Cu2+-induced modification of the kinetics of A beta(1-42) channels

We found that the amyloid peptide A(1-42) is capable of interacting with membrane and forming heterogeneous ion channels in the absence of any added Cu²⁺ or biological redox agents that have been reported to mediate A(1-42) toxicity. The A(1-42)-formed cation channel was inhibited by Cu²⁺ in cis solution ([Cu²⁺]_cis) in a voltage- and concentration-dependent manner between 0 and 250 µM. The [Cu²⁺]_cis-induced channel inhibition is fully reversible at low concentrations between 50 and 100 µM [Cu²⁺]_cis and partially reversible at 250 µM [Cu²⁺]_cis. The inhibitory effects of [Cu²⁺]_cis between 50 and 250 µM on the channel could not be reversed with addition of Cu²⁺-chelating agent clioquinol (CQ) at concentrations between 64 and 384 µM applied to the cis chamber. The effects of 200-250 µM [Cu²⁺]_cis on the burst and intraburst kinetic parameters were not fully reversible with either wash or 128 µM [CQ]_cis. The kinetic analysis of the data indicate that Cu²⁺-induced inhibition was mediated via both desensitization and an open channel block mechanism and that Cu²⁺ binds to the histidine residues located at the mouth of the channel. It is proposed that the Cu²⁺-binding site of the A(1-42)-formed channels is modulated with Cu²⁺ in a similar way to those of channels formed with the prion protein fragment PrP(106-126), suggesting a possible common mechanism for Cu²⁺ modulation of A and PrP channel proteins linked to neurodegenerative diseases. neurodegenerative diseases; transitional metals; ion channel pathologies; membrane injuries; calcium homeostasis     

Purification, Properties and Phosphorylation of Anaerobically Induced Enolase in Echinochloa phyllopogon and E. crus-pavonis

Enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11 [EC] ) activityis differentially induced by anoxia in the flood-tolerant speciesE. phyllopogon (Stev.) Koss and the flood-intolerant speciesE. crus-pavonis (H.B.K.) Schult. To examine the regulation ofenolase at the protein level, we purified the enzyme from bothspecies to near homogeneity and compared their physico-chemicaland catalytic properties. Enolase purified from E. phyllopogonexhibits optimal activity at pH 7.0, a K_m of 80 µM for2-PGA, a Q₁₀ of 1.97 and an E_a of 12.3 kcal mol^-1. Similarly,enolase from E. crus-pavonis exhibits optimal activity at pH7.0, a K_m of 50 µM for 2-PGA, a Q₁₀ of 2.04 and an E_aof 12.9 kcal mol^-1. The enzyme from both species is thermostable(100% active after 15 min, 50°C) and is a homodimer of 52.5kDa subunits as resolved by SDS-PAGE and immunoblotting. E.phyllopogon enolase was phosphorylated in vitro using either[     

Neuronal swelling and surface area regulation: elevated intracellular calcium is not a requirement

Neurons aremechanically robust. During prolonged swelling, molluscan neurons cantriple their apparent membrane area. They gain surface area andcapacitance independent of extracellular Ca concentration([Ca]_e), but it isunknown if an increase in intracellular Ca concentration([Ca]_i) isnecessary. If Ca for stimulating exocytosis is unnecessary, it ispossible that swelling-induced membrane tension changes directlytrigger surface area readjustments. If, however, Ca-mediated but nottension-mediated membrane recruitment is responsible for surface areaincreases, swelling neurons should sustain elevated levels of[Ca]_i. The purpose ofthis investigation is to determine if the[Ca]_i in swellingneurons attains levels high enough to promote exocytosis and if anysuch increase is required. Lymnaeaneurons were loaded with the Ca concentration indicator fura 2. Calibration was performed in situ using 4-bromo-A-23187 and Ca-ethyleneglycol-bis(-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), with free Ca concentration ranging from 0 to 5 µM. Swelling perturbations (medium osmolarity reduced to 25% for 5 min)were done at either a standard[Ca]_e or very low[Ca]_e level (0.9 mM or0.13 µM, respectively). In neither case did the[Ca]_i increase tolevels that drive exocytosis. We also monitored osmomechanically drivenmembrane dynamics [swelling, then formation and reversal ofvacuole-like dilations (VLDs)] with the[Ca]_i clamped below 40 nM via1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). [Ca]_idid not change with swelling, and VLD behavior was unaffected,consistent with tension-driven,[Ca]_i-independent surface area adjustments. In addition, neurons with[Ca]_i clamped at 0.1 µM via an ionophore could produce VLDs. We conclude that, undermechanical stress, neuronal membranes are compliant by virtue ofsurface area regulatory adjustments that operate independent of[Ca]_i. The findingssupport the hypothesis that plasma membrane area is regulated in partby membrane tension.

     

SH oxidation coordinates subunits of rat brain ryanodine receptor channels activated by calcium and ATP

We have reported that ryanodine receptor (RyR) channels display three different responses to cytoplasmic free Ca²⁺ concentration ([Ca²⁺]) depending on their redox state (Marengo JJ, Hidalgo C, and Bull R. Biophys J 74: 1263–1277, 1998), with low, moderate, and high maximal fractional open times (P_o). Activation by ATP of single RyR channels from rat brain cortex was tested in planar lipid bilayers with 10 or 0.1 µM cytoplasmic [Ca²⁺]. At 10 µM [Ca²⁺], low-P_o channels presented lower apparent affinity to activation by ATP [[ATP] for half-maximal activation (K_aATP) = 422 µM] than moderate-P_o channels (K_aATP = 82 µM). Oxidation of low-P_o channels with thimerosal or 2,2'-dithiodipyridine (DTDP) gave rise to moderate-P_o channels and decreased K_aATP from 422 to 82 µM. At 0.1 µM cytoplasmic [Ca²⁺], ATP induced an almost negligible activation of low-P_o channels. After oxidation to high-P_o behavior, activation by ATP was markedly increased. Noise analysis of single-channel fluctuations of low-P_o channels at 10 µM [Ca²⁺] plus ATP revealed the presence of subconductance states, suggesting a conduction mechanism that involves four independent subchannels. On oxidation the subchannels opened and closed in a concerted mode. subconductance states; calcium ion release channels; calcium ion regulation; thimerosal; 2,2'-dithiodipyridine     

Characterization of regulatory mechanisms and states of human organic cation transporter 2

Polyspecific organic cation transporters (OCTs) have a large substrate binding pocket with different interaction domains. To determine whether OCT regulation is substrate specific, suitable fluorescent organic cations were selected by comparing their uptake in wild-type (WT) human embryonic kidney (HEK)-293 cells and in HEK-293 cells stably transfected with hOCT2. N-amidino-3,5-diamino-6-chloropyrazine-carboxamide (amiloride) and 4-[4-(dimethylamino)-styryl]-N-methylpyridinium (ASP) showed concentration-dependent uptake in hOCT2 at 37°C. After subtraction of unspecific uptake determined in WT at 37°C or in hOCT2 at 8°C saturable specific uptake of both substrates was measured. K_m values of hOCT2-mediated uptake of 95 µM amiloride and 24 µM ASP were calculated. Inhibition of amiloride and ASP uptake by several organic cations was also measured [IC₅₀ (in µM) for amiloride and ASP, respectively, tetraethylammonium (TEA) 98 and 30, cimetidine 14 and 26, and tetrapentylammonium (TPA) 7 and 2]. Amiloride and ASP uptake were significantly reduced by inhibition of Ca²⁺/CaM complex (–55 ± 5%, n = 10 and –63 ± 2%, n = 15, for amiloride and ASP, respectively) and stimulation of PKC (–54 ± 5%, n = 14, and –31 ± 6%, n = 26) and PKA (–16 ± 5%, n = 16, and –18 ± 4%, n = 40), and they were increased by inhibition of phosphatidylinositol 3-kinase (+28 ± 6%, n = 8, and +55 ± 17%, n = 16). Inhibition of Ca²⁺/CaM complex resulted in a significant decrease of V_max (160–99 photons/s) that can be explained in part by a reduction of the membrane-associated hOCT2 (–22 ± 6%, n = 9) as determined using FACScan flow cytometry. The data indicate that saturable transport by hOCT2 can be measured by the fluorescent substrates amiloride and ASP and that transport activity for both substrates is regulated similarly. Inhibition of the Ca²⁺/CaM complex causes changes in transport capacity via hOCT2 trafficking. organic cation transport; fluorescence measurement; 4-[4-(dimethylamino)-styryl]-n-methylpyridinium; amiloride     

Rates of Photosynthesis in Characean Cells: I. PHOTOSYNTHETIC 14CO2 FIXATION BY NITELLA TRANSLUCENS

A study has been made of photosynthetic ¹⁴CO₂ fixation by isolated‘mature’ internodes of Nitella translucens. Experimentalconditions were similar to those used in studies of the ionicrelations of these cells. Maximum rates of photosynthesis were33–40µµmoles CO₂, fixed per cm² of surfacearea per second (equivalent to 12–15 /xmoles fixed permg chlorophyll per hour). ^l4CO₂ fixation was inhibited to thedark level by 3(3,4,dichlorophenyl)-1, 1-dimethylurea (at 0-6µM or 10µM) and by the uncoupler carbonyl cyanide-m-chlorophenylhydrazone(SµM). The presence of imidazole or ammonium sulphate(both of which uncouple ATP production in vitro) did not resultin an inhibition of 14CO2 fixation. These results are discussedin relation to published work on solute uptake by Nitella translucens.During photosynthesis there was rapid movement of ¹⁴C-labelledorganic compounds out of the chloroplasts. ¹⁴C-labelled sucrose,ammo-acids, and sugar phosphates were found in samples of vacuolarsap.     

Acclimation of Lolium temulentum to enhanced carbon dioxide concentration

Acclimation of single plants of Lolium temulentum to changing[CO₂] was studied on plants grown in controlled environmentsat 20°C with an 8 h photoperiod. In the first experimentplants were grown at 135 µ;mol m^–2 s^–1 photosyntheticphoton flux density (PPFD) at 415µl l^–1 or 550µll^–1 [CO₂] with some plants transferred from the lowerto the higher [CO₂] at emergence of leaf 4. In the second experimentplants were grown at 135 and 500 µmol m^–2 s^–1PPFD at 345 and 575 µl l^–1 [CO₂]. High [CO₂] during growth had little effect on stomatal density,total soluble proteins, chlorophyll a content, amount of Rubiscoor cytochrome f. However, increasing [CO₂] during measurementincreased photosynthetic rates, particularly in high light.Plants grown in the higher [CO₂] had greater leaf extension,leaf and plant growth rates in low but not in high light. Theresults are discussed in relation to the limitation of growthby sink capacity and the modifications in the plant which allowthe storage of extra assimilates at high [CO₂]. Key words: Lolium, carbon dioxide, photosynthesis, growth, stomatal density     

Functional interaction of the K-Cl cotransporter (KCC1) with the Na-K-Cl cotransporter in HEK-293 cells

We have studiedthe regulation of the K-Cl cotransporter KCC1 and its functionalinteraction with the Na-K-Cl cotransporter. K-Cl cotransporter activitywas substantially activated in HEK-293 cells overexpressing KCC1(KCC1-HEK) by hypotonic cell swelling, 50 mM external K, andpretreatment with N-ethylmaleimide(NEM). Bumetanide inhibited ⁸⁶Rbefflux in KCC1-HEK cells after cell swelling [inhibition constant (K_i) ~190µM] and pretreatment with NEM(K_i ~60 µM).Thus regulation of KCC1 is consistent with properties of the red cellK-Cl cotransporter. To investigate functional interactions between K-Cland Na-K-Cl cotransporters, we studied the relationship between Na-K-Clcotransporter activation and intracellular Cl concentration([Cl]_i). Without stimulation, KCC1-HEK cells had greater Na-K-Cl cotransporter activitythan controls. Endogenous Na-K-Cl cotransporter of KCC1-HEK cells wasactivated <2-fold by low-Cl hypotonic prestimulation, compared with10-fold activation in HEK-293 cells and >20-fold activation in cellsoverexpressing the Na-K-Cl cotransporter (NKCC1-HEK). KCC1-HEK cellshad lower resting[Cl]_i than HEK-293cells; cell volume was not different among cell lines. We found a steeprelationship between[Cl]_i and Na-K-Clcotransport activity within the physiological range, supporting aprimary role for [Cl]_iin activation of Na-K-Cl cotransport and in apical-basolateral crosstalk in ion-transporting epithelia.     

Effects of some SH-inhibitors and EDTA on flowering in Lemna perpusilla 6746

Lemna perpusilla 6746, a short-day duckweed, flowered undercontinuous illumination on M-sucrose medium containing CuSO₄,AgNO₃ and HgCl₂, which are SH-inhibitors. The optimum concentrationsof CuSO₄, AgNO₃ and HgCl₂ were 5, 1 and 20 µM, respectively.Other metal ions tested were ineffective, but at least two otherSHinhibitors, potassium ferricyanide and iodoacetamide, alsoinduced long-day flowering at the concentrations of 0.1-1 µM. Adding 50 µM EDTA to the medium prevented the effect ofcupric ion, but not that of other SH-inhibitors. EDTA at 200µM induced some long-day flowering when added to a mediumwith no SH-inhibitors. It also permitted some flowering whenadded together with cupric ion, and accelerated flowering inthe presence of the other SHinhibitors listed above. EDTA andSH-inhibitor effects appeared to be additive. (Received May 25, 1973; )     

Kinetic Characterization of Two Phosphate Uptake Systems with Different Affinities in Suspension-Cultured Catharanthus roseus Protoplasts

We studied the kinetics of inorganic phosphate (P₁) uptake from0.1–1,000 µM P₁ by protoplasts from suspension-culturedcells of Catharanthus roseus (L.) G. Don. Concentration dependenceof [³²P]P₁ uptake revealed two kinetically different uptakesystems, a high-affinity system and a low-affinity system, withK_m values of 3.0 and 47 µM, respectively. Protoplastsfrom cells grown in P_i-rich media had a medium level of thelow-affinity activity and a very low level of the high-affinityactivity. It appeared low-affinity system is expressed constitutively,while the high-affinity system is regulated by the availabilityof P_i. When cells grown in a P_i-rich media were transferredto P_i-depleted media, the high-affinity activity increased significantlyafter 2 d, but the low-affinity activity was barely changed.Upon addition of 10 mM P_i, the high level of the high-affinityactivity fell to almost undetectable level in 1d. Both uptakesystems exhibited maximum activity between pH 5 and 6. ¹ Present address: Tokyo Research Laboratories, Kyowa HakkoKogyo Co., Ltd., 3-6-6 Asahi-cho, Machida, Tokyo, 194 Japan.     

Nitric oxide regulates oxygen uptake and hydrogen peroxide release by the isolated beating rat heart

Isolated rat heart perfused with 1.5-7.5µM NO solutions or bradykinin, which activates endothelial NOsynthase, showed a dose-dependent decrease in myocardial O₂uptake from 3.2 ± 0.3 to 1.6 ± 0.1 (7.5 µM NO, n = 18,P < 0.05) and to 1.2 ± 0.1 µM O₂ · min¹ · gtissue¹ (10 µM bradykinin, n = 10,P < 0.05). Perfused NO concentrations correlated with aninduced release of hydrogen peroxide (H₂O₂) inthe effluent (r = 0.99, P < 0.01). NO markedlydecreased the O₂ uptake of isolated rat heart mitochondria(50% inhibition at 0.4 µM NO, r = 0.99,P < 0.001). Cytochrome spectra in NO-treated submitochondrial particles showed a double inhibition of electron transfer at cytochrome oxidase and between cytochrome b andcytochrome c, which accounts for the effects in O₂uptake and H₂O₂ release. Most NO was bound tomyoglobin; this fact is consistent with NO steady-state concentrationsof 0.1-0.3 µM, which affect mitochondria. In the intact heart,finely adjusted NO concentrations regulate mitochondrial O₂uptake and superoxide anion production (reflected byH₂O₂), which in turn contributes to thephysiological clearance of NO through peroxynitrite formation.

     

Differentiating Day from Night Effects of High Ambient [CO2] on the Gas Exchange and Growth of Xanthium strumarium L. Exposed to Salinity Stress

Sodium chloride, at a concentration of 88 mol m^-3in half strengthHoagland nutrient solution, increased dry weight per unit areaofXanthium strumarium L. leaves by 19%, and chlorophyll by 45%compared to plants grown without added NaCl at ambient (350µmol mol^-1) CO₂concentration. Photosynthesis, per unitleaf area, was almost unaffected. Even so, over a 4-week period,growth (dry weight increment) was reduced in the salt treatmentby 50%. This could be ascribed to a large reduction in leafarea (>60%) and to an approx. 20% increase in the rate ofdark respiration (Rd). Raising ambient [CO₂] from zero to 2000 µmol mol^-1decreasedRd in both control and salinized plants (by 20% at 1000, andby 50% at 2000 µmol mol^-1CO₂concentration) compared toRd in the absence of ambient CO₂. High night-time [CO₂] hadno significant effect on growth of non-salinized plants, irrespectiveof day-time ambient [CO₂]. Growth reduction caused by salt wasreduced from 51% in plants grown in 350 µmol mol^-1throughoutthe day, to 31% in those grown continuously in 900 µmolmol^-1[CO₂]. The effect of [CO₂] at night on salinized plants depended onthe daytime CO₂concentration. Under 350 µmol mol^-1day-time[CO₂], 900 µmol mol^-1at night reduced growth over a 4-weekperiod by 9% (P <0.05) and 1700 µmol mol^-1reduced itby 14% (P <0.01). However, under 900 µmol mol^-1day-time[CO₂], 900vs . 350 µmol mol^-1[CO₂] at night increasedgrowth by 17% (P <0.01). It is concluded that there is both a functional and an otiose(functionless) component to Rd, which is increased by salt.Under conditions of low photosynthesis (such as here, in thelow day-time [CO₂] regime) the otiose component is small andhigh night-time [CO₂] partly suppresses functional Rd, therebyreducing salt tolerance. In plants growing under conditionswhich stimulate photosynthesis (e.g. with increased daytime[CO₂]), elevated [CO₂] at night suppresses mainly the otiosecomponent of respiration, thus increasing growth. Consequently,in regions of adequate water and sunlight, the predicted furtherelevation of the world atmospheric [CO₂] may increase plantsalinity tolerance. Xanthium strumarium ; respiration; photosynthesis; salt stress; sodium chloride; carbon dioxide; atmosphere     

Measurements of Photosynthetic Sulfide Oxidation by Chlorobium Using a Sulfide Ion Selective Electrode

A simple technique is described for using a sulfide sensitiveelectrode to measure the photooxidation of H₂S by a green sulfurbacterium, Chlorobium limicola forma thiosulfatophilum. Sulfidephotooxidation occurred only in the presence of bicarbonateat concentrations greater than 0.1 mM. This implies that therate-limiting carboxylating enzyme for CO₂ fixation in Chlorobiumhas a relatively low affinity for CO₂ compared to ribulose-1,5-biphosphatecarboxylase. Carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone(FCCP), an uncoupler of photophosphorylation, delays sulfideoxidation for about 15 sec after the onset of illumination at2 µM and is completely inhibitory at 10 µM. Theseeffects can be explained by the ATP requirement for CO₂ fixation.When the photooxidation of H₂S was prevented by 10 µMFCCP, a photoevolution of H₂S was observed. (Received December 24, 1981; Accepted September 10, 1982)     

O-Benzylhydroxylamine: An Inhibitor of Phenylpropanoid Metabolism in Plants

O-Benzylhydroxylamine (OBHA) is a potent inhibitor of phenylalanineammonialyase (PAL, EC 4.3.1.5 [EC] ) and phenylpropanoid metabolismas evidenced by its effects on three plant species [soybean(Glycine max (L.) Merr.), buckwheat (Fagopyrum esculentum Moench.),and mung bean (Vigna radiata L.)]. When supplied to roots, OBHA(10^–5 M) did not significantly inhibit light- or dark-growthof soybean seedlings, but reduced (25%) soluble hydroxyphenoliccompound accumulation in light-grown axes. Higher concentrations(510^–5 M) of OBHA caused reductions (25%) in axis freshweight of light-grown seedlings (72 h), but did not lower axisweight of dark-grown seedlings. Anthocyanin accumulation inhypocotyls of intact mung bean seedlings was reduced by 25%after 3 days light growth after treatment with OBHA (10^–5M) via root feeding. Anthocyanin content of excised, etiolatedbuckwheat hypocotyls floated on solutions of OBHA (10^–5M) and incubated in the light for 24 h was reduced by 40%. L-Phenylalanineand t-cinnamic acid, intermediates of phenylpropanoid metabolism,were able to partially reverse this inhibition in buckwheat.Extractable PAL activity (specific activity basis) in soybeanaxes was substantially reduced (20% in dark, 40% in light) asearly as 24 h after root feeding with OBHA (10^–5 M). Reductionof PAL activity (specific activity or per axis basis) by OBHAcompared to control levels, continued throughout a time courseof 96 h. Kinetic studies on soybean PAL revealed a K_m of 1.1mM for L-phenylalanine and an apparent K_i of 3.5 µM forOBHA. (Received May 31, 1985; Accepted August 6, 1985)     

Nitric oxide relaxes human myometrium by a cGMP-independent mechanism

The role of intracellular guanosine 3',5'-cyclicmonophosphate concentration([cGMP]_i) in nitricoxide (NO)-mediated relaxations in the uterus has become controversial.We found the NO donor S-nitroso-L-cysteine(CysNO) to potently (IC₅₀ = 30 nM)inhibit spontaneous contractions in the nonpregnant human myometrium. CysNO treatment increased[cGMP]_i significantly(P < 0.001), and this increase wasblocked by the guanylyl cyclase inhibitors methylene blue (10 µM) orLY-83583 (1 µM); however, pretreatment with these guanylyl cyclaseinhibitors failed to block CysNO-mediated relaxations. IntracellularcAMP concentrations were not altered by treatment of tissues with 10 µM CysNO. Incubation with the cGMP analogs 8-bromo-cGMP or-phenyl-1,N²-etheno-cGMPdid not significantly affect spontaneous contractility. Pretreatment oftissues with charybdotoxin [a calcium-dependent potassium channel(BK) blocker] completely reversed CysNO-induced relaxations. Weconclude that NO is a potent inhibitor of spontaneous contractileactivity in the nonpregnant human uterus and that, although guanylylcyclase and BK activities are increased by NO, increases in[cGMP]_i are notrequired for NO-induced relaxations in this tissue.

     

Photosynthetic Carbon Sources of Stream Macrophytes

Rates of photosynthesis of four submerged stream macrophyteswere examined under varying pH and composition of inorganiccarbon species. Callitriche stagnatis and Sparganium simplexused only CO₂ for photosynthesis. Potamogeton crispus and P.pectinatus used HCO₃ in addition to CO₂, but with much lowerefficiency. The photosynthetic rates at air equilibrium anda total inorganic carbon concentration of 5.0 mM were 2–3times lower than maximum rates at CO2 saturation for the HCO₃users and 10–14 times lower for the CO₂ users. The CO₂compensation point of entire plants of Callitriche (2.5 µM)and Sparganium (6.0µM) was well below the equilibriumconcentration (15 µM). and the low saturation points (250–500µM) also pointed to efficient use of CO2. Callitricheand Sparganium compete successfully with HCO₃^– users inhardwater streams, which have a higher exchange and generationcapacity of CO2 than stagnant and more soft waters. Rates ofphotosynthesis of Potamogeton crispus and P. pectinatus decreasedat high pH. Depending on the two alternative hypotheses forHCO₃use, this decline can be explained by CO₃^––inhibition of HCO₃^– uptake or by increasing capacity tobuffer H+efflux from the plant. Habitats subject to high pH,e. g. small ponds with dense vegetation, may have a strong selectionfor efficient mechanisms of HCO₃ use. Key words: Photosynthesis, Macrophytes, Carbon-source     

Ethylene Biosynthesis and Xylogenesis in Lactuca Pith Explants Cultured In Vitro in the Presence of Auxin and Cytokinin: The Effect of Ethylene Precursors and Inhibitors

The possible involvement of ethylene in the induction of xylemdifferentiation was studied in lettuce (Lactuca saliva L. cv.Romaine) pith parenchyma explants. The addition of the ethyleneprecursors L-methionine (0.25 µM), S-adenosylmethionine(25 µM) and 1-aminocyclopropane-l-carboxylic acid (0.01µM), or the ethylene-releasing agent 2-chloroethylphosphonicacid (1.0 µM), to a standard IAA-kinetin-containing mediumenhanced xylogenesis compared to control explants cultured inthe absence of these compounds. In the presence of the ethyleneinhibitors aminoethoxyvinylglycine, Co(NO₃)₂ and AgNO₃, xylogenesiswas inhibited. Inhibition of xylogenesis by aminoethoxyvinylglycine(75 µM), Co(NO₃)₂ (50 µM) and AgNO₃ (6.0 µM)was reversed by exogenous 1-aminocyclopropane-l-carboxylic acid(0.01 µM), 2-chloroethylphosphonic acid (5.0 µM)and L-methionine (0.25 µM), respectively. Ethylene productionby explants cultured on media containing L-methionine or 1-aminocyclopropane-l-carboxylicacid was greater than the biosynthesis of ethylene by explantscultured in the absence of these compounds. The incorporationof 2-chloroethylphosphonic acid into the culture medium resultedin higher rates of ethylene production compared to explantscultured on the IAA-kinetin medium. The presence of either aminoethoxyvinylglycineor Co(NO₃)₂ inhibited ethylene production by explants culturedon the IAA-kinetin medium. The data support the hypothesis thatethylene plays a positive role in the initiation of xylem differentiation. Key words: Xylogenesis, Differentiation, Ethylene, IAA, Kinetin, Lactuca sativa     

Total and Polar Lipid Biosynthesis during Crotalaria juncea Linn. Pollen Tube Growth: Effect of Gibberellic Acid, Indoleacetic Acid and (2-Chloroethyl)-phosphonic Acid

Gibberellic acid (GA₃) at 58 µM, indoleacetic acid (IAA)at 29 µM, and (2-chloroethyl) phosphonic acid (Ethephon)at 70 µM promoted pollen tube growth in Crotalaria junceapollen suspension cultures both in water and basal medium. GA₃stimulated [ l-¹⁴C]acetate incorporation into total lipids inboth media, whereas IAA enhanced incorporation in water culturesonly. On the contrary, Ethephon reduced the label in total lipidswhen supplemented in basal medium. Based on [l-¹⁴C lacetateincorporation into different phospho- and glycolipids, it isproposed that these growth regulators have a definite role inthe biosynthesis of lipid components of the membranes.     

Nitric oxide induces [Ca2+]i oscillations in pituitary GH3 cells: involvement of IDR and ERG K+ currents

The role of nitric oxide (NO) in the occurrence of intracellular Ca²⁺ concentration ([Ca²⁺]_i) oscillations in pituitary GH₃ cells was evaluated by studying the effect of increasing or decreasing endogenous NO synthesis with L-arginine and nitro-L-arginine methyl ester (L-NAME), respectively. When NO synthesis was blocked with L-NAME (1 mM) [Ca²⁺]_i, oscillations disappeared in 68% of spontaneously active cells, whereas 41% of the quiescent cells showed [Ca²⁺]_i oscillations in response to the NO synthase (NOS) substrate L-arginine (10 mM). This effect was reproduced by the NO donors NOC-18 and S-nitroso-N-acetylpenicillamine (SNAP). NOC-18 was ineffective in the presence of the L-type voltage-dependent Ca²⁺ channels (VDCC) blocker nimodipine (1 µM) or in Ca²⁺-free medium. Conversely, its effect was preserved when Ca²⁺ release from intracellular Ca²⁺ stores was inhibited either with the ryanodine-receptor blocker ryanodine (500 µM) or with the inositol 1,4,5-trisphosphate receptor blocker xestospongin C (3 µM). These results suggest that NO induces the appearance of [Ca²⁺]_i oscillations by determining Ca²⁺ influx. Patch-clamp experiments excluded that NO acted directly on VDCC but suggested that NO determined membrane depolarization because of the inhibition of voltage-gated K⁺ channels. NOC-18 and SNAP caused a decrease in the amplitude of slow-inactivating (I_DR) and ether-à-go-go-related gene (ERG) hyperpolarization-evoked, deactivating K⁺ currents. Similar results were obtained when GH₃ cells were treated with L-arginine. The present study suggests that in GH₃ cells, endogenous NO plays a permissive role for the occurrence of spontaneous [Ca²⁺]_i oscillations through an inhibitory effect on I_DR and on I_ERG. voltage-gated potassium channels; ether-à-go-go-related gene potassium channels; slow-inactivating outward currents; fast-inactivating outward currents     

The Isolation and Characterization of a Cytochrome b6f Complex from the Cyanobacterium Spirulina sp.

A cytochrome b₆f complex was isolated and purified from Spirulinasp. The complex was solubilized with n-heptyl ß-D-thioglucosideand chromatographed on a DEAE-Toyopearl 650M column. The purifiedcomplex contained a small amount of chlorophyll and carotenoid.At least four polypeptides were present in the complex: cytochromef (29 kDa), cytochrome b₆(23 kDa), iron-sulfur protein (ISP,23 kDa), and a 17 kDa polypeptide. Each polypeptide was separatedfrom the complex treated with 2-mercaptoethanol or urea. Theabsorption spectra of cytochrome b₆ and cytochrome f were similarto those of Anabaena and spinach as expected. The complex wasactive in supporting ubiquinol-cytochrome c oxidoreductase activity.Fifty percent inhibition of the activity was accomplished by1 µM dibromothymoquinone (DBMIB). The K_m values for ubiquinol-2and cytochrome c (horse heart) were 5.7 µM and 7.4 µM,respectively. (Received August 15, 1988; Accepted November 14, 1988)