Effects of Acriflavine on the Formation of the Plasma Membrane of Escherichia coli

When cells of acriflavine-sensitive (acrA) and acriflavine-resistant(acrA⁺) Escherichia coli K-12 strains were treated with a ratherhigh concentration (100 µg ml^-1) of acriflavine in mediumthat had been adjusted to pH 8.1, distinct whirlpool-like structuresderived from the plasma membrane appeared not only in the acrAcells but also in the acrA⁺ cells. Chemical analysis was performedto determine the lipid composition of the cells by thin-layerchromatography on silica gel and gas-liquid chromatography.The amount of total fatty acids was significantly higher inthe acrA cells than in the acrA⁺ cells, when cells were culturedin the presence of acriflavine. This difference seems to becaused by the greater accumulation of unsaturated fatty acids(palmitoleic and cis-vaccenic acid) in the acrA mutant cellsthan in the acrA⁺ cells and by the acceleration of this accumulationas a result of the presence of the dye. A comparison of phospholipidcontents between the acrA and acrA⁺ cells cultured under acriflavine-freeconditions showed that the former cells contained more phosphatidylethanolamine(PE) and, in particular, more cardiolipin (CL) than the lattercells. However, the situation was reversed in the case of phosphatidylglycerol(PG). Addition of acriflavine to the medium led to a markedincrease in levels of PE and CL in both acrA and acrA⁺ cellsbut an increase in levels of PG was found only in the acrA⁺cells. (Received October 13, 1992; Accepted May 31, 1993)     

Modulation of BK(Ca) channel activity by fatty acids: structural requirements and mechanism of action

To determinethe mechanism of fatty acid modulation of rabbit pulmonary arterylarge-conductance Ca²⁺-activated K⁺(BK_Ca) channel activity, we studied effects of fatty acidsand other lipids on channel activity in excised patches withpatch-clamp techniques. The structural features of the fatty acidrequired to increase BK_Ca channel activity (or averagenumber of open channels, NP_o) were identified tobe the negatively charged head group and a sufficiently long (C > 8) carbon chain. Positively charged lipids like sphingosine, which havea sufficiently long alkyl chain (C  8), produced a decrease inNP_o. Neutral and short-chain lipids did notalter NP_o. Screening of membrane surface chargewith high-ionic-strength bathing solutions (330 mM K⁺ or130 mM K⁺, 300 mM Na⁺) did not alter themodulation of the BK_Ca channel NP_oby fatty acids and other charged lipids, indicating that channelmodulation is unlikely to be due to an alteration of the membraneelectric field or the attraction of local counterions to the channel.Fatty acids and other negatively charged lipids were able to modulate BK_Ca channel activity in bathing solutions containing 0 mMCa²⁺, 20 mM EGTA, suggesting that calcium is not requiredfor this modulation. Together, these results indicate that modulationof BK_Ca channels by fatty acids and other charged lipidsmost likely occurs by their direct interaction with the channel proteinitself or with some other channel-associated component.

     

Effects of cadmium on the behaviour of citric acid in isolated tomato xylem cell walls

Effects of cadmium on the sorption of citric acid In isolatedxylem cell walls were Investigated. 2.5 nM to 9.5 mM [1.5–¹⁴]crticacid solutions were perfused through columns of xylem cell wallmaterial, isolated from tomato plants (Lycoperslcon esculentumMill, cv. Tiny Tim). The anion exchange potential of the column was estimated byamino acid analysis as approximately 46 meq dm whereas the apparentanion exchange capacity (AEC) was estimated as 1.65±0.1810^–4(citric acId units). This low AEC was attributed toa ‘zipper’ effect, a mutual screening of fixed R^–and A⁺ charges. Pre-loading with ¹¹⁵Cd²⁺ did not affect citric acid sorption,indicating the absence of Cd-effects on the availability offixed A⁺ charges, and the absence of the formation of effectiveR^–-Cd²⁺ and Donnan tree space (DFS) (Cd(cit)H₂]⁺ complexes. Simultaneous application of both citric acid and ¹¹⁵Cd²⁺,⁴⁵Ca²⁺or ²⁸Mg²⁺ resufted in increased sorption of citric acid, probablydue to capacity improvement rather than changes in valence-dependentanion sorption; this may be due to the presence of bulk (M(cit)H₂]⁺,held in the column as [M(cit)H₂]⁺ after protonation in the DFS.Sorption of citric acid was greatest in the presence of Ca²⁺which was discussed in the light of the differences betweenCa, Cd and Mg in their characteristics as co-ordinative M-complexes of citric acid. The overall results indicate the potentialimportance of the presence of metal ions for the xylem transportbehaviour of organic acids in plants. Key words: Cadmium, citric acid, ion exchange, ligand exchange, tomato, xylem cell walls     

Metabolism of Slices of the Tomato Stem

The rate of respiration of tomato stem slices varied considerably,the highest values (Qo₂, 2–3) being obtained for plantsin a good nutritional state, and the lowest (Qo₂, 1) in starvedplants. The respiratory quotient of 1.0 remained constant. Glucose fermentation was found to follow both glycolytic andalcohol fermentation pathways, the ratio of ethyl alcohol: lacticacid being 6.6:1. Fermentation seems to take place accordingto the Embden-Meyerhof scheme, as shown by the presence of someof these enzymes operative in this scheme and by inhibitionexperiments. In the presence of oxygen there was no formationof alcohol or lactic acid. Pyruvate added to tomato stem slices was metabolized by directoxidation to acetic acid and by dismutation to lactic and aceticacids and CO₂ The metabolism of acetic acid was demonstratedby its condensation with oxaloacetic acid to form citrate, thisbeing the second time that synthesis of citric acid by thismechanism has been found in plants. The presence of aconitase,of isocitric dehydrogenase, of succinic dehydrogenase, and ofmalic dehydrogenase, as well as the inhibition of respirationby malonic acid, favour the hypothesis that oxidation of carbohydratein tomato stem slices proceeds via the citric acid cycle. Thepossibility of an auxiliary route, the malic acid oxidationpathway, also was demonstrated. Tomato stem tissue anaerobicallysplit malic acid into glycolic acid. The further oxidation ofglycolic, glyoxylic, and formic acid was demonstrated. In experiments with C¹⁴-labelled acetate and butyrate a dilutionof the C¹⁴-labelled acids was found after incubation indicatingnew formation of these acids and of active participation offatty acid metabolism in the metabolic activities of the tissue. With the exception of alanine, added amino acids produced adefinite increase in O₂ uptake without extra formation of ammonia. Experimental demonstration of the possibility of electron transportfrom substrate to molecular oxygen in respiration via polyphenoloridase was provided by the attainment in a tomato tissue homogenateof a coupled oxidation-reduction between -ketoglutarate andcatechol with DPN and tyrosinase as the catalysts. The presenceof cytochrome oxidase was also demonstrated. Thus both systemspossibly may take part in the respiration of tomato stem slices.     

Compositions and Positional Distributions of Fatty Acids in Phospholipids from Leaves of Chilling-Sensitive and Chilling-Resistant Plants

The compositions and positional distributions of fatty acidsin the major leaf phospholipids of phosphatidylglycerol, phosphatidylcholineand phosphatidylethanolamine were analyzed by gas-liquid chromatographyand enzymic hydrolysis, and chilling-sensitive and chilling-resistantplants were comparcd with respect to the relative contents ofpalmitic and trans-³-hexadecenoic acids in the separated phospholipids.A distinct difference between these plants was found in thefatty acid compositions of phosphatidylglycerol, in which thesum of palmitic and trans-³-hexadecenoic acids ranged from 60to 78% of the total fatty acids in 8 species of chilling-sensitiveplants, and from 50 to 57% in 11 species of chilling-resistantplants. The only exception among the chilling sensitive plantsin this respect was the tomato, in which the sum of palmiticand trans-³-hexadecenic acids in phosphatidylglycerol amountedto 54%. The fatty acid compositions and the positional distributionsof fatty acids in phosphatidylglycerol suggest that the occurrenceof high proportions of dipalmitoyl and 1-palmitoyl-2-(trans-³-hexadecenoyl)species in this lipid is correlated with the susceptibilityto chilling of the leaves of higher plants. In the compositionsand positional distributions of fatty acids in phosphatidylcholineand phosphatidylethanolamine, no difference was found betweenthe chilling-sensitive and chilling-resistant plants. ¹ Present address: Department of Biology, Faculty of Science,Universityof Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan. (Received May 21, 1982; Accepted June 25, 1982)     

Potent block of inactivation-deficient Na+ channels by n-3 polyunsaturated fatty acids

A voltage-gated, small, persistent Na⁺ current (I_Na) has been shown in mammalian cardiomyocytes. Hypoxia potentiates the persistent I_Na that may cause arrhythmias. In the present study, we investigated the effects of n-3 polyunsaturated fatty acids (PUFAs) on I_Na in HEK-293t cells transfected with an inactivation-deficient mutant (L409C/A410W) of the -subunit (hH1) of human cardiac Na⁺ channels (hNav1.5) plus ₁-subunits. Extracellular application of 5 µM eicosapentaenoic acid (EPA; C20:5n-3) significantly inhibited I_Na. The late portion of I_Na (I_{Na late}, measured near the end of each pulse) was almost completely suppressed. I_Na returned to the pretreated level after washout of EPA. The inhibitory effect of EPA on I_Na was concentration dependent, with IC₅₀ values of 4.0 ± 0.4 µM for I_Na peak (I_{Na peak}) and 0.9 ± 0.1 µM for I_{Na late}. EPA shifted the steady-state inactivation of I_{Na peak} by –19 mV in the hyperpolarizing direction. EPA accelerated the process of resting inactivation of the mutant channel and delayed the recovery of the mutated Na⁺ channel from resting inactivation. Other polyunsaturated fatty acids, docosahexaenoic acid, linolenic acid, arachidonic acid, and linoleic acid, all at 5 µM concentration, also significantly inhibited I_Na. In contrast, the monounsaturated fatty acid oleic acid or the saturated fatty acids stearic acid and palmitic acid at 5 µM concentration had no effect on I_Na. Our data demonstrate that the double mutations at the 409 and 410 sites in the D1–S6 region of hH1 induce inactivation-deficient I_Na and that n-3 PUFAs inhibit mutant I_Na. human cardiac sodium channel     

Studies on cutin-esterase II. Characteristics of cutin-esterase from Botrytis cinerea and its activity on tomato-cutin

The activity of cutin-esterase, cutinase, was detected in themycelial homogenate of Botrytis cinerea cultured in a peptone-sucrosemedium at 25°C for 7 days. The crude enzyme solution wasprepared from the homogenate by centrifugation at 106,600xg,treatment with (NH₄)₂SO₄ at 70% saturation, and dialysis against0.01 M phosphate buffer. The optimum pH, temperature and assayduration for enzyme activity were 5.0, 25°C and 18 hr, respectively.Specific activity was 255 mµmoles/mg protein/18 hr aspalmitate under optimum conditions. 83% of the activity waslost by heating the enzyme solution (pH 7.6) for 4 min at 95°C.Palmitic, stearic, oleic, 9, 10-dihydroxystearic or linoleic,dihydroxyeicosanoic and octadecanedioic acids were recognizedin the enzymic hydrolysate of tomato-cutin using gas-liquidchromatography. Among these fatty acids, palmitic, oleic andoctadecanedioic acids were readily liberated by the enzyme,but dihydroxyeicosanoic acid, the major component of tomato-cutin,was isolated only in small amounts. The enzyme is, therefore,an exo-type cutinase which hydrolyses minor side chains of fattyacids bound to the major structure of cutin. Cutinesterase mayfacilitate cuticular invasion by fungi as a result of reductionin mechanical strength of the cuticle by the enzyme ¹Biological Laboratory, Research Department, Nihon Noyaku Co.,Ltd., Kawachinagano, Osaka, Japan (Received June 16, 1970; )     

Ammonium release by zooplankton in suspensions of heat-killed algae and an evaluation of the flow-cell method

The maximum excretion rate of NH₄ (39 nmol mg dry wt^–1h^–1) was directly measured for Daphnia pulex by measuringNH₄ accumulation in bottles containing D. pulex and dense, satiatingsuspensions of heat-killed algae. Ammonium release rates inthe algal suspensions were compared to those of individual animalsremoved from the suspension and placed in flow cells. Ammoniumrelease rate, R (nmol mg dry wt^–1 h^–1). in the flowcell decreased very rapidly with time, t (min), after removalaccording to the relation R = 26 + 25e^–0.16t. Ammoniumexcretion obtained by the flow cell method after extrapolationto time zero was not significantly different from that obtainedin the bottles. The considerable experiment-to-experiment variationin NH₄ excretion was in large part correlated (r² = 0.73) withthe feeding rate on the algae.     

Modulation of K+ channels by arachidonic acid in T84 cells. I.Inhibition of the Ca2+-dependent K+ channel

TheCl secretory response ofcolonic cells to Ca²⁺-mediatedagonists is transient despite a sustained elevation of intracellular Ca²⁺. We evaluated the effects ofsecond messengers proposed to limit Ca²⁺-mediatedCl secretion on thebasolateral membrane,Ca²⁺-dependentK⁺ channel(K_Ca) in colonic secretorycells, T84. Neither protein kinase C (PKC) nor inositoltetrakisphosphate (1,3,4,5 or 3,4,5,6 form) affectedK_Ca in excised inside-out patches.In contrast, arachidonic acid (AA; 3 µM) potently inhibitedK_Ca, reducingNP_o, the productof number of channels and channel open probability, by 95%. Theapparent inhibition constant for this AA effect was 425 nM. AAinhibited K_Ca in the presence ofboth indomethacin and nordihydroguaiaretic acid, blockers of thecyclooxygenase and lipoxygenase pathways. In the presence of albumin,the effect of AA on K_Ca wasreversed. A similar effect of AA was observed onK_Ca during outside-out recording.We determined also the effect of thecis-unsaturated fatty acid linoleate,the trans-unsaturated fatty acidelaidate, and the saturated fatty acid myristate. At 3 µM, all ofthese fatty acids inhibited K_Ca,reducing NP_o by 72-86%. Finally, the effect of the cytosolic phospholipaseA₂ inhibitorarachidonyltrifluoromethyl ketone(AACOCF₃) on thecarbachol-induced short-circuit current(I_sc) responsewas determined. In the presence ofAACOCF₃, the peakcarbachol-inducedI_sc response wasincreased ~2.5-fold. Our results suggest that AA generation inducedby Ca²⁺-mediated agonists maycontribute to the dissociation observed between the rise inintracellular Ca²⁺ evoked by theseagonists and the associatedCl secretory response.

     

Oxidative Damages and Repair in Euglena gracilis Exposed to Ozone. I. SH Groups and Lipids

Malondialdehyde, a product of lipid oxidation, increased graduallywhen Euglena gracilis cells were bubbled with 240 µ1.liter^–1ozone (delivery rate of 1µmolO₃.min^–1) for 120 min.Simultaneously, the sulfhydryl group content decreased by 36%during the treatment, which was mainly due to oxidation of proteinsulfhydryl groups. The molar amount of SH groups oxidized was3 times higher than that of fatty acid oxidized, indicatingthat sulfhydryl groups were more accessible or more easily oxidizedby O₃ than fatty acids. When Euglena cells were allowed to recoverunder autotrophic growth conditions following O₃ treatment,viable cells were incapable of dividing during the first 5 hof the recovery period but regenerated SH groups nearly to thecontrol level. The increase of SH content during this periodpreceded the resumption of cell division and the restorationof normal growth. These results suggest that the regenerationof SH groups by Euglena cells is a part of a mechanism involvedin the repair of oxidative damage caused by ozone and is anessential step for the initiation of cell division. (Received July 20, 1987; Accepted December 14, 1987)     

The Resolution by HPLC of RS-[2-14C]Me 1', 4'-cis-Diol of Abscisic acid and the Metabolism of (-)-R- and (+)-S-Abscisic Acid

The R- and S-enantiomers of racemic [2-¹⁴C]Me 1', 4'-cis-diolof abscisic acid have been separated by high performance liquidchromatography on an optically-active Pirkle column. R-[2-¹⁴C]-and S-[2-¹⁴C]abscisic acids, formed from the Me 1', 4'-cis-diolby oxidation and alkyline hydrolysis were fed to tomato shootsand the extracts analysed by reversed phase high performanceliquid chromatography. R-[2-¹⁴C]abscisic acid formed mainlythe abscisic acid glucose ester (ABAGE), abscisic acid l'-glucoside(ABAGS) and an uncharacterized conjugate. Dihydrophaseic acid4'-B-D-glucoside, the major metabolite of RS-abscisic acid intomato shoots, was found to be derived virtually exclusivelyfrom the natural, S-abscisic acid. Phaseic acid and conjugatesof abscisic acid were also found as products of the naturallyoccurring enantiomer. The resolution method was used to measurethe relative proportions of R and S enantiomers in the freeacid liberated from conjugates formed from RS-[2-¹⁴C]ABA fedto shoots. The ratios show an excess of the R-enantiomer: 5.8:1, ABAGE; 29.4: 1, ABAGE; 8.3: 1 for an uncharacterized conjugateand 6.1: 1 for the residual free [2-¹⁴C]ABA. Key words: ABA, HPLC, Tomato     

Effects of fatty acids on BK channels in GH(3) cells

Ca²⁺-activated K⁺ (BK) channels inGH₃ cells are activated by arachidonic acid (AA). Becausecytosolic phospholipase A₂ can produce other unsaturatedfree fatty acids (FFA), we examined the effects of FFA on BK channelsin excised patches. Control recordings were made at several holdingpotentials. The desired FFA was added to the bath solution, and thevoltage paradigm was repeated. AA increased the activity of BK channelsby 3.6 ± 1.6-fold. The cis FFA, palmitoleic, oleic,linoleic, linolenic, eicosapentaenoic, and the triple bond analog ofAA, eicosatetraynoic acid, all increased BK channel activity, whereasstearic (saturated) or the trans isomers elaidic,linolelaidic, and linolenelaidic had no effect. The cisunsaturated FFA shifted the open probability vs. voltage relationshipsto the left without a change in slope, suggesting no change in thesensitivity of the voltage sensor. Measurements of membrane fluidityshowed no correlation between the change of membrane fluidity and thechange in BK channel activation. In addition, AA effects on BK channelswere unaffected in the presence of N-acetylcysteine.Arachidonyl-CoA, a membrane impermeable analog of AA, activateschannels when applied to the cytosolic surface of excised patches,suggesting an effect of FFAs from the cytosolic surface of BK channels.Our data imply a direct interaction between cis FFA and theBK channel protein.

     

Effect of concentration on albumin diffusion in lung interstitium

The transport of macromolecules through the lung interstitiumdepends on both bulk transport of fluid and diffusion. In the presentstudy, we studied the diffusion of albumin. Isolated rabbit lungs wereinflated with silicon rubber via airways and blood vessels, and twochambers were bonded to the sides of a 0.5-cm-thick slab that encloseda vessel with an intersititial cuff. One chamber was filled with eitheralbumin solution (2 or 5 g/dl) containing tracer¹²⁵I-albumin or with tracer¹²⁵I-albumin alone; the other wasfilled with Ringer solution. Unbound ¹²⁵I was removed from the tracerby dialysis before use. The chamber with Ringer solution was placed inthe well of a NaI(Tl) scintillation detector. Diffusion oftracer through the interstitium was measured continuously for 60 h.Tracer mass (M) showed a time(t) delay followed by an increase toa steady-state flow(dM/dtconstant). Albumin diffusion coefficient(D) was given byL²/(6T),where T was the time intercept of thesteady-stateM-t line at zero M, andL was interstitial length.Interstitial cuff thickness-to-vessel radius ratio(Th₀/R)was estimated by using Fick's law for steady-state diffusion. BothD andTh₀/Rwere independent of albumin concentration.D averaged 6.6 × 10⁷cm²/s, similar to the freeD for albumin. Values ofTh₀/Raveraged 0.047 ± 0.024 (SD), near the values measuredhistologically. Thus pulmonary interstitial constituents offered norestriction to the diffusion of albumin.

     

Acyl-CoA Synthetase in Maturing Safflower Seeds

Acyl-CoA synthetase in maturing seeds of safflower (Carthamustinctorius) was membranebound, and the highest specific activitywas associated with microsomes. Activity absolutely dependedon the concentrations of fatty acid, CoA, ATP and Mg²⁺. Theapparent K_m values were 4.2 µM for oleate, 24 µMfor CoA, and 250 µM for ATP. The optimum pH of the reactionwas 7.5. Triacsin C, a potent inhibitor of the animal and bacterialacyl-CoA synthetase, was ineffective for the safflower enzyme.The enzyme utilized C₁₆ and C₁₈ long-chain fatty acids preferentially,while medium-chain and very-long-chain fatty acids were poorsubstrates. The order of specificity for native fatty acidswas linoleate > oleate=palmitate > stearate. Althoughactivity per seed varied during seed maturation, it was enoughto account for the rate of triacylglycerol synthesis in vivo. (Received February 2, 1993; Accepted March 3, 1993)     

2-Trans-Abscisic Acid Biosynthesis and Metabolism of ABA-Aldehyde and Xanthoxin in Wild Type and the aba Mutant of Arabidopsis thaliana

Abscisic acid (ABA) and 2-trans-ABA (t-ABA) biosynthesis werestudied in wild type Landsberg erecta and the three allelicaba mutants of Arabidopsis thaliana (L.) Heynh., which are impairedin epoxy-carotenoid biosynthesis. Labelling experiments with¹⁸O₂and mass spectrometric analysis of [¹⁸O]ABA and its catabolitesABA-glucose ester (ABA-GE) and phaseic acid (PA), and t- ABAand t-ABA-GE, showed that t-ABA biosynthesis was less affectedthan ABA biosynthesis by mutations at the ABA locus. The aba-4allele caused the most severe impairment of ABA biosynthesiscompared with the other two mutant alleles aba-1 and aba-3,yet aba-4 plants synthesized as much t-ABA as wild type Landsbergerecta plants. Feeding experiments with RS- [²H₆]ABA-aldehydeisomers and unlabelled xanthoxin isomers suggest that t-xanthoxinand t-ABA-aldehyde are precursors to ABA and t-ABA in Arabidopsis Key words: ABA-alcohol, ABA-aldehyde, ABA-glucose ester, ¹⁸O₂ labelling, phaseic acid     

Studies on the Mechanisms of Action of the Antitranspirant Phenylmercuric Acetate, and its Penetration into the Mesophyll

Experiments have examined the effect of phenylmercuric acetate(PMA) on the guard cells of Commelina communis. In one series,PMA was supplied to the leaf surface; after different time intervalsthe epidermis was removed and the ability of the stomata toopen was determined. In the other series, different concentrationsof PMA were included in the medium used for inccubating epidermalstrips with which ion-stimulated stomatal opening was assayed.At concentrations of 10^-54 M and above the effect of PMA wassevere and the structural integrity of the guard cells was affected;they were unable to accumulate neutral red. At concentrationsarpound 10^-6 M the guard cells were less affected and PMA broughtabout a transient stimulation of stomatal opening by releasingsubsidiary-cell turgor pressure. A solution of 5 x 10^-4 M PMA applied to leaves reduced by halfthe photosynthetic ¹⁴CO₂ incorporation into C. communis mesophyll.In Zea mays it increased the CO₂ compensation point and alsothe resistance to diffusion in the gas phase (R_A, but therewas a proportionately greater increase in the apparent liquidphase resistance (R_t). This direct inhibition of mesophyll photosynthesisundermines one of the major objectives of applying anatitranspirants,and for this reason it is suggested that PMA is unsuitable forgeneral application to crops.     

Changes in Endogenous Gibberellin Levels in Tulipa Bulblets during Ontogeny

The endogenous gibberellin activity of Tulipa gesneriana cv.Apeldoorn bulbiets of field-grown mother bulbs was determinedduring ontogeny by paper and gas-liquid chromatography and thedwarf pea bioassay. It was shown that the gibberellin activityof bulblets increased dramatically in November-March and declinedsharply in April-July. The increase in gibberellin-like substanceswas considered to be derived primarily by synthesis within thebulblets with possible contributions via translocation fromthe mother bulb scales, roots and shoots. Gibberellin A₁₃ wastentatively identified by gas-liquid chromatography but theother components of the bulbiet extracts remained unidentified.     

Chemical Composition of Bleeding Xylem Sap from Kiwifruit Vines

A study of the chemical composition and charge balance was madeof bleeding xylem sap collected from excised one-year-old extensionshoots of healthy, Mn-deficient, Mn-toxic and Zn-deficient kiwifruitvines (Actinidia deliciosa (A. Chev.) C. F. Liang et A. R. Ferguson)immediately prior to leafburst. The exudates were analysed formacronutrient cations and anions, trace elements, amino acids,organic acids and sugars. Major charged species measured wereCa (13.3 mM), K (8.9 mM), Mg (5.6 mM), malate (12.5 mM) andphosphate (5.8 mM). Glutamine (12 mM) was the predominant Ncarrier identified, accounting for 58 per cent of the totalN followed by NO₂^–-N (4.5 per cent), NH₄⁺-N (3.5 per cent)and arginine-N (2.9 per cent). Approximately 22 per cent ofthe N was in a hydrolysable proteinaceous fraction comprisingmainly glutamine and glutamate. Eighteen free proteinaceousamino acids were idetified in sap, the most abundant being glutamine,glutamic acid, valine, isoleucine and phenylalanine. Computersimulation of the chemical composition predicted that in additionto hydrated cations, ion pairs formed between inorganic components(SO₄^2–, HPO₄^2–, H₂PO₄^–) and cations (Ca²⁺,Mg²⁺, Mn²⁺), plus metal-organic ligand complexes (Ca Malate,Zn Malate, FeCit, CuHis, CuGln) are important species involvedin translocation. The solubility product of hydroxyapatite wasexceeded in all exudates although in vitro precipitation wasnot observed. To achieve electroneutrality with the componentsmeasured, however, formation of precipitate precursors (hydroxyapatitenuclei) had to be assumed. Irregularities in Mn nutrition (butnot Zn) were clearly indicated by the elemental compositionof exudate suggesting the use of sap analysis as a possiblepre-season indicator of nutritional status for this species. Actinidia deliciosa (A. Chev.) C. F. Liang et A. R. Ferguson, kiwifruit, xylem sap composition, trace metals, amino acids, organic acids     

Direct Evaluation of Effects of Fatty-Acid Unsaturation on the Thermal Properties of Photosynthetic Activities, as Studied by Mutation and Transformation of Synechocystis PCC6803

Glycerolipids of thylakoid membranes isolated from the cyanobacteriumSynechocystis PCC6803 contained high levels of dienoic and trienoicC₁₈ fatty acids, in addition to saturated C₁₆ and monoenoicC₁₈ fatty acids. A mutant (Fadl2) of this cyanobacterium wasdefective in the desaturation of C₁₈ fatty acids at the ¹² position,and its thylakoid membranes lacked trienoic acids and containeda very reduced level of dienoic acids. A derivative strain ofFadl2 (Fadl2/desA), which had been transformed with a gene fordesaturation at the ¹² position, fully recovered the abilityto desaturate the fatty acids in the glycerolipids of thylakoidmembranes. The thermal properties of the photosynthetic activitiesof the mutant and the transformant were compared with thoseof the wild-type strain. Despite great diversity in the extentof unsaturation of fatty acids between the wild-type, Fadl2,and Fad12/desA strains, no significant differences were foundeither in the temperature dependence of photosynthesis or inthe heat stability of photosynthetic, photosystem II and photosystemI activities. These results demonstrate that the trienoic fattyacids and, probably, the dienoic acids of the lipids in thethylakoid membrane do not affect the thermal properties of theabove-mentioned activities of photosynthesis. ³Permanent address: Institute of Plant Physiology, BiologicalResearch Center of Hungarian Academy of Sciences, H-6701 Szeged,P.O. Box 521, Hungary (Received August 9, 1990; Accepted December 7, 1990)     

Estimation of ammonium regeneration efficiencies associated with bacterivory in pelagic food webs via a 15N tracer method

Phagotrophic protists are major components of pelagic food webs,both as consumers of bacterial and phytoplankton cells, andas regenerators of inorganic nutrients. In this study, we estimatedthe efficiency of ammonium regeneration by protists feedingon bacteria within natural plank-tonic assemblages, using a¹⁵N tracer method, in which the excretion of ¹⁵N-labeled ammoniumdue to grazing on ¹⁵N pre-labeled bacteria was followed overtime. We tested this approach in experiments based on the additionof heat-killed ¹⁵N-labeled bacteria to laboratory cultures andto samples of coastal seawater. During two experiments, variationin abundance of bacterivores and bacterioplankton resulted innon-constant grazing rates. Deterministic computer models thatused abundance of bacteria and protists as variables were developedto estimate best-fit values of grazing mortality (g, h^–1)and of ammonium regeneration efficiency (R_E, fraction of theinitial ¹⁵N label in added bacteria which is released as ammonium).Estimated ammonium R_E were 0.30–0.35 for one trophic linksystems with both a monospecific culture and a mixed speciesassemblage of bacterivorous flagellates. R_E was higher for multi-trophicstep food webs: 0.60 for 5 µm pre-screened coastal seawaterand 0.90 for whole coastal seawater.