet (1990)