Fruit-specific lectins from banana and plantain

 One of the predominant proteins in the pulp of ripe bananas (Musa acuminata L.) and plantains (Musa spp.) has been identified as a lectin. The banana and plantain agglutinins (called BanLec and PlanLec, respectively) were purified in reasonable quantities using a novel isolation procedure, which prevented adsorption of the lectins onto insoluble endogenous polysaccharides. Both BanLec and PlanLec are dimeric proteins composed of two identical subunits of 15 kDa. They readily agglutinate rabbit erythrocytes and exhibit specificity towards mannose. Molecular cloning revealed that BanLec has sequence similarity to previously described lectins of the family of jacalin-related lectins, and according to molecular modelling studies has the same overall fold and three-dimensional structure. The identification of BanLec and PlanLec demonstrates the occurrence of jacalin-related lectins in monocot species, suggesting that these lectins are more widespread among higher plants than is actually believed. The banana and plantain lectins are also the first documented examples of jacalin-related lectins, which are abundantly present in the pulp of mature fruits but are apparently absent from other tissues. However, after treatment of intact plants with methyl jasmonate, BanLec is also clearly induced in leaves. The banana lectin is a powerful murine T-cell mitogen. The relevance of the mitogenicity of the banana lectin is discussed in terms of both the physiological role of the lectin and the impact on food safety. Received: 1 December 1999 / Accepted: 31 January 2000     

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     

Symplastic communication between the central cell and the egg apparatus cells in the embryo sac of Torenia fournieri Lind. before and during fertilization

 Various membrane-impermeable, water-soluble fluorescent tracers with different molecular weights were microinjected into the central cell of the embryo sac of Torenia fournieri Lind. before and during fertilization. Before anthesis, there was high symplastic permeability between the central cell and the egg apparatus cells. In this stage, fluorescent tracers up to 10 kDa could pass from the central cell into the egg apparatus cells, whereas those with larger molecular weight remained in the central cell. As the embryo sac matured, symplastic permeability decreased such that 2 d after anthesis only tracers less than 3 kDa could spread from the central cell into the egg cell. There appeared to be no symplastic permeability between the primary endosperm and zygote after fertilization, since tracers as small as 521 Da could not pass into the zygote in about half of the microinjected embryo sacs. This is the first report of a change in cell-to-cell communication among the cells of the female germ unit before and after fertilization. Received: 16 December 1999 / Accepted: 4 February 2000     

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.     

Life and death near a windy oasis

We propose a simple experiment to study delocalization and extinction in inhomogeneous biological systems. The nonlinear steady state for, say, a bacteria colony living on and near a patch of nutrient or favorable illumination (“oasis”) in the presence of a drift term (“wind”) is computed. The bacteria, described by a simple generalization of the Fisher equation, diffuse, divide A→A + A, die A→ 0, and annihilate A + A→ 0. At high wind velocities all bacteria are blown into an unfavorable region (“desert”), and the colony dies out. At low velocity a steady state concentration survives near the oasis. In between these two regimes there is a critical velocity at which bacteria first survive. If the “desert” supports a small nonzero population, this extinction transition is replaced by a delocalization transition with increasing velocity. Predictions for the behavior as a function of wind velocity are made for one and two dimensions. Received: 3 August 1998 / Revised version: 17 July 1999 / Published online: 4 July 2000     

Transgenic Nicotiana tabacum and Arabidopsis thaliana plants overexpressing allene oxide synthase

 Allene oxide synthase (AOS), encoded by a single gene in Arabidopsis thaliana (L.) Heynh., catalyzes the first step specific to the octadecanoid pathway. Enzyme activity is very low in control plants, but is upregulated by wounding, octadecanoids, ethylene, salicylate and coronatine (D. Laudert and E.W. Weiler, 1998, Plant J 15: 675–684). In order to study the consequences of constitutive expression of AOS on the level of jasmonates, a complete cDNA encoding the enzyme from A. thaliana was constitutively expressed in both  A. thaliana and tobacco (Nicotiana tabacum L.). Overexpression of AOS did not alter the basal level of jasmonic acid; thus, output of the jasmonate pathway in the unchallenged plant appears to be strictly limited by substrate availability. In wounded plants overexpressing AOS, peak jasmonate levels were 2- to 3-fold higher compared to untransformed plants. More importantly, the transgenic plants reached the maximum jasmonate levels significantly earlier than wounded untransformed control plants. These findings suggest that overexpression of AOS might be a way of controlling defense dynamics in higher plants. Received: 10 February 2000 / Accepted: 11 March 2000     

Diuretic potential of energy drinks

Summary. Recent literature suggests that both caffeine and taurine can induce diuresis and natriuresis in rat and man. Although they act via different cellular mechanisms, their diuretic actions might be additive. This is of considerable interest, as several commercially available energy drinks contain both substances. In this study we examined the possible diuretic effects of caffeine and taurine in a cross-over-design in which 12 healthy male volunteers received each of 4 different test drinks (750 ml of energy drink containing 240 mg caffeine and 3 g taurine, the three other test drinks either lacked caffeine, taurine or both) after restraining from fluids for 12 h. Mixed model analyses demonstrated that urinary output and natriuresis were significantly increased by caffeine (mean differences 243 ml and 27 mmol; both p < 0.001) and that there were no such effects of taurine (mean differences 59 ml and −4 mmol). Additionally, urinary osmolarity at baseline was significantly related to the urinary output (p < 0.001). Urine osmolarity values at baseline and in the 6 h urine collection did not differ significantly between treatments. Taken together, our study demonstrates that diuretic and natriuretic effects of the tested energy drink were largely mediated by caffeine. Taurine played no significant role in the fluid balance in moderately dehydrated healthy young consumers. Consequently, the diuretic potential of energy drinks will not differ significantly from other caffeine containing beverages.     

Identification of a signal peptide for oryzacystatin-I

 A previously unidentified extension of an open reading frame from the genomic DNA of Japonica rice (Oryza sativa L.) encoding oryzacystatin-I (OC-I; access. M29259, protein ID AAA33912.1) has been identified as a 5′ gene segment coding for the OC-I signal peptide. The signal peptide appears to direct a pre-protein (SPOC-I; Accession No. AF164378) to the endoplasmic reticulum, where it is processed into the mature form of OC-I. The start codon of SPOC-I begins 114 bp upstream from that previously published for OC-I. A putative proteolytic site, which may yield a mature OC-I approximately 12 residues larger than previously described, has been identified within SPOC-I between Ala-26 and Glu-27. The signal peptide sequence was amplified by polymerase chain reaction using genomic DNA from O. sativa seedlings and ligated to the 5′ end of the truncated OC-I gene at the endogenous SalI site. Partially purified protein extracts from Escherichia coli expressing SPOC-I reacted with polyclonal antibodies raised against OC-I and revealed a protein of the expected molecular weight (15,355 Da). In-vitro translation of SPOC-I in the presence of microsomal membranes yielded a processed product approximately 2.7 kDa smaller than the pre-protein. Nicotiana tabacum L. cv. Xanthi plants independently transformed with the SPOC-I gene processed SPOC-I and accumulated the mature form of OC-I (approximately 12.6 kDa), which co-migrated with natural, mature OC-I extracted from rice seed when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Received: 29 July 1999 / Accepted: 25 August 1999     

Isolation and characterisation of cell wall polysaccharides from cocoa (Theobroma cacao L.) beans

 Cell wall material (CWM) was prepared from sun-dried cocoa (Theobroma cacao L.) bean cotyledons before and after fermentation. The monosaccharide composition of the CWM was identical for unfermented and fermented beans. Polysaccharides of the CWM were solubilised by sequential extraction with 0.05 M trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA), 0.05 M Na₂CO₃, and 1 M, 4 M and 8 M KOH. The non-cellulosic sugar composition for each fraction was similar for unfermented and fermented samples, indicating that fermentation caused no significant modification of the structural features of individual cell wall polysaccharides. Pectic polysaccharides accounted for 60% of the cell wall polysaccharides but only small amounts could be solubilised in solutions of CDTA, Na₂CO₃, and 1 M and 4 M KOH. The bulk of the pectic polysaccharides were solubilised in 8 M KOH and were characterised by a rhamnogalacturonan backbone heavily substituted with side-chains of 5-linked arabinose and 4-linked galactose. Linkage analysis indicated the presence of additional acidic polysaccharides, including a xylogalacturonan and a glucuronoxylan. Cellulose, xyloglucan and a galactoglucomannan accounted for 28%, 8% and 3% of the cell wall polysaccharides, respectively. It is concluded that the types and structural features of cell wall polysaccharides in cocoa beans resemble those found in the parenchymatous tissue of many fruits and vegetables rather than those reported for many seed storage polysaccharides. Received: 29 May 1999 / Accepted: 19 October 1999     

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     

Turing instabilities in general systems

We present necessary and sufficient conditions on the stability matrix of a general n(≥2)-dimensional reaction-diffusion system which guarantee that its uniform steady state can undergo a Turing bifurcation. The necessary (kinetic) condition, requiring that the system be composed of an unstable (or activator) and a stable (or inhibitor) subsystem, and the sufficient condition of sufficiently rapid inhibitor diffusion relative to the activator subsystem are established in three theorems which form the core of our results. Given the possibility that the unstable (activator) subsystem involves several species (dimensions), we present a classification of the analytically deduced Turing bifurcations into p (1 ≤p≤ (n− 1)) different classes. For n = 3 dimensions we illustrate numerically that two types of steady Turing pattern arise in one spatial dimension in a generic reaction-diffusion system. The results confirm the validity of an earlier conjecture [12] and they also characterise the class of so-called strongly stable matrices for which only necessary conditions have been known before [23, 24]. One of the main consequences of the present work is that biological morphogens, which have so far been expected to be single chemical species [1–9], may instead be composed of two or more interacting species forming an unstable subsystem. Received: 21 September 1999 / Revised version: 21 June 2000 / Published online: 24 November 2000     

Affinity for inorganic carbon of Gracilaria tenuistipitata cultured at low and high irradiance

Regulation by irradiance level of the mechanism for dissolved inorganic carbon (DIC) acquisition was examined in the red macroalga Gracilaria tenuistipitata Zhang et Xia. For this purpose, affinity for external DIC, carbonic anhydrase (CA; EC 4.2.1.1) activity and content of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) were determined in thalli grown at 45 and 500 μmol photons m⁻² s⁻¹. Oxygen evolution rates declined by 50% when the medium pH was changed from 8.1 to 8.7, and the pH compensation point attained was ca. 9.2. These characteristics were unaffected by the light treatments. In contrast, photosynthetic conductance for DIC at pH 8.7 was doubled in thalli grown at high irradiance compared with those grown at low irradiance (to 0.74 × 10⁻⁶ from 0.33 × 10⁻⁶ m s⁻¹). Photosynthetic rates at saturating DIC concentration were also higher by 60% in thalli grown at high irradiance. These differences could not be attributed to changes in the use of external DIC, since external CA activity did not vary. Although the irradiance level did not modify the pool size of Rubisco, Rubisco content expressed on a chlorophyll a basis was almost doubled at high irradiance. These results likely indicate that the internal transport of DIC towards the active-site of Rubisco, rather than the external use of DIC, is enhanced in the thalli grown at high irradiance. Received: 7 June 1999 / Accepted: 16 October 1999     

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     

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.     

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     

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     

Artificially increased ascorbate content affects zeaxanthin formation but not thermal energy dissipation or degradation of antioxidants during cold-induced photooxidative stress in maize leaves

Infiltrating detached maize (Zeamays L.) leaves with L-galactono-1,4-lactone (L-GAL) resulted in a 4-fold increase in the content of leaf ascorbate. Upon exposure to high irradiance (1000 μmol photons m⁻² s⁻¹) at 5 °C, L-GAL leaves de-epoxidized the xanthophyll-cycle pigments faster than the control leaves; the maximal ratio of de-epoxidized xanthophyll-cycle pigments to the whole xanthophyll-cycle pool was the same in both leaf types. The elevated ascorbate content, together with the faster violaxanthin de-epoxidation, did not affect the degree of photoinhibition and the kinetics of the recovery from photoinhibition, assayed by monitoring the maximum quantum efficiency of photosystem II primary photochemistry (F_v/F_m). Under the experimental conditions, the thermal energy dissipation seems to be zeaxanthin-independent since, in contrast to the de-epoxidation, the decrease in the efficiency of excitation-energy capture by open photosystem II reaction centers (F_v′/F_m′) during the high-irradiance treatment at low temperature showed the same kinetic in both leaf types. This was also observed for the recovery of the maximal fluorescence after stress. Furthermore, the elevated ascorbate content did not diminish the degradation of pigments or α-tocopherol when leaves were exposed for up to 24 h to high irradiance at low temperature. Moreover, a higher content of ascorbate appeared to increase the requirement for reduced glutathione. Received: 20 May 1999 / Accepted: 29 October 1999     

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     

The time-profile of the PBMC HSP70 response to in vitro heat shock appears temperature-dependent

Summary. Heat shock proteins (HSPs) are synthesised by cells subsequent to a stress exposure and are known to confer protection to the cell in response to a second challenge. HSP induction and decay are correlated to thermotolerance and may therefore be used as a biomarker of thermal history. The current study tested the temperature-dependent nature of the heat shock response and characterised its time profile of induction. Whole blood from 6 healthy males (Age: 26 ± (SD) 2 yrs; Body mass 74.2 ± 3.8 kgs; VO_2max: 49.1 ± 4.0 ml·kg⁻¹·min⁻¹) were isolated and exposed to in vitro heat shock (HS) at 37, 38, 39, 40, and 41 °C for a period of 90 min. After HS the temperature was returned to 37 °C and intracellular HSP70 was quantified from the leukocytes at 0, 2, 4, and 6 h after heat treatment. The concentration of HSP70 was not different between temperatures (P > 0.05), but the time-profile of HSP70 synthesis appeared temperature-dependent. At control (37 °C) and lower temperatures (38–39 °C) the mean HSP70 concentration increased up to 4 h post HS (P < 0.05) and then returned towards baseline values by 6 h post HS. With in vitro hyperthermic conditions (40–41 °C), the time-profile was characterised by a sharp rise in HSP70 levels immediately after treatment (P < 0.05 for 40 °C at 0 h), followed by a progressive decline over time. The results suggest a temperature-dependent time-profile of HSP70 synthesis. In addition, the temperature at which HSP70 is inducted might be lower than 37 °C.     

Cloning and expression of UDP-glucose: flavonoid 7-O-glucosyltransferase from hairy root cultures of Scutellaria baicalensis

 A cDNA encoding UDP-glucose: baicalein 7-O-glucosyltransferase (UBGT) was isolated from a cDNA library from hairy root cultures of Scutellaria baicalensis Georgi probed with a partial-length cDNA clone of a UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) from grape (Vitis vinifera L.). The heterologous probe contained a glucosyltransferase consensus amino acid sequence which was also present in the Scutellaria cDNA clones. The complete nucleotide sequence of the 1688-bp cDNA insert was determined and the deduced amino acid sequences are presented. The nucleotide sequence analysis of UBGT revealed an open reading frame encoding a polypeptide of 476 amino acids with a calculated molecular mass of 53 094 Da. The reaction product for baicalein and UDP-glucose catalyzed by recombinant UBGT in Escherichia coli was identified as authentic baicalein 7-O-glucoside using high-performance liquid chromatography and proton nuclear magnetic resonance spectroscopy. The enzyme activities of recombinant UBGT expressed in  E. coli were also detected towards flavonoids such as baicalein, wogonin, apigenin, scutellarein, 7,4′-dihydroxyflavone and kaempferol, and phenolic compounds. The accumulation of UBGT mRNA in hairy roots was in response to wounding or salicylic acid treatments. Received: 8 September 1999 / Accepted: 4 October 1999