Biosynthesis of aryl carotenoids: Inhibitor studies of chlorobactene biosynthesis in Chlorobium limicola f. thiosulfatophilum

The biosynthesis of the aryl carotenoid, chlorobactene, was examined in the green sulfur bacterium, Chlorobium limicola f. thiosulfatophilum. Nicotine, which was used to inhibit carotenoid cyclization, caused the accumulation of the acyclic carotenoid, lycopene. Cells reincubated in fresh medium, after removal of nicotine, synthesized chlorobactene more readily from newly synthesized lycopene rather than from the pool of lycopene accumulated during nicotine inhibition. When the cells were reincubated in the presence of diphenylamine, which inhibited de novo carotenogenesis, a portion of the lycopene which had accumulated during nicotine inhibition was converted into chlorobactene. There was no evidence that neurosporene, rather than lycopene, was the precyclization intermediate. The involvement of -carotene as the cyclic precursor of chlorobactene also was shown. The pathway for chlorobactene biosynthesis is discussed in terms of a possible arrangement of the enzymes involved in carotenoid biosynthesis.Abbreviations DPA Diphenylamine - TLC Thin layer chromatography     

Superoxide dismutase restores endothelium-dependent arteriolar dilatation during acute infusion of nicotine

We previouslyshowed [Am. J. Physiol. 272 (Heart Circ. Physiol. 41):H2337-H2342, 1997] that nicotine impairsendothelium-dependent arteriolar dilatation. However, mechanisms thataccounted for the effect of nicotine on endothelium-dependentvasodilatation were not examined. Thus the goal of this study was toexamine the role of oxygen radicals in nicotine-induced impairment of arteriolar reactivity. We measured diameter of cheek pouch resistance arterioles (~50 µm diameter) in response to endothelium-dependent (ACh and ADP) and -independent (nitroglycerin) agonists before andafter infusion of vehicle or nicotine in the absence or presence ofsuperoxide dismutase. ACh, ADP, and nitroglycerin produced dose-relateddilatation of cheek pouch arterioles before infusion of vehicle ornicotine. Infusion of vehicle, in the absence or presence of superoxidedismutase (150 U/ml), did not alter endothelium-dependent or-independent arteriolar dilatation. In contrast, infusion of nicotine(2 µg · kg¹ · min¹)impaired endothelium-dependent, but not -independent, arteriolar dilatation. In addition, the effect of nicotine onendothelium-dependent vasodilatation was reversed by topicalapplication of superoxide dismutase. We suggest that nicotine impairsendothelium-dependent arteriolar dilatation via an increase in thesynthesis/release of oxygen-derived free radicals.

     

Differing temporal roles of Ca2+ and cAMP in nicotine-elicited elevation of tyrosine hydroxylase mRNA

The involvement of cAMP- andCa²⁺-mediated pathways in theactivation of tyrosine hydroxylase (TH) gene expression by nicotine wasexamined in PC-12 cells. ExtracellularCa²⁺ and elevations inintracellular Ca²⁺ concentration([Ca²⁺]_i)were required for nicotine to increase TH mRNA. The nicotine-elicited rapid rise in[Ca²⁺]_iwas inhibited by blockers of either L-type or N-type, and to a lesserextent P/Q-, but not T-type, voltage-gatedCa²⁺ channels. With continualnicotine treatment,[Ca²⁺]_ireturned to basal levels within 3-4 min. After a lag of~5-10 min, there was a smaller elevation in[Ca²⁺]_ithat persisted for 6 h and displayed different responsiveness toCa²⁺ channel blockers. This secondphase of elevated[Ca²⁺]_iwas blocked by an inhibitor of store-operatedCa²⁺ channels, consistent with theobserved generation of inositol trisphosphate.1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM), when added before or 2 h after nicotine,prevented elevation of TH mRNA. Nicotine treatment significantly raised cAMP levels. Addition of the adenylyl cyclase inhibitor2',5'-dideoxyadenosine (DDA) prevented thenicotine-elicited phosphorylation of cAMP response element bindingprotein. DDA also blocked the elevation of TH mRNA only when addedafter the initial transient rise in [Ca²⁺]_iand not after 1 h. This study reveals that several temporal phases areinvolved in the induction of TH gene expression by nicotine, each ofthem with differing requirements forCa²⁺ and cAMP.

     

Down-Regulation of the Plasmalemma H+ Pump Activity by Nicotine-Induced Intracellular Alkalinization: a Balance between Base Accumulation, Biochemical pH-Stat Response and Intracellular pH Increase

Nicotine was used to induce an intracellular alkalinizationin Elodea densa leaves in order to study the regulation of theplasmalemma H⁺ pump activity by alkaline intracellular pH values.Nicotine was found to enter the cells rapidly in the unchargedform and to induce a significant intracellular pH increase,measured either directly as cell sap pH or as vacuolar and cytoplasmicpH by calculation from the distribution at equilibrium of labelledpH probes. The nicotine-induced alkalinization was associatedwith a progressive decrease in K⁺ uptake. A strong inhibitionof net H⁺ efflux was also evident in the presence of K⁺ in theexternal medium, whereas no nicotine effect on net H⁺ effluxwas detected in the absence of K⁺ (in spite of the larger accumulationof nicotine in the tissue) in agreement with a down-regulationof the activity of the K⁺-dependent plasmalemma H⁺-ATPase byalkaline intracellular pH values. The increase in vacuolar pHresulting from nicotine accumulation was small compared to thebase load calculated from the vacuolar buffer capacity and theintracellular dissociation of nicotine. Conversely, the nicotine-inducedincrease in cytoplasmic pH was considerably larger than expectedon the basis of the cytoplasmic buffer capacity and of the theoreticalaccumulation of nicotine in the experimental conditions adopted.A balance sheet between nicotine accumulation, intracellularalkalinization and malate system response was drawn up, andthe seeming discrepancies observed were discussed. (Received August 11, 1997; Accepted November 21, 1997)     

Periplasmic location of dissimilatory nitrate and nitrite reductases in a denitrifying phototrophic bacterium, Rhodopseudomonas sphaeroides forma sp. denitrificans

The localization of dissimilatory nitrate and nitrite reductasesof a denitrifying phototrophic bacterium, Rhodopseudomonas sphaeroidesforma sp. denitrificans, was investigated. Nitrate and nitritereductases were located in the periplasmic space of the bacteriumgrown anaerobically in the presence of nitrate either in lightor in darkness. Chromatophores showed nitrate and nitrite reductaseactivities when dithionite-reduced benzyl viologen was an electrondonor; this suggests that the enzymes were trapped inside thevesicles. ¹Present address: Japanese Red Cross Central Blood Center, Hiroo4-1-31, Shibuyaku, Tokyo 150, Japan. ²Present address: Plant Growth Laboratory, University of California,Davis, California 95616, U.S.A. (Received November 7, 1979; )     

Malformin negates the Inhibition of Ethrel-induced Leaf Abscission by AgNO3

In light, malformin completely abolished the ability of Ag⁺to inhibit Ethrel-induced leaf abscission from cuttings of Vignaradiata, even though Ag⁺ was applied 24 hr before malformin.Malformin itself did not induce abscission in the light. However,Ag⁺ was active on cuttings which had been pre-treated with malforminfor 2 days in the light. No evidence was obtained to suggestreaction between malformin and Ag⁺. In the dark, Ag⁺ had noeffect on stimulation of leaf abscission by malformin. (Received March 7, 1981; Accepted May 12, 1981)     

Amino Acid-Sugar Linkages in Rice Coleoptile Cell Walls

The nature of amino acid-sugar linkages in cell walls was investigatedin a monocotyledonous tissue, rice coleoptiles. The molar ratiosof aspartic acid, threonine, and serine in cell walls were decreasedby hydrazinolysis in coleoptiles grown both on and under water.The molar ratios of threonine and serine were decreased alsoby a NaOHNaBH₄ treatment, while the alanine content was increased,and -aminobutyric acid was not formed. The cell walls were treated with NaOH in the presence of NaB³H₄,hydrolyzed, then divided into amino acid and sugar fractions.Two distinct radioactive peaks were detected in the thin-layerchromatography of the amino acid fractions. One was identifiedas alanine derived from glycosylated serine; the other was confirmedto be an oxidation product of glucosaminitol. There was justone ³H-labeled product in the sugar fractions, galactitol. Theseresults suggest the presence of serine-O-galactose and asparagine-N-N-acetylglucosamine linkages in rice coleoptile cell walls. The existence of glucosamine linked to amino acids was furthersupported by the incorporation of ¹⁴C-glucosamine into cellwalls. These linkages were also detected in the cell walls ofa dicotyledonous tissue, Vicia epicotyls. (Received April 2, 1981; Accepted June 24, 1981)     

Phytoplankton population dynamics of a small reservoir: effect of intermittent mixing on phytoplankton succession and the growth of blue-green algae

The seasonal succession of the phytoplankton of a small reservoir(Guelph Lake, Ontario) in the spring-summer of 1982 was comparedto that in 1981. Mixing of the deeper waters occurred severaltimes throughout the summer in 1982 but not in 1981. The waterat 10 m became anoxic for only 2 weeks in late July in 1982.In contrast, in 1981, the water at 10 m became anoxic at thebeginning of July and remained so until mid-September. The phytoplanktondynamics observed in 1982 did not follow the typical progressionfrom spring diatoms to summer species adapted to survive understratified conditions, as in 1981. Diatoms were present throughoutthe summer in higher amounts in 1982 than in 1981. The mostobvious difference in the two summers was the much greater abundanceof Aphanizomenon flow-aquae in 1982. Other blue-green algaeincluding Microcystis aeruginosa, Gomphosphasria lacustris,and Lyngbya birgei appeared earlier on in 1982, but did notimmediately increase in abundance as in 1981. In 1982, ratesof phytoplankton community change were low in May and June andincreases were observed in mid-July, early August, late Augustand late September. In contrast, in 1981, the rate of communitychange increased in late May, mid-June, early July and lateJuly and remained low throughout August and September. ¹Present address: Department of Zoology, University of Alberta,Edmonton, Alberta T6G 2E1, Canada. ²Present address: CSIRO Division of Fisheries Research, G.P.O.Box 1538, Hobart, Tasmania 7001, Australia     

Relation between Sex Expression Types and Cotyledon Etiolation of Cucumber in vitro I. On the Role of Ethylene Evolved from Seedlings

Cucumber seedlings, when cultured in vitro, showed differencesin cotyledon etiolation rates among cultivars with differentgenetic backgrounds for sex expression. The chlorophyll contentin gynoecious cultivars (acr^F/acr^F) decreased rapidly whilethat in monoecious ones (acr⁺/acr⁺) decreased more slowly, andthat in mono-gynoecious ones (acr¹/acr¹) decreased at an intermediaterate. Etiolation was suppressed even in early-etiolating cultivarswhen the flask remained unsealed or endogenously evolved ethylenewas removed. Cotyledon etiolation was enhanced even in late-etiolatingcultivars when ethephon was added to the flask. The rate ofetiolation corresponded to the ethylene concentration in theflask; much more ethylene was detected in early-etiolating cultivarsthan in late-etiolating ones. Ethylene accumulation is one of the important factors involvedin the cotyledon etiolation observed in in vitro cultures. Thedifference in etiolation rates among seedlings with differentgenetic backgrounds for sex expression corresponds to theirability for ethylene evolution, in the order of acr^F>acr¹>acr⁺. (Received January 6, 1981; Accepted March 23, 1981)     

Changes in Endogenous Ribosyl-trans-zeatin and IAA Levels in Relation to the Proliferation of Tobacco Crown Gall Cells

Changes in the contents of major endogenous plant hormones intobacco crown gall cells, namely IAA and ribosyl-trans-zeatin,during cell growth were examined using HPLC and ¹⁴C-labeledplant hormones. The content of IAA was high at the early logarithmicstage, while that of ribosyl-trans-zeatin was high at the middlelogarithmic stage. This suggests that cell growth is affectedfirst by IAA, then by ribosyl-trans-zeatin. ³ Present address: Department of Agricultural Chemistry, TottoriUniversity, Koyama, Tottori 680, Japan (Received July 13, 1981; Accepted September 11, 1981)     

A Role for the acrA Gene in the Protection of Escherichia coli Cells against Excess Sodium

The acrA gene determines the sensitivity of Escherichia coliK-12 cells to acriflavine, which is one of the acridine dyesand which effectively eliminates certain plasmids from the bacterialcells. The acriflavine-sensitivity mutation leads to instabilityof plasmids, such as sex (F)- and drug-resistance (R)-factorsand to loss of a membrane protein with molecular weight about60 kDa (Nakamura 1974, 1976, Nakamura et al. 1975, 1981). Wehave found that cells with a mutant acrA gene were also moresensitive to an excess of sodium ions in the medium than werethe wild-type acrA⁺ cells (Nakamura 1977). The product of theacrA gene hindered the accumulation of sodium by the cells.Although mutations in theproA or proB genes, which determinethe synthesis of the enzymes y-glutamyl phosphate reductaseand y-glutamyl kinase, respectively, in the proline-biosyntheticpathway also led to sensitivity to and accumulation of excesssodium, the presence of the acrA⁺ allele decreased both theseparameters. (Received November 1, 1989; Accepted April 27, 1990)     

Stimulation by Ethylene of Mitochondrial Development in Wounded Sweet Potato Root Tissue

Ethylene (about 100 µl per liter) markedly stimulatedincreases in respiratory, Cyt c oxidase and succinate dehydrogenaseactivities of the crude mitochondrial fraction as well as mitochondrialmembrane protein during aging of sliced sweet potato root tissue,indicating that it stimulated mitochondrial development in woundedtissue. It had such an effect even when slices were pre-agedin its absence for 1 day and thereafter aged in its presence.The mitochondrial inner membrane from slices aged in ethylene-containingair was denser than that from fresh slices, while the membranefrom slices aged in ethylene-free air was lighter. Chloramphenicolcompletely inhibited the increase in Cyt c oxidase activitywhether slices were aged in the presence or absence of ethylene.Cycloheximide did not inhibit the increase in slices aged inethylene-free air, but did by 50% in those aged in ethylene-containingair. ¹ This work was supported in part by a Grant-in-Aid (No. 411308)for Scientific Research from the Ministry of Education, Scienceand Culture, Japan. (Received April 4, 1981; Accepted July 7, 1981)     

Effect of a Growth Inhibitor on the Hydroxyproline Level in Cell Wall of Zea Primary Roots

Evidence supporting the view that there is an inverse relationshipbetween the hydroxyproline-protein level in the cell wall andthe ability of a cell to undergo rapid cell elongation was obtained.A growth inhibitor extracted from Zea primary roots acceleratedthe incorporation of radioactivity derived from ¹⁴C-prolineinto the sodium lauryl sulfate (SLS)-insoluble cell wall fraction.However, the inhibitor had no effect on the ratio of hydroxyprolineto proline that was incorporated into the SLS-insoluble fraction. We have discussed what this growth inhibitor may mean in thegeotropic curvature of Zea primary roots. ¹ Present address: Faculty of Education, University of Yamagata,Yamagata 990, Japan (Received May 9, 1981; Accepted August 8, 1981)     

The production of microbial-regulatory materials by isolated aspen tissue

Isolated aspen (Populus tremuloides) callus tissue was foundto secrete materials which resulted in an initial stimulation,followed by an inhibition, of the growth of an Agrobacteriumspecies. Isolated aspen tissue also produced starch digestionmaterials which were not identical to the antimicrobial materials.The antimicrobial material was primarily bactericidal. A progressiverelease of antimicrobial materials was found as the weight ofthe tissue increased. The presence of dimethylsulfoxide in theculture medium did not influence the secretion of antimicrobialmaterials under the conditions employed. Attempts to isolateand characterize the antimicrobial materials have been unsuccessful. ¹Supported in part by the Pioneering Research Program of theBoard of Trustees of the Institute of Paper Chemistry, Appleton,Wisconsin, acting on behalf of a group of sponsoring pulp andpaper companies. ²Portions of this study were presented in partial fullfillmentof the requirements of the Master of Arts degree. Present address:Department of Biological Sciences, University of California,Santa Barbara, California, U.S.A. (Received December 10, 1970; )     

Mating Type Specific Inhibition of Gametic Differentiation of Chlamydomonas reinhardtii by Tunicamycin

The effect of tunicamycin, an inhibitor of glycosylation ofproteins, on the gametic differentiation of Chlamydomonas reinhardtiiwas studied. When mt⁺ cells were treated with tunicamycin duringgametogenesis, the acquisition of agglutinability on their flagellawas completely inhibited. However, no significant inhibitionwas observed when mt^– cells were treated with tunicamycinduring gametic induction. The agglutinability of the fully competentgametes of mt⁺ cells decreased sharply after about 4 hr of incubationwith tunicamycin and was lost completely after 8 hr. These resultsindicate that the gametic flagellar membrane of the mt⁺ cellmay acquire glycoproteins with tunicamycin sensitive sugar chains,the halflife of which is about 6 hr. (Received August 11, 1981; Accepted October 7, 1981)     

L-Leucine Transport in Isolated Protoplasts of Vinca Suspension Culture: Characterization of Uptake

The uptake of L-leucine into Vinca protoplasts was studied undervarious conditions. The uptake was highly pH-dependent, withthe optimal pH between 3.0 and 4.0. The uptake was also energydependent, since azide, 2,4-dinitrophenol (DNP), carbonyl cyanidem-chlorophenyl hydrazone (CCCP), and iodoacetate inhibited theuptake. Oligomycin, N,N'-dicycIohexyI carbodiimide (DCCD) andvanadate, but not ouabain, inhibited the uptake, suggestingthat ATPase for H⁺ electrogenic extrusion was necessary to theuptake of L-leucine. The uptake showed stereospecificity, butwas partially inhibited by other L-amino acids. A kinetic studyof the uptake showed that the uptake was multiphasic with threesaturable phases and one unsaturable phase which occurred atconcentrations of L-leucine over 1 mM. The K_m values of thethree affinity sites were 1.4 x 10^–3 M, 1.3 x 10^–4M, 4.3 x 10^–5 M; the maximum velocity values were 3.3x 10^–8, 4.5 x 10^–9, 1.8 x 10_–9 mol/10 min/4x 10⁶ cells. (Received April 18, 1981; Accepted August 25, 1981)     

Ti Plasmid Homologous Sequences Present in Tissues from Agrobacterium Plasmid-transformed Petunia Protoplasts

Suspension cell protoplasts of albino Petunia hybrida have beentransformed by isolated Agrobacterium tumefaciens Ti plasmid.Uptake of octopine Ti plasmid (pTiACH5) into protoplasts wasstimulated by poly-L-ornithine and polyethylene glycol (PEG).The frequency and efficiency of transformation of protoplaststo phytohormone autotrophy was compared using the two uptakeagents with various concentrations of plasmid. Transformationwas most efficient with PEG-mediated uptake, 5 µg of Tiplasmid per 10⁶ protoplasts giving a frequency of 6?10^–5.Octopine was not synthesised in any of the transformants afterthe second subculture on hormone-free medium. DNA-DNA hybridisationshowed the presence of DNA homologous to the T-DNA region ofpTiACH5 in all clones analysed. (Received November 9, 1981; Accepted January 29, 1982)     

Effects of Abscisic Acid and Cytoplasmic pH on Potassium and Chloride Efflux in Arabidopsis thaliana Seedlings

The effects of ABA, isobutyric acid (IBA) and nicotine on K⁺and Cl⁺ efflux were studied in Arabidopsis thaliana seedlings,and the role of pH_cyt, and E_m in the regulation of the effluxof these ions was discussed. The data show that treatments withIBA and nicotine influenced in opposite directions the effluxof either K⁺ or Cl^–: K⁺ efflux was increased by nicotineand reduced in the presence of IBA, whereas Cl^– effluxwas stimulated by IBA and decreased by nicotine treatment. Underall the conditions tested ABA induced cytoplasmic acidificationand inhibition of K⁺ and Cl^– net efflux. Experiments aimedto estimate the individual contribution of pH_cyt and E_m in modulatingK^– efflux indicated that, within the range of acidic pH_cytvalues, a regulation of K⁺ efflux was imposed by pH_cyt on thecontrol exerted by E_m, the efflux being inhibited by lower pH_cytvalues. Conversely, in the alkaline side of pH_cyt K⁺ effluxseemed linked only to the E_m values. These results are consistentwith the hypothesis that the decrease in K⁺ efflux observedin non-stomatal tissues in the presence of ABA may be mediatedby the cytoplasmic acidification induced by the hormone. (Received August 6, 1996; Accepted January 19, 1997)     

Comparison of Some Catalytic Properties of the Purified and Membrane-Bound Reduced Nicotinamide Adenine Dinucleotide-Cytochrome o Reductase of Vitreoscilla

The NADH oxidase system of Vitreoscilla was localized in themembrane as shown by three assays of respiratory activity: NADHoxidation, oxygen consumption and NADH dehydrogenase activityusing p-iodonitrotetrazolium violet (INT) as electron acceptor.The purified metalloflavoprotein component of the NADH-cytochromeo reductase system catalyzes the aerobic reduction of impurecytochrome o but not of the pure cytochrome. The purified enzymeexhibited hyperbolic kinetics with NADH when impure cytochromeo was used as electron acceptor but sigmoid kinetics with TNTas electron acceptor. The kinetics of INT reduction became morehyperbolic when assayed in the presence of impure cytochromeo and the Hill coefficient decreased from 1.8 to 1.1. In contrast,both the NADH-INT reductase and NADH oxidase activities of membranevesicles showed hyperbolic kinetics with NADH. The NADH oxidaseactivity of membrane vesicles was subject to substrate inhibitionat NADH concentrations greater than 150 µM which was notobserved for the NADH-cytochrome o reductase activity of thepurfied enzyme under aerobic conditions. Respiratory inhibitorssuch as rotenone, cyanide, and salicylhydroxamic acid inhibitedthe oxidation of NADH by membrane vesicles with oxygen as electronacceptor but not with INT as electron acceptor. These comparativestudies of some catalytic properties of the purified and membranebound enzyme provided evidence that the NADH-cytochrome o oxidasesystem of Vitreoscilla consists of cytochrome o, the flavoproteinreductase, and at least one other component which may regulatethe catalytic properties of the reductase. ¹ This work was supported by National Science Foundation GrantPCM77-15915 and Public Health Service Grant GM20006. This paperwas submitted by V.G.P. in partial fulfillment of the requirementsfor the Ph.D. degree of Illinois Institute of Technology. ² Present address: Department of Biological Science, NorthwesternUniversity, Evanston, Illinois 60201, U.S.A. (Received July 28, 1981; Accepted November 9, 1981)     

Phytochemical studies on tobacco alkaloids XIV. The occurrence and properties of putrescine N-methyltransferase in tobacco roots

Putrescine N-methyltransferase, a new enzyme catalyzing theformation of N-methylputrescine from putrescine and S-adenosyl-L-methioninewas found in roots of tobacco plants. The enzyme was purified30-fold from crude extracts of tobacco roots. NMethylputrescinewas identified as the reaction product by comparison with theauthentic compound. The enzyme had a pH optimum between pH 8and 9, and a molecular weight of about 60,000, as determinedby gel filtration. Km values for putrescine and 5-adenosyl-L-methioninewere 4.0 x 10^–4 M and 1.1 x 10^–4 M, respectively.Enzyme activity was inhibited by N-chloromercuribenzoate andAg⁺. No cofactors were required. Of the various substrates tested,only putrescine served as a methyl acceptor. The enzyme waslocalized exclusively in the roots and its activity was greadyenhanced by decapitation. The presence of putrescine N-methyltransferase in tobacco rootsstrongly suggests that N-methylputrescine participates as anintermediate in nicotine biosynthesis. (Received March 2, 1971; )