Taurine release in developing mouse hippocampus is modulated by glutathione and glutathione derivatives

Summary. Glutathione (reduced form GSH and oxidized form GSSG) constitutes an important defense against oxidative stress in the brain, and taurine is an inhibitory neuromodulator particularly in the developing brain. The effects of GSH and GSSG and glycylglycine, γ-glutamylcysteine, cysteinylglycine, glycine and cysteine on the release of [³H]taurine evoked by K⁺-depolarization or the ionotropic glutamate receptor agonists glutamate, kainate, 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) were now studied in slices from the hippocampi from 7-day-old mouse pups in a perfusion system. All stimulatory agents (50 mM K⁺, 1 mM glutamate, 0.1 mM kainate, 0.1 mM AMPA and 0.1 mM NMDA) evoked taurine release in a receptor-mediated manner. Both GSH and GSSG significantly inhibited the release evoked by 50 mM K⁺. The release induced by AMPA and glutamate was also inhibited, while the kainate-evoked release was significantly activated by both GSH and GSSG. The NMDA-evoked release proved the most sensitive to modulation: L-Cysteine and glycine enhanced the release in a concentration-dependent manner, whereas GSH and GSSG were inhibitory at low (0.1 mM) but not at higher (1 or 10 mM) concentrations. The release evoked by 0.1 mM AMPA was inhibited by γ-glutamylcysteine and cysteinylglycine, whereas glycylglycine had no effect. The 0.1 mM NMDA-evoked release was inhibited by glycylglycine and γ-glutamylcysteine. In turn, cysteinylglycine inhibited the NMDA-evoked release at 0.1 mM, but was inactive at 1 mM. Glutathione exhibited both enhancing and attenuating effects on taurine release, depending on the glutathione concentration and on the agonist used. Both glutathione and taurine act as endogenous neuroprotective effectors during early postnatal life. Authors’ address: Prof. Simo S. Oja, Brain Research Center, Medical School, FI-33014 University of Tampere, Finland     

Inoculation and nitrate alter phytohormone levels in soybean roots: differences between a supernodulating mutant and the wild type

 The levels of different cytokinins, indole-3-acetic acid (IAA) and abscisic acid (ABA) in roots of Glycine max [L.] Merr. cv. Bragg and its supernodulating mutant nts382 were compared for the first time. Forty-eight hours after inoculation with Bradyrhizobium, quantitative and qualitative differences were found in the root's endogenous hormone status between cultivar Bragg and the mutant nts382. The six quantified cytokinins, ranking similarly in each genotype, were present at higher concentrations (30–196% on average for isopentenyl adenosine and dihydrozeatin riboside, respectively) in mutant roots. By contrast, the ABA content was 2-fold higher in Bragg, while the basal levels of IAA [0.53 μmol (g DW)⁻¹, on average] were similar in both genotypes. In 1 mM NO₃ ⁻-fed Bragg roots 48 h post-inoculation, IAA, ABA and the cytokinins isopentenyl adenine, and isopentenyl adenosine quantitatively increased with respect to uninoculated controls. However, only the two cytokinins increased in the mutant. High NO₃ ⁻ (8 mM) markedly reduced root auxin concentration, and neither genotypic differences nor the inoculation-induced increase in auxin concentration in Bragg was observed under these conditions. Cytokinins and ABA, on the other hand, were little affected by 8 mM NO₃ ⁻. Root IAA/cytokinin and ABA/cytokinin ratios were always higher in Bragg relative to the mutant, and responded to inoculation (mainly in Bragg) and nitrate (both genotypes). The overall results are consistent with the auxin-burst-control hypothesis for the explanation of autoregulation and supernodulation in soybean. However, they are still inconclusive with respect to the inhibitory effect of NO₃ ⁻. Received: 16 April 1999 / Accepted: 13 December 1999     

Inhibition of glutathione synthesis reduces chilling tolerance in maize

 The role of glutathione (GSH) in protecting plants from chilling injury was analyzed in seedlings of a chilling-tolerant maize (Zea mays L.) genotype using buthionine sulfoximine (BSO), a specific inhibitor of γ-glutamylcysteine (γEC) synthetase, the first enzyme of GSH synthesis. At 25 °C, 1 mM BSO significantly increased cysteine and reduced GSH content and GSH reductase (GR: EC 1.6.4.2) activity, but interestingly affected neither fresh weight nor dry weight nor relative injury. Application of BSO up to 1 mM during chilling at 5 °C reduced the fresh and dry weights of shoots and roots and increased relative injury from 10 to almost 40%. Buthionine sulfoximine also induced a decrease in GR activity of 90 and 40% in roots and shoots, respectively. Addition of GSH or γEC together with BSO to the nutrient solution protected the seedlings from the BSO effect by increasing the levels of GSH and GR activity in roots and shoots. During chilling, the level of abscisic acid increased both in controls and BSO-treated seedlings and decreased after chilling in roots and shoots of the controls and in the roots of BSO-treated seedlings, but increased in their shoots. Taken together, our results show that BSO did not reduce chilling tolerance of the maize genotype analyzed by inhibiting abscisic acid accumulation but by establishing a low level of GSH, which also induced a decrease in GR activity. Received: 9 November 1999 / Accepted: 17 February 2000     

Discrepancy between nitrate reduction rates in intact leaves and nitrate reductase activity in leaf extracts: What limits nitrate reduction in situ?

Nitrate reductase (NR) activity in spinach leaf extracts prepared in the presence of a protein phosphatase inhibitor (50 μM cantharidine) was measured in the presence of Mg²⁺ (NRact) or EDTA (NRmax), under substrate saturation. These in-vitro activities were compared with nitrate reduction rates in leaves from nitrate-sufficient plants. Spinach leaves containing up to 60 μmol nitrate per g fresh weight were illuminated in air with their petiole in water. Their nitrate content decreased with time, permitting an estimation of nitrate reduction in situ. The initial rates (1–2 h) of nitrate consumption were usually lower than NRact, and with longer illumination time (4 h) the discrepancy grew even larger. When leaves were fed through their petiole with 30 mM nitrate, initial in-situ reduction rates calculated from nitrate uptake and consumption were still lower than NRact. However, nitrate feeding through the petiole maintained the in situ-nitrate reduction rate for a longer time. Initial rates of nitrate reduction in situ only matched NRact when leaves were illuminated in 5% CO₂. In CO₂-free air or in the dark, both NRact and in-situ nitrate reduction decreased, but NRact still exceeded in-situ reduction. More extremely, under anoxia or after feeding 5-amino-4-imidazole carboxyamide ribonucleoside in the dark, NR was activated to the high light level; yet in spite of that, nitrate reduction in the leaf remained very low. It was examined whether the standard assay for NRact would overestimate the in-situ rates due to a dissociation of the inactive phospho-NR-14-3-3 complex after extraction and dilution, but no evidence for that was found. In-situ NR obviously operates below substrate saturation, except in the light at high ambient CO₂. It is suggested that in the short term (2 h), nitrate reduction in situ is mainly limited by cytosolic NADH, and cytosolic nitrate becomes limiting only after the vacuolar nitrate pool has been partially emptied. Received: 19 June 1999 / Accepted: 12 October 1999     

Analytical electron microscopical investigations on the apoplastic pathways of lanthanum transport in barley roots

 In transmission electron microscopy studies, lanthanum ions have been used as electron-opaque tracers to delineate the apoplastic pathways for ion transport in barley (Hordeum vulgare L.) roots. To localize La³⁺ on the subcellular level, e.g. in cell walls and on the surface of membranes, electron-energy-loss spectroscopy and electron-spectroscopic imaging were used. Seminal and nodal roots were exposed for 30 min to 1 mM LaCl₃ and 10 mM LaCl₃, respectively. In seminal roots, possessing no exodermis, La³⁺ diffusion through the apoplast was stopped by the Casparian bands of the endodermis. In nodal roots with an exodermis, however, La³⁺ diffusion through the cortical apoplast had already stopped at the tight junctions of the exodermal cell walls resembling the Casparian bands of the endodermis. Therefore, we conclude that in some specialized roots such as the nodal roots of barley, the physiological role of the endodermis is largely performed by the exodermis. Received: 28 July 1999 / Accepted: 24 February 2000     

Intraprotoplasmic and wall-localised formation of arabinoxylan-bound diferulates and larger ferulate coupling-products in maize cell-suspension cultures

Maize (Zea mays L.) cell cultures incorporated radioactivity from [¹⁴C]cinnamate into hydroxycinnamoyl-CoA derivatives and then into polysaccharide-bound feruloyl residues. Within 5–20 min, the CoA pool had lost its ¹⁴C by turnover and little or no further incorporation into polysaccharides then occurred. The system was thus effectively a pulse–chase experiment. Kinetics of radiolabelling of diferulates (also known as dehydrodiferulates) varied with culture age. In young (1–3 d) cultures, polysaccharide-bound [¹⁴C]feruloyl- and [¹⁴C]diferuloyl residues were both detectable within 1 min of [¹⁴C]cinnamate feeding. Thus, feruloyl residues were dimerised <1 min after their attachment to polysaccharides. For at least the first 2.3 h after [¹⁴C]cinnamate feeding, polysaccharide-bound [¹⁴C]diferuloyl residues remained almost constant at ≈7% of the total polysaccharide-bound [¹⁴C]ferulate derivatives. Since feruloyl residues are attached to polysaccharides <1 min after the biosynthesis of the latter, and >10 min before secretion, the data show that extensive feruloyl coupling occurred intra-protoplasmically. Exogenous H₂O₂ (1 mM) caused little additional feruloyl coupling; therefore, wall-localised coupling may have been peroxidase-limited. In older (e.g. 4 d) cultures, less intraprotoplasmic coupling occurred: during the first 2.5 h, polysaccharide-bound [¹⁴C]diferuloyl residues were a steady 1.4% of the total polysaccharide-bound [¹⁴C]ferulate derivatives. In contrast to the situation in younger cultures, exogenous H₂O₂ induced a rapid 4- to 6-fold increase in all coupling products, indicating that coupling in the walls was H₂O₂-limited. In both 2- and 4-d-old cultures, polysaccharide-bound ¹⁴C-trimers and larger coupling products exceeded [¹⁴C]diferulates 3- to 4-fold, but followed similar kinetics. Thus, although all known dimers of ferulate can now be individually quantified, it appears to be trimers and larger products that make the major contribution to cross-linking of wall polysaccharides in cultured maize cells. We argue that feruloyl arabinoxylans that are cross-linked before and after secretion are likely to loosen and tighten the cell wall, respectively. The consequences for the control of cell expansion and for the response of cell walls to an oxidative burst are discussed. Received: 19 January 2000 / Accepted: 13 April 2000     

Nitric oxide is involved in taurine release in the mouse brain stem under normal and ischemic conditions

Summary. Nitric oxide (NO) has been shown to regulate neurotransmitter release in the brain; both inhibitory and excitatory effects have been seen. Taurine is essential for the development and survival of neural cells and protects them under cell-damaging conditions. In the brain stem, it regulates many vital functions such as cardiovascular control and arterial blood pressure. Now we studied the effects of the NO-generating compounds hydroxylamine (HA), S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) on the release of preloaded [³H]taurine under normal and ischemic conditions in slices prepared from the mouse brain stem from developing (7-day-old) to young adult (3-month-old) mice. In general, the effects of NO on the release were somewhat complex and difficult to explain, as expected from the multifunctional role of NO in the central nervous system. The basal initial release under normal conditions was enhanced by the NO donors 5 mM HA and 1.0 mM SNAP at both ages, but SNP was inhibitory in developing mice. The release was markedly enhanced by K⁺ stimulation. The effects of HA, SNAP and SNP on the basal release were not antagonized by the NO synthase inhibitor N^G-nitro-L-arginine (L-NNA, 1.0 mM), demonstrating that mechanisms other than NO synthesis are involved. Taurine release in developing mice in the presence of SNP was reduced by the inhibitor of soluble guanylate cyclase, 1H-(1,2,3)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), indicating the possible involvement of cGMP. In normoxia, N-methyl-D-aspartate (NMDA, 1.0 mM) enhanced the SNAP- and HA-evoked taurine release in developing mice and the HA-evoked release in adults. In ischemia, both K⁺ stimulation and NMDA potentiated the NO-induced release, particularly in the immature mice, probably without the involvement of the NO synthase or cGMP. The substantial release of taurine in the developing brain stem evoked by NO donors together with NMDA might represent signs of important mechanisms against excitotoxicity which protect the brain stem under cell-damaging conditions. Authors’ address: Prof. Pirjo Saransaari, Brain Research Center, Medical School University of Tampere, Tampere, FIN-3 3014, Finland     

Elimination kinetics of L-alanyl-L-glutamine in ICU patients

Summary. A randomised, double blind, placebo-controlled study was performed giving 0.5 g · kg⁻¹ · day⁻¹ of undiluted alanyl-glutamine (20%) or saline in a peripheral vein during 4 hours in ICU patients (n = 20). During the infusion period a steady state in plasma concentration was reached for alanyl-glutamine, but not for alanine, glutamine or glutamate. On the other hand there was no accumulation of any of the amino acids, as the pre-infusion concentrations were reached within 8 hours after the end of infusion. The half-life of the dipeptide was 0.26 hours (range, 0.15–0.63 h). The distribution volume of alanyl-glutamine was larger than the extracellular water volume, indicating a rapid hydrolysis of the dipeptide. There was no detectable alanyl-glutamine in the urine of any of the patients. All patients had excretion of small amounts of amino acids in urine, but the renal clearance of alanine, glutamine and glutamate were not different between the two groups.     

The effect of taurine depletion on the contractile properties and fatigue in fast-twitch skeletal muscle of the mouse

Summary. Taurine as well as tauret (retinyliden taurine) levels were measured in locust Locusta migratoria compound eyes. HPLC measurements revealed relatively low taurine levels (1.9 ± 0.16 mM) in dark-adapted eyes. Glutamate, aspartate and glycine levels were 2.0 ± 0.2, 2.7 ± 0.4 and 3.0 ± 0.37 mM, respectively, while GABA was present only in trace amounts. After about 4 h of light adaptation at 1500–2000 lx, amino acid levels in the compound eye were as follows: taurine, 1.8 ± 0.17 mM; glutamate, no change at 2.1 ± 0.2 mM; aspartate sharply increased to 4.7 ± 0.7 mM; glycine slightly decreased to 2.8 ± 0.3 mM; and GABA trace levels. In the compound eye of locust Locusta migratoria, the existence of endogenous tauret in micro-molar range was established. In the dark, levels were several times higher compared with compound eye after light adaptation 1500 lx for 3 h, as estimated by TLC in combination with spectral measurements. Existence of tauret in compound eye is of special interest because in the compound eye, rhodopsin regeneration is based on photoregeneration.     

Dietary γ-aminobutyric acid affects the brain protein synthesis rate in young rats

Summary. The purpose of this study was to determine whether the γ-aminobutyric acid (GABA) affects the rate of brain protein synthesis in male rats. Two experiments were done on five or three groups of young rats (5 wk) given the diets containing 20% casein administrated 0 mg, 25 mg, 50 mg, 100 mg or 200 mg/100 g body weight GABA dissolved in saline by oral gavage for 1 day (d) (Experiment 1), and given the diets contained 0%, 0.25% or 0.5% GABA added to the 20% casein diet (Experiment 2) for 10 d. The plasma concentration of growth hormone (GH) was the highest in rats administrated 50 mg and 100 mg/100 g body weight GABA. The concentration of serum GABA increased significantly with the supplementation groups. The fractional (Ks) rates of protein synthesis in brain regions, liver and gastrocnemius muscle increased significantly with the 20% casein + 0.25% GABA diet and still more 20% casein + 0.5% GABA compared with the 20% casein diet. In brain regions, liver and gastrocnemius muscle, the RNA activity [g protein synthesized/(g RNA·d)] significantly correlated with the fractional rate of protein synthesis. The RNA concentration (mg RNA/g protein) was not related to the fractional rate of protein synthesis in any organ. Our results suggest that the treatment of GABA to young male rats are likely to increase the concentrations of plasma GH and the rate of protein synthesis in the brain, and that RNA activity is at least partly related to the fractional rate of brain protein synthesis.     

In vitro biosynthesis of 1,4-β-galactan attached to rhamnogalacturonan I

 The biosynthesis of galactan was investigated using microsomal membranes isolated from suspension-cultured cells of potato (Solanum tuberosum L. var. AZY). Incubation of the microsomal membranes in the presence of UDP-[¹⁴C]galactose resulted in a radioactive product insoluble in 70% methanol. The product released only [¹⁴C]galactose upon acid hydrolysis. Treatment of the product with Aspergillus niger endo-1,4-β-galactanase released 65–70% of the radioactivity to a 70%-methanol-soluble fraction. To a minor extent, [¹⁴C]galactose was also incorporated into proteins, however these galactoproteins were not a substrate for Aspergillus niger endo-1,4-β-galactanase. Thus, the majority of the ¹⁴C-labelled product was 1,4-β-galactan. Compounds released by the endo-1,4-β-galactanase treatment were mainly [¹⁴C]galactose and [¹⁴C]galactobiose, indicating that the synthesized 1,4-β-galactan was longer than a trimer. In vitro synthesis of 1,4-β-galactan was most active with 6-d-old cells, which are in the middle of the linear growth phase. The optimal synthesis occurred at pH 6.0 in the presence of 7.5 mM Mn²⁺. Aspergillus aculeatus rhamnogalacturonase A digested at least 50% of the labelled product to smaller fragments of approx. 14 kDa, suggesting that the synthesized [¹⁴C]galactan was attached to the endogenous rhamnogalacturonan I. When rhamnogalacturonase A digests of the labelled product were subsequently treated with endo-1,4-β-galactanase, radioactivity was not only found as [¹⁴C]galactose or [¹⁴C]galactobiose but also as larger fragments. The larger fragments were likely the [¹⁴C]galactose or [¹⁴C]galactobiose still attached to the rhamnogalacturonan backbone since treatment with β-galactosidase together with endo-1,4-β-galactanase digested all radioactivity to the fraction eluting as [¹⁴C]galactose. The data indicate that the majority of the [¹⁴C]galactan was attached directly to the rhamnose residues in rhamnogalacturonan I. Thus, isolated microsomal membranes contain enzyme activities to both initiate and elongate 1,4-β-galactan sidechains in the endogenous pectic rhamnogalacturonan I. Received: 24 June 1999 / Accepted: 30 August 1999     

Enhancing effect of taurine on CYP7A1 mRNA expression in Hep G2 cells

Summary. Taurine has been reported to enhance cholesterol 7α-hydroxylase (CYP7A1) mRNA expression in animal models. However, no in vitro studies of this effect have been reported. The Hep G2 human hepatoma cell line has been recognized as a good model for studying the regulation of human CYP7A1. This work characterizes the effects of taurine on CYP7A1 mRNA levels of Hep G2 cells in a dose- and time-dependent manner. In the dose-dependent experiment, Hep G2 cells were treated with 0, 2, 10 or 20 mM taurine in the presence or absence of cholesterol 0.2 mM for 48 h. In the time-dependent experiment, Hep G2 cells were treated with 0 or 20 mM taurine for 4, 24 and 48 h with and without cholesterol 0.2 mM. Our data revealed that taurine showed time- and dose-response effects on CYP7A1 mRNA levels in Hep G2 cells. However, glycine – a structural analogue of taurine – did not have an effect on CYP7A1 gene expression. These results show that, in agreement to previous studies on animal models, taurine induces the mRNA levels of CYP7A1 in Hep G2 cells, which could enhance cholesterol conversion into bile acids. Also, Hep G2 cell line may be an appropriate model to study the effects of taurine on human cholesterol metabolism.     

Decrease of phosphoribulokinase activity by antisense RNA in transgenic tobacco: definition of the light environment under which phosphoribulokinase is not in large excess

 To test the hypothesis that the contribution of phosphoribulokinase (PRK) to the control of photosynthesis changes depending on the light environment of the plant, the response of transgenic tobacco (Nicotiana tabacum L.) transformed with antisense PRK constructs to irradiance was determined. In plants grown under low irradiance (330 μmol m⁻² s⁻¹) steady-state photosynthesis was limited in plants with decreased PRK activity upon exposure to higher irradiance, with a control coefficient of PRK for CO₂ assimilation of 0.25 at and above 800 μmol m⁻² s⁻¹. The flux control coefficient of PRK for steady-state CO₂ assimilation was zero, however, at all irradiances in plant material grown at 800 μmol m⁻² s⁻¹ and in plants grown in a glasshouse during mid-summer (alternating shade and sun 300–1600 μmol m⁻² s⁻¹). To explain these differences between plants grown under low and high irradiances, Calvin cycle enzyme activities and metabolite content were determined. Activities of PRK and other non-equilibrium Calvin cycle enzymes fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase and ribulose-1,5-bisphosphate carboxylase-oxygenase were twofold higher in plants grown at 800 μmol m⁻² s⁻¹ or in the glasshouse than in plants grown at 330 μmol m⁻² s⁻¹. Activities of equilibrium enzymes transketolase, aldolase, ribulose-5-phosphate epimerase and isomerase were very similar under all growth irradiances. The flux control coefficient of 0.25 in plants grown at 330 μmol m⁻² s⁻¹ can be explained because low ribulose-5-phosphate content in combination with low PRK activity limits the synthesis of ribulose-1,5-bisphosphate. This limitation is overcome in high-light-grown plants because of the large relative increase in activities of sedoheptulose-1,7-bisphosphatase and fructose-1,6-bisphosphatase under these conditions, which facilitates the synthesis of larger amounts of ribulose-5-phosphate. This potential limitation will have maintained evolutionary selection pressure for high concentrations of PRK within the chloroplast. Received: 15 November 1999 / Accepted: 27 January 2000     

Relationship between NO synthesis, arginine transport, and intracellular arginine levels in vascular smooth muscle cells

Summary. The present study was designed to evaluate the relevance of arginine transport in nitric oxide (NO) synthesis in vascular smooth muscle cells. For this purpose, NO synthesis and arginine transport (system B^0,+ and y⁺) were evaluated in cells treated with IL-1β or angiotensin II (Ang II). In addition, the effects of 5 mM lysine and glutamine, competitive inhibitors of systems y⁺ and B^0,+ respectively, were examined. L-arginine transport was estimated with ³H-labelled arginine and NO was determined with the Griess reagent. These studies were done in control conditions, arginine-starved cells, and in cells incubated in media containing 10 mM arginine. Our data indicate that induction of NO biosynthesis by IL-1β depends on external arginine when cells are arginine-depleted for 24 hours. The concentration of arginine producing half maximal activation of NO synthesis in arginine-depleted cells ([arginine]_i < 10 μM) was 41.1 ± 18 μM. By contrast, in normal culture conditions, NO synthesis occurred independently of arginine transport. Neither 5 mM lysine or glutamine which abolished arginine transport through systems y⁺ and B^0,+, respectively, reduced nitrite release in cells incubated in normal media. This suggests that the relevance of arginine uptake to NO synthesis depends on the status of intracellular arginine pools. Intracellular arginine concentrations were not affected by the stimulation of NO production using IL-1β or its inhibition using Ang II, but were markedly reduced by arginine starvation for 48 h. Aspartate levels were also reduced by arginine-depletion, but were not affected in cells incubated with 10 mM arginine. By contrast, glutamate levels were reduced in arginine-starved cells and were increased in cells incubated in arginine-supplemented medium. Ornithine levels were markedly increased by arginine supplementation. Altogether, these findings indicate that NO synthesis is normally independent of membrane transport. However in arginine-depleted cells, membrane transport is essential for NO synthesis. It is concluded that arginine transport is required for the long-term maintenance of intracellular arginine pools. Received February 7, 1999; Accepted June 21, 1999     

Fast track to the trichome: induction of N-acyl nornicotines precedes nicotine induction in Nicotiana repanda

 Nicotiana repanda Wildenow ex Lehmann acylates nornicotine in its trichomes to produce N-acyl-nornicotine (NacNN) alkaloids which are dramatically more toxic than nicotine is to the nicotine-adapted herbivore, Manduca sexta. These NacNNs, like nicotine, were induced by methyl jasmonate (MeJA) and wounding, but the 2-fold increase in NacNN pools was much faster (within 6 h) than the MeJA-induced increase in nornicotine pools (24 h to 4 d), its parent substrate. When ¹⁵NO₃ ⁻ pulse-chase experiments with intact and induced plants were used to follow the incorporation of ¹⁵N into alkaloids in different plant parts over the plant's lifetime, it was found that the root nicotine pool was most rapidly labeled, followed by the shoot nornicotine and NacNN pools. After 3 d, 3.12% of ¹⁵N acquired was in nicotine (0.93%), nornicotine (0.32%) and NacNNs (1.73%) while only 0.14% was in anabasine. Once NacNNs are externalized to the leaf surface, they are not readily re-distributed within the plant and are lost with senescing leaves. The wound- and MeJA-induced N-acylation of nornicotine is independent of induced changes in nornicotine pools and the rapidity of the response suggests its importance in defense against herbivores. Received: 3 July 1999 / Accepted: 17 September 1999     

The application of atomic force microscopy to topographical studies and force measurements on the secreted adhesive of the green alga Enteromorpha

Atomic force microscopy (AFM) enables the topographical structure of cells and biological materials to be resolved under natural (physiological) conditions, without fixation and dehydration artefacts associated with imaging methods in vacuo. It also provides a means of measuring interaction forces and the mechanical properties of biomaterials. In the present study, AFM has been applied for the first time to the study of the mechanical properties of a natural adhesive produced by a green plant cell. Swimming spores of the green alga Enteromorpha linza (L.) J. Ag. (7–10 μm) secrete an adhesive glycoprotein which provides firm anchorage to the substratum. Imaging of the adhesive in its hydrated state revealed a swollen gel-like pad, approximately 1 μm thick, surrounding the spore body. Force measurements revealed that freshly released adhesive has an adhesion strength of 173 ± 1.7 mN m⁻¹ (mean ± SE; n=90) with a maximum value for a single adhesion force curve of 458 mN m⁻¹. The adhesive had a compressibility (equivalent to Young's modulus) of 0.54 × 10⁶ ± 0.05 × 10⁶ N m⁻² (mean ± SE; n=30). Within minutes of release the adhesive underwent a progressive `curing' process with a 65% reduction in mean adhesive strength within an hour of settlement, which was also reflected in a reduction in the average length of the adhesive polymer strands (polymer extension) and a 10-fold increase in Young's modulus. Measurements on the spore surface itself revealed considerably lower adhesion-strength values but higher polymer-extension values than the adhesive pad, which may reflect the deposition of different polymers on this surface as a new cell wall is formed. The study demonstrates the value of AFM to the imaging of plant cells in the absence of fixation and dehydration artefacts and to the characterisation of the mechanical properties of plant glycoproteins that have potential utility as adhesives. Received: 22 February 2000 / Accepted: 20 April 2000     

Inactivation of DNA replication origins by the cell cycle regulator, trigonelline, in root meristems of Lactuca sativa

 The effects of trigonelline (TRG) on the cell cycle in root meristems of Lactuca sativa L. were examined in the knowledge that TRG is a cell cycle regulator that causes cell arrest in G2, and prevents ligation of replicons in S-phase. The hypothesis was tested that continuous exposure to TRG would perturb DNA replication which, in turn, would lengthen the cell cycle and impair root elongation. Using DNA fibre autoradiography, mean replicon size was 31 and 13 μm in the TRG (3 mM) and control treatments, respectively. Trigonelline also resulted in a lengthening of both S-phase and the cell cycle and a decrease in primary root elongation. Hence, replicon inactivation was responsible for the protracted S-phase. Trigonelline treatment also resulted in a 1.6-fold increase in fork rate (13.8 μm h⁻¹) compared with the control (8.4 m h⁻¹). The faster fork rate in the larger replicons is in accord with the highly significant positive relationship already established between fork rate and replicon size for various unrelated higher plants. Received: 11 October 1999 / Accepted: 23 December 1999     

Osmotically evoked shrinking of guard-cell protoplasts causes vesicular retrieval of plasma membrane into the cytoplasm

The dye FM1-43 was used alone or in combination with measurements of the membrane capacitance (C_m) to monitor membrane changes in protoplasts from Viciafaba L. guard cells. Confocal images of protoplasts incubated with FM1-43 (10 μM) at constant ambient osmotic pressure (π_o) revealed in confocal images a slow internalisation of FM1-43-labelled membrane into the cytoplasm. As a result of this process the relative fluorescence intensity of the cell interior (f_FM,i) increased with reference to the total fluorescence (f_FM,t) by 7.4 × 10⁻⁴ min⁻¹. This steady internalisation of dye suggests the occurrence of constitutive endocytosis under constant osmotic pressure. Steady internalisation of FM1-43 labelled membrane caused a prominent staining of a ring-like structure located beneath the plasma membrane. Abrupt elevation of π_o by 200 mosmol kg⁻¹ caused, over the first minutes of incubation, a rapid internalisation of FM1-43 fluorescence into the cytoplasm concomitant with a decrease in cell perimeter. Within the first 5 min the cell perimeter decreased by 7.9%. Over the same time f_FM,i/f_FM,t increased by 0.13, reflecting internalisation of fluorescent label into the cytoplasm. Combined measurements of C_m and total fluorescence of a protoplast (f_FM,p) showed that an increase in π_o evoked a decrease in C_m but no change in f_FM,p. This means that surface contraction of the protoplast is due to retrieval of excess membrane from the plasma membrane and internalisation into the cytoplasm. Further inspection of confocal images revealed that protoplast shrinking was only occasionally associated with internalisation of giant vesicles (median diameter 2.7 μm) with FM1-43-labelled membrane. But, in all cases, osmotic contraction was correlated with a diffuse distribution of FM1-43 label throughout the cytoplasm. From this, we conclude that endocytosis of small vesicles into the cytoplasm is the obligatory process by which cells accommodate an osmotically driven decrease in membrane surface area. Received: 4 May 1999 / Accepted: 19 August 1999     

Intracellular chloroplast photorelocation in the moss Physcomitrella patens is mediated by phytochrome as well as by a blue-light receptor

 The light-induced intracellular relocation of chloroplasts was examined in red-light-grown protonemal cells of the moss Physcomitrella patens. When irradiated with polarized red or blue light, chloroplast distribution in the cell depended upon the direction of the electrical vector (E-vector) in both light qualities. When the E-vector was parallel to the cross-wall (i.e. perpendicular to the protonemal axis), chloroplasts accumulated along the cross-wall; however, no accumulation along the cross-wall was observed when the E-vector was perpendicular to it (i.e. parallel to the protonemal axis). When a part of the cell was irradiated with a microbeam of red or blue light, chloroplasts accumulated at or avoided the illumination point depending on the fluence rate used. Red light of 0.1–18 W m⁻² and blue light of 0.01–85.5 W m⁻² induced an accumulation response (low-fluence-rate response; LFR), while an avoidance response (high-fluence-rate response; HFR) was induced by red light of 60 W m⁻² or higher and by blue light of 285 W m⁻². The red-light-induced LFR and HFR were nullified by a simultaneous background irradiation of far-red light, whereas the blue-light-induced LFR and HFR were not affected at all by this treatment. These results show, for the first time, that dichroic phytochrome, as well as the dichroic blue-light receptor, is involved in the chloroplast relocation movement in these bryophyte cells. Further, the phytochrome-mediated responses but not the blue-light responses were revealed to be lost when red-light-grown cells were cultured under white light for 2 d. Received: 7 September 1999 / Accepted: 15 October 1999     

L-NAME administration prevents the inhibition of nucleotide hydrolysis by rat blood serum subjected to hyperargininemia

Summary. The main objective of the present study was to evaluate the in vivo and in vitro effect of Arg on serum nucleotide hydrolysis. The action of N^ω-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, on the effects produced by Arg was also examined. Sixty-day-old rats were treated with a single or a triple (with an interval of 1 h between each injection) intraperitoneal injection of saline (group I), Arg (0.8 g/kg) (group II), L-NAME (2.0 mg/kg or 20 mg/kg) (group III) or Arg (0.8 g/kg) plus L-NAME (2.0 mg/kg or 20 mg/kg) (group IV) and were killed 1 h later. The present results show that a triple Arg administration decreased ATP, ADP and AMP hydrolysis. Simultaneous injection of L-NAME (20 mg/kg) prevented such effects. Arg in vitro did not alter nucleotide hydrolysis. It is suggested that in vivo Arg administration reduces nucleotide hydrolysis in rat serum, probably through nitric oxide or/and peroxynitrite formation. Both are first authors.