The FAD-Enzyme Monodehydroascorbate Radical Reductase Mediates Photoproduction of Superoxide Radicals in Spinach Thylakoid Membranes

The photoreduction of dioxygen in spinach thylakoid membraneswas enhanced about 10-fold by the FAD-enzyme monodehydroascorbateradical (MDA) reductase at 1µM. The primary photoreducedproduct of dioxygen catalyzed by MDA reductase was the superoxideradical, as evidenced by the inhibition of photoreduction ofCytc by superoxide dismutase. The apparent K_m for dioxygen ofthe MDA reductase-dependent photoreduction of dioxygen was 100µM,higher by one order of magnitude than that observed with thylakoidmembranes only. Glutathione reductase, ferredoxin-NADP⁺ reductase,and glycolate oxi-dase also mediated the photoproduction ofsuperoxide radicals in thylakoid membranes at rates similarto those with MDA reductase. Among these flavoenzymes, MDA reductaseis the most likely mediator stimulating the photoreduction ofdioxygen in chloroplasts; its function in the protection fromphotoinhibition under excess light is discussed. (Received February 24, 1998; Accepted May 19, 1998)     

Induction of the H+ Release from Thylakoid Membranes by Illumination in the Presence of Protonophores at High Concentrations

The generally observed light-induced uptake of protons intothe thylakoid lumen is diminished by adding protonophores. Insteadof the H⁺ uptake, the release of protons was observed duringillumination in the presence of various protonophores at highconcentrations, namely, 1 µM nigericin, 10 µM carbonylcyanidem-chlorophenylhydrazone or 30 µM gramicidin. An uncoupler,NH₄C1 (4 mM), and a detergent, Triton X-100 (0.02%), also inducedthe H⁺ release but a K⁺ ionophore, valinomycin, did not. Theamount of H⁺ released reached about 100 nmol H⁺ (mg Chl)^–1at pH 7.5 under continuous illumination. The rate of the H⁺release was similar to that of the conventional H⁺ uptake butits dark relaxation was much slower than that of the H⁺ uptake.We compared the H⁺ release in protonophore-added thylakoidswith the previously reported H⁺ release in coupling factor 1(CF₁-depleted thylakoids. The H⁺ release in thylakoids withnigericin showed similar characteristics to that in CF₁-depletedthylakoids in terms of their responses to pH, phenazine methosulfateand light intensity. Both types of H⁺ release were relativelyinsensitive to DCMU and were stimulated somewhat by DCMU atlow concentrations (around 200 nM). Nigericin did not inhibitthe superoxide dismutase activity of the membranes. These resultsindicate that the H⁺ release in protonophore-added thylakoidsand that in CF₁ depleted thylakoids involve the same mechanismand that water-derived protons from PS II that result from animpairment of the activity of superoxide dismutase, as previouslyproposed, are not involved. Judging from the rate of electronflow and the lumenal acidification under the illumination, weconclude that the H⁺ release is a light-dependent scalar processwhich can be observed in thylakoid membranes with high H⁺ permeability.The H⁺ release of this type was not observed in mitochondriafrom rat liver or in chromatophores from Rhodobacter sphaeroides. (Received November 29, 1990; Accepted June 27, 1991)     

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     

Purification and Characterization of Assimilatory Nitrate Reductase from the Cyanobacterium Plectonema boryanum

Assimilatory nitrate reductase (NR) was solubilized by acetonetreatment from Plectonema boryanum and was purified 7,700-foldby heat treatment, ammonium sulfate fractionation and chromatographyon DEAE-Sephacel and Sephadex G-150. Purified NR had a specificactivity of 85 µmol NO₂^– formed min^–1 mg^–1protein. The enzyme retained both ferredoxin (Fd)- and methylviologen (MV)-linked NR activities throughout the purificationprocedure. Molecular weight was 80,000. The pH optimum was 10.5in the MV-assay and 8.5 when assayed with enzymatically reducedFd as the electron donor. Apparent Km values for nitrate andMV were 700 µM and 2,500µM in the MVassay and 55µM and 75 µM for nitrate and Fd in the Fd-assay.The enzyme was inhibited by thiol reagents and metal-chelatingreagents. (Received October 1, 1982; Accepted March 8, 1983)     

Relationship between biomass and enzymatic activity of a bloom-forming dinoflagellate (Dinophyceae) in southern Chile (41{degrees} S): a field approach

In a coastal area of southern Chile (41° S), the major ammoniumassimilating enzyme glutamine synthetase (GS) was detected ina green dinoflagellate bloom during April 2003. High chlorophylla concentrations (1000 µg L^–1) attributable to Gymnodiniumcf. chlorophorum in surface waters were associated with highand very low nitrate reductase activities. Coincident with thebloom, dissolved inorganic nitrogen concentrations were nearthe detection limit (NO₃^– + NH₄⁺ <0.5 µM). SinceGS correlates with the use of ammonium as an external nitrogensource, we suggest that GS activity seems to be a good indicatorof ammonium utilization in a period dominated by a single dinoflagellatespecies.     

DCEBIO stimulates Cl- secretion in the mouse jejunum

We investigated the effects of 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one(DCEBIO) on the Cl^– secretory response of the mouse jejunum using the Ussing short-circuit current (I_sc) technique. DCEBIO stimulated a concentration-dependent, sustained increase in I_sc (EC₅₀ 41 ± 1 µM). Pretreating tissues with 0.25 µM forskolin reduced the concentration-dependent increase in I_sc by DCEBIO and increased the EC₅₀ (53 ± 5 µM). Bumetanide blocked (82 ± 5%) the DCEBIO-stimulated I_sc consistent with Cl^– secretion. DCEBIO was a more potent stimulator of Cl^– secretion than its parent molecule, 1-ethyl-2-benzimidazolinone. Glibenclamide or NPPB reduced the DCEBIO-stimulated I_sc by >80% indicating the participation of CFTR in the DCEBIO-stimulated I_sc response. Clotrimazole reduced DCEBIO-stimulated I_sc by 67 ± 15%, suggesting the participation of the intermediate conductance Ca²⁺-activated K⁺ channel (IK_Ca) in the DCEBIO-activated I_sc response. In the presence of maximum forskolin (10 µM), the DCEBIO response was reduced and biphasic, reaching a peak response of the change in I_sc of 43 ± 5 µA/cm² and then falling to a steady-state response of 17 ± 10 µA/cm² compared with DCEBIO control tissues (61 ± 6 µA/cm²). The forskolin-stimulated I_sc in the presence of DCEBIO was reduced compared with forskolin control tissues. Similar results were observed with DCEBIO and 8-BrcAMP where adenylate cyclase was bypassed. H89, a PKA inhibitor, reduced the DCEBIO-activated I_sc, providing evidence that DCEBIO increased Cl^– secretion via a cAMP/PKA-dependent manner. These data suggest that DCEBIO stimulates Cl^– secretion of the mouse jejunum and that DCEBIO targets components of the Cl^– secretory mechanism. 1-ethyl-2-benzimidazolinone; forskolin; glibenclamide; clotrimazole; H89     

The Requirement of Selenium for the Growth of Marine Coccolithophorids, Emiliania huxleyi, Gephyrocapsa oceanica and Helladosphaera sp. (Prymnesiophyceae)

Marine coccolithophorids, Emiliania huxleyi, Gephyrocapsa oceanicaand Helladosphaera sp. were found to require selenium for theirgrowth. The optimum concen trations were 1–10 nM for SeO₂and SeO₃^2– and above 1 µM for SeO₂^4– Duringthe depletion of selenium algal growth was strongly suppressedaccompanied with the decrease in net photosynthesis. (Received November 18, 1998; Accepted April 16, 1999)     

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.     

Characterization of vectorial chloride transport pathways in the human pancreatic duct adenocarcinoma cell line HPAF

Pancreatic duct cells express a Ca²⁺-activated Cl^- conductance (CaCC), upregulation of which may be beneficial to patients with cystic fibrosis. Here, we report that HPAF, a human pancreatic ductal adenocarcinoma cell line that expresses CaCC, develops into a high-resistance, anion-secreting epithelium. Mucosal ATP (50 µM) caused a fourfold increase in short-circuit current (I_sc), a hyperpolarization of transepithelial potential difference (from -4.9 ± 0.73 to -8.5 ± 0.84 mV), and a fall in resistance to less than one-half of resting values. The effects of ATP were inhibited by mucosal niflumic acid (100 µM), implicating an apical CaCC in the response. RT-PCR indicated expression of hClC-2, hClC-3, and hClC-5, but surprisingly not hCLCA-1 or hCLCA-2. K⁺ channel activity was necessary to maintain the ATP-stimulated I_sc. Using a pharmacological approach, we found evidence for two types of K⁺ channels in the mucosal and serosal membranes of HPAF cells, one activated by chlorzoxazone (500 µM) and sensitive to clotrimazole (30 µM), as well as one blocked by clofilium (100 µM) but not chromanol 293B (5 µM). RT-PCR indicated expression of the Ca²⁺-activated K⁺ channel KCNN4, as well as the acid-sensitive, four transmembrane domain, two pore K⁺ channel, KCNK5 (hTASK-2). Western blot analysis verified the expression of CLC channels, as well as KCNK5. We conclude that HPAF will be a useful model system for studying channels pertinent to anion secretion in human pancreatic duct cells. Ussing chamber; short-circuit current; RT-PCR; immunoblot     

Attachment of CuZn-Superoxide Dismutase to Thylakoid Membranes at the Site of Superoxide Generation (PSI) in Spinach Chloroplasts: Detection by Immuno-Gold Labeling After Rapid Freezing and Substitution Method

In chloroplasts O^–₂ is photoproduced via the univalentreduction of O₂ in PSI even under conditions that are favorablefor photosynthesis. The photogenerated O^–₂ is disproportionatedto H₂O₂ and O₂ in a reaction that is catalyzed by superoxidedismutase (SOD). The H₂O₂-scavenging ascorbate peroxidase isbound to the thylakoid membranes at or near the PSI reactioncenter [Miyake and Asada (1992) Plant Cell Physiol. 33: 541],and the primary product of oxidation in the peroxidase-catalyzedreaction, the monodehydroascorbate radical, is photoreducedto ascorbate in PSI in a reaction mediated by ferredoxin [Miyakeand Asada (1994) Plant Cell Physiol. 35: 539]. Therefore, SODshould be localized at or near the PSI complex. We report herethe microcompartmentalization of the chloroplastic CuZn-SODon the stromal-faces of thylakoid membranes where the PSI-complexis located. Spinach leaves were fixed and substituted by a rapidfreezing and substitution method that allows visualization ofintact chloroplasts. The embedded sections were immuno labeledwith the antibody against CuZn-SOD by the immunogold method.About 70% of the immunogold particles were found within 5 nmfrom the surface of the stromal-faces of thylakoid membranes.Of these particles, about 40% were found at the ends and marginsof the grana thylakoids and 60% were found on the stromal sideof the stromal thylakoids. From these results, the local concentrationof CuZn-SOD on the stroma-facing surfaces of the thylakoid membraneswas estimated to be about 1 mM. The effect of the microcompartmentalizationof CuZn-SOD on the scavenging of superoxide radicals is discussed. (Received November 25, 1994; Accepted February 23, 1995)     

Beta-adrenergic stimulation does not activate Na+/Ca2+ exchange current in guinea pig, mouse, and rat ventricular myocytes

The effect of -adrenergic stimulation on cardiac Na⁺/Ca²⁺ exchange has been controversial. To clarify the effect, we measured Na⁺/Ca²⁺ exchange current (I_NCX) in voltage-clamped guinea pig, mouse, and rat ventricular cells. When I_NCX was defined as a 5 mM Ni²⁺-sensitive current in guinea pig ventricular myocytes, 1 µM isoproterenol apparently augmented I_NCX by 32%. However, this increase was probably due to contamination of the cAMP-dependent Cl^– current (CFTR-Cl^– current, I_CFTR-Cl), because Ni²⁺ inhibited the activation of I_CFTR-Cl by 1 µM isoproterenol with a half-maximum concentration of 0.5 mM under conditions where I_NCX was suppressed. Five or ten millimolar Ni²⁺ did not inhibit I_CFTR-Cl activated by 10 µM forskolin, an activator of adenylate cyclase, suggesting that Ni²⁺ acted upstream of adenylate cyclase in the -adrenergic signaling pathway. Furthermore, in a low-extracellular Cl^– bath solution, 1 µM isoproterenol did not significantly alter the amplitude of Ni²⁺-sensitive I_NCX at +50 mV, which is close to the reversal potential of I_CFTR-Cl. No change in I_NCX amplitude was induced by 10 µM forskolin. When I_NCX was activated by extracellular Ca²⁺, it was not significantly affected by 1 µM isoproterenol in guinea pig, mouse, or rat ventricular cells. We concluded that -adrenergic stimulation does not have significant effects on I_NCX in guinea pig, mouse, or rat ventricular myocytes. cystic fibrosis transmembrane conductance regulator; nickel ion     

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.     

Distinct K+ conductive pathways are required for Cl- and K+ secretion across distal colonic epithelium

Secretion of Cl^– and K⁺ in the colonic epithelium operates through a cellular mechanism requiring K⁺ channels in the basolateral and apical membranes. Transepithelial current [short-circuit current (I_sc)] and conductance (G_t) were measured for isolated distal colonic mucosa during secretory activation by epinephrine (Epi) or PGE₂ and synergistically by PGE₂ and carbachol (PGE₂ + CCh). TRAM-34 at 0.5 µM, an inhibitor of K_Ca3.1 (IK, Kcnn4) K⁺ channels (H. Wulff, M. J. Miller, W. Hänsel, S. Grissmer, M. D. Cahalan, and K. G. Chandy. Proc Natl Acad Sci USA 97: 8151–8156, 2000), did not alter secretory I_sc or G_t in guinea pig or rat colon. The presence of K_Ca3.1 in the mucosa was confirmed by immunoblot and immunofluorescence detection. At 100 µM, TRAM-34 inhibited I_sc and G_t activated by Epi (4%), PGE₂ (30%) and PGE₂ + CCh (60%). The IC₅₀ of 4.0 µM implicated involvement of K⁺ channels other than K_Ca3.1. The secretory responses augmented by the K⁺ channel opener 1-EBIO were inhibited only at a high concentration of TRAM-34, suggesting further that K_Ca3.1 was not involved. Sensitivity of the synergistic response (PGE₂ + CCh) to a high concentration TRAM-34 supported a requirement for multiple K⁺ conductive pathways in secretion. Clofilium (100 µM), a quaternary ammonium, inhibited Cl^– secretory I_sc and G_t activated by PGE₂ (20%) but not K⁺ secretion activated by Epi. Thus Cl^– secretion activated by physiological secretagogues occurred without apparent activity of K_Ca3.1 channels but was dependent on other types of K⁺ channels sensitive to high concentrations of TRAM-34 and/or clofilium. epinephrine; prostaglandin E₂; cholinergic; Kcnn4; TRAM-34; clofilium     

Ultrastructural Responses of the LichenBryoria fuscescensto Simulated Acid Rain and Heavy Metal Deposition

Effects of simulated acid rain and heavy metal deposition onthe ultrastructure of the lichenBryoria fuscescens(Gyeln.) Brodoand Hawksw. were examined in a field study conducted in northernFinland. Lichens were exposed to simulated rain containing twolevels of a mixture of copper (Cu²⁺) and nickel (Ni²⁺) ionsalone or in combination with acid rain (H₂SO₄) at pH 3 over2 months in addition to ambient rainfall. The algal and fungalcomponents responded differently to pH and there was an interactionwith metal toxicity. The algal partner was the most sensitiveto acid rain and heavy metal combinations and had more degeneratecells than the fungal partner. Damage was apparent in chloroplastsand mitochondria, where thylakoid and mitochondrial cristaewere swollen. The fungal partner was the more sensitive to highconcentrations of metal ions in the absence of acidity, suggestingan ameliorating interaction between the metals and acidity.For algae, critical metal concentrations in the thallus were>50 µg g^-1for Cu and >7 µg g^-1for Ni in thepresence of acidity, and >20 µ g g^-1for Ni in the absenceof acidity. Detrimental effects of heavy metals on fungal ultrastructurewere seen when thallus metal concentrations exceeded 400 µgg^-1for Cu and 100 µg g^-1d. wt for Ni. The results suggestthat acid wet deposition containing metal ions may reduce survivalof lichens in northern environments.Copyright 1998 Annals ofBotany Company. Copper, nickel, sulphuric acid, ultrastructure,Bryoria fuscescens(Gyeln.) Brodo and Hawksw., epiphytic lichen, air pollution.     

Intermediate-conductance Ca2+-activated K+ channel is expressed in C2C12 myoblasts and is downregulated during myogenesis

We report here the expression in C₂C₁₂ myoblasts of the intermediate-conductance Ca²⁺-activated K⁺ (IK_Ca) channel. The IK_Ca current, recorded under perforated-patch configuration, had a transient time course when activated by ionomycin (0.5 µM; peak current density 26.2 ± 3.7 pA/pF; n = 10), but ionomycin (0.5 µM) + 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (100 µM) evoked a stable outward current (28.4 ± 8.2 pA/pF; n = 11). The current was fully inhibited by charybdotoxin (200 nM), clotrimazole (2 µM), and 5-nitro-2-(3-phenylpropylamino)benzoic acid (300 µM), but not by tetraethylammonium (1 mM) or D-tubocurarine (300 µM). Congruent with the IK_Ca channel, elevation of intracellular Ca²⁺ in inside-out patches resulted in the activation of a voltage-insensitive K⁺ channel with weak inward rectification, a unitary conductance of 38 ± 6 pS (at negative voltages), and an IC₅₀ for Ca²⁺ of 530 nM. The IK_Ca channel was activated metabotropically by external application of ATP (100 µM), an intracellular Ca²⁺ mobilizer. Under current-clamp conditions, ATP application resulted in a membrane hyperpolarization of 35 mV. The IK_Ca current downregulated during myogenesis, ceasing to be detectable 4 days after the myoblasts were placed in differentiating medium. Downregulation was prevented by the myogenic suppressor agent basic FGF (bFGF). We also found that block of the IK_Ca channel by charybdotoxin did not inhibit bFGF-sustained myoblast proliferation. These observations show that in C₂C₁₂ myoblasts the IK_Ca channel expression correlates inversely with differentiation, yet it does not appear to have a role in myoblast proliferation. ATP; cell proliferation     

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.

     

Thylakoid Membrane-Bound, NADPH-Specific Pyridine Nucleotide Dehydrogenase Complex Mediates Cyclic Electron Transport in the Cyanobacterium Synechocystis sp. PCC 6803

The donation of electrons from NADPH to the intersystem chain,as monitored by an increase in Chl fluorescence, occurred inthe isolated thylakoid membranes of Synechocystis PCC 6803.The stimulation by NADPH of the methyl viologen-dependent photoreductionof dioxygen and of the reduction of P700⁺ after photooxidationin the presence of DCMU also confirmed the donation of electronsfrom NADPH to the electron carriers in the intersystem. Thesereactions were sensitive to rotenone, capsaicin, l-(2-thenoyl)-3,3,3-trifluoroacetoneand HgCl₂ but not to antimycin A or flavone. In contrast tothe thylakoid membranes from the wild type, those from a mutant,designated M55, in which a gene of a subunit of the pyridinenucleotide dehydrogenase complex (NDH) had been inactivated,did not show evidence of such reactions. These results supportour previous hypothesis that the transport of electrons fromNADPH to the intersystem chain is mediated by NDH [Mi et al.(1994) Plant Cell Physiol. 35: 163] and indicate the bindingof an NADPH-specific NDH to the thylakoid membranes. The Chlfluorescence was quenched transiently by addition of ferredoxinand NADP₊ to the thylakoid membranes but showed a subsequentincrease. This result suggests the reduction of plastoquinoneby the photoreduced NADP₊ and initiation of the NADPH-mediatedcyclic flow of electrons around PSI. Furthermore, a similarresponse of Chl fluorescence was observed upon the additionof ferredoxin only, demonstrating the ferredoxin-dependent cyclicflow of electrons. Both pathways of cyclic electron transportwere inhibited by rotenone, and were not detected in the NDH-defectedthylakoid membranes from M55, indicating the participation ofthe NDH complex. These results confirm that, in Synechocystis,the thylakoid-bound NDH complex mediates the ferredoxin-dependentcyclic electron flow, as well as the NADPH-dependent cyclicelectron flow. (Received November 24, 1994; Accepted March 16, 1995)     

Relation between Ultra-Violet Spectral Change and Nucleotide Binding of the Chloroplast Coupling Factor 1

Properties of the nucleotide binding sites on chloroplast couplingfactor 1 (CF₁) were studied by equilibrium dialysis and UV spectroscopy.From our direct binding studies, we identified at least fourkinds of ADP binding sites on CF₁; a barely dissociable ADPbinding site (site A), a slowly exchangeable high affinity sitewith dissociation constant (Kd) 0.021 µM (site B), anotherslowly exchangeable high affinity site with Kd 1.6 µM(site C) and several low affinity (Kd {small tilde}30 µM)sites. The Kd values for sites B and C of the other nucleotidestested were 0.5 µM and 16 µM (GDP), 8 µM and34 µM (CDP), 17 µM and 20 µM (UDP) and 1.4µM and 1.4 µM (PP₁). From a comparison of the observed UV spectral change and theamount of nucleotide bound to these sites, as calculated fromthe above Kd values, we concluded that the nucleotide bindingto site B or G induces UV spectral changes that are almost thesame in shape and magnitude. The estimated difference molarabsorption coefficient () was 3.4?10³M^–1_ADP cm^–1for ADP at 278 nm. Our conclusions were strengthened by thegood agreement between the observed spectra and the calculatedspectra (derived from the Kd and values of ADP and GDP) whenADP and GDP were added together to CF₁. The cause of the unusual behavior of GDP in the UV differencespectrum which was unexplained in our previous report was shownto be competition between the GDP added and previously boundADP at sites B and C; this distorted the real spectrum inducedby GDP. (Received October 3, 1983; Accepted February 13, 1984)     

Apoplastic Solute Concentrations of Organic Acids and Mineral Nutrients in the Leaves of Several Fagaceae

Ion chromatographic methods determined organic acids and mainnutrient minerals in the apoplastic solution from leaves ofseveral Fagaceae (Quercus ilex L., Quercus cerris L., Quercusvirgiliana (Ten.) Ten, and Fagus sylvatica L.). The anions oforganic acids found in high amounts (250 to 650 µM) werequinate, malate, and oxalate. Lactate, pyruvate, formate andacetate were detected in relatively low amounts with concentrationsbetween 20 and 200 µM. The total concentration of organicacids in the apoplastic sap ranged between 1.5 and 2 mM. Thetotal concentration of inorganic cations (K⁺, Mg²⁺, NH₄⁺, Ca²⁺,Na⁺) and anions (C1^–, NO₃^–, SO^2–₄ and PO^3–₄)in the apoplastic sap varied between 5 and 10 mM, and 0.35 and1.8 mM, respectively. We conclude that the concentration oforganic acid ions in the leaf apoplast depends mainly on theexchange with the leaf cells and is influenced by the electrochemicalgradient between the symplast and the apoplast in relation tothe water potential of the leaf. The determination of formateand acetate in the apoplastic compartment of leaves lend weightto the argument that the production of these acids by treesis a important emission source to the atmosphere. (Received June 9, 1998; Accepted April 8, 1999)     

p38 mitogen-activated protein kinase inhibits calcium-dependent chloride secretion in T84 colonic epithelial cells

We havepreviously shown that Ca²⁺-dependent Clsecretion across intestinal epithelial cells is limited by a signalingpathway involving transactivation of the epidermal growth factorreceptor (EGFR) and activation of ERK mitogen-activated protein kinase (MAPK). Here, we have investigated a possible role for p38 MAPK inregulation of Ca²⁺-dependent Cl secretion.Western blot analysis of T₈₄ colonic epithelial cells revealed that the muscarinic agonist carbachol (CCh; 100 µM)stimulated phosphorylation and activation of p38 MAPK. The p38inhibitor SB-203580 (10 µM) potentiated and prolonged short-circuitcurrent (I_sc) responses to CCh acrossvoltage-clamped T₈₄ cells to 157.4 ± 6.9% of thosein control cells (n = 21; P < 0.001).CCh-induced p38 phosphorylation was attenuated by the EGFR inhibitortyrphostin AG-1478 (0.1 nM-10 µM) and by the Src family kinaseinhibitor PP2 (20 nM-2 µM). The effects of CCh on p38phosphorylation were mimicked by thapsigargin (TG; 2 µM), whichspecifically elevates intracellular Ca²⁺, and wereabolished by the Ca²⁺ chelator BAPTA-AM (20 µM), implyinga role for intracellular Ca²⁺ in mediating p38 activation.SB-203580 (10 µM) potentiated I_sc responses toTG to 172.4 ± 18.1% of those in control cells (n = 18; P < 0.001). When cells were pretreated withSB-203580 and PD-98059 to simultaneously inhibit p38 and ERK MAPKs,respectively, I_sc responses to TG and CCh weresignificantly greater than those observed with either inhibitor alone.We conclude that Ca²⁺-dependent agonists stimulate p38 MAPKin T₈₄ cells by a mechanism involving intracellularCa²⁺, Src family kinases, and the EGFR. CCh-stimulated p38activation constitutes a similar, but distinct and complementary,antisecretory signaling pathway to that of ERK MAPK.