Characterization of natural variation for zinc, iron and manganese accumulation and zinc exposure response in Brassica rapa L.

Brassica rapa L. is an important vegetable crop in eastern Asia. The objective of this study was to investigate the genetic variation in leaf Zn, Fe and Mn accumulation, Zn toxicity tolerance and Zn efficiency in B. rapa. In total 188 accessions were screened for their Zn-related characteristics in hydroponic culture. In experiment 1, mineral assays on 111 accessions grown under sufficient Zn supply (2 μM ZnSO₄) revealed a variation range of 23.2–155.9 μg g⁻¹ dry weight (d. wt.) for Zn, 60.3–350.1 μg g⁻¹ d. wt. for Fe and 20.9–53.3 μg g⁻¹ d. wt. for the Mn concentration in shoot. The investigation of tolerance to excessive Zn (800 μM ZnSO₄) on 158 accessions, by using visual toxicity symptom parameters (TSPs), identified different levels of tolerance in B. rapa. In experiment 2, a selected sub-set of accessions from experiment 1 was characterized in more detail for their mineral accumulation and tolerance to excessive Zn supply (100 μM and 300 μM ZnSO₄). In this experiment Zn tolerance (ZT) determined by relative root or shoot dry biomass varied about 2-fold. The same six accessions were also examined for Zn efficiency, determined as relative growth under 0 μM ZnSO₄ compared to 2 μM ZnSO₄. Zn efficiency varied 1.8-fold based on shoot dry biomass and 2.6-fold variation based on root dry biomass. Zn accumulation was strongly correlated with Mn and Fe accumulation both under sufficient and deficient Zn supply. In conclusion, there is substantial variation for Zn accumulation, Zn toxicity tolerance and Zn efficiency in Brassica rapa L., which would allow selective breeding for these traits.     

Changes in the total content of iron,copper, and zinc in serum,heart, liver,spleen, and skeletal muscle tissues of rats infected withTrypanosoma cruzi

The effects of theTrypanosoma cruzi infection on the total content of the essential trace elements iron (Fe), copper (Cu), and zinc (Zn) in serum, heart, liver, spleen, and skeletal muscle were determined in “Wistar” rats inoculated with reticulotropic “Y” strain trypanosoms (Tryps) in their slender blood form. The 250 rats were divided in two groups of 80 rats (L-1 and L-2) and one of 90 (C) used as controls. L-1 and L-2 were inoculated with 2×10⁵ and 5×10² Tryps, respectively. Ten rats of the C group were killed the inoculation day (i), and ten rats of each group chosen at random were killed and blood parasitemia determined at 5, 10, 15, 20, 25, 30, 60, and 90 post-i days covering the infection acute-phase myocarditis. Previously cryohomogenized and lyophilized tissues were digested in an HNO₃ ⁻ H₂O₂ mixture with the aid of a microwave oven, and the elements Fe, Cu, and Zn were determined by Flame Atomic Absorption Spectrometry. Generally, more intense changes were observed in the L-1 group. Serum Fe and Zn levels are lower and Cu levels higher in groups L-1 and L-2 than in C. However, Fe is not significantly sequestered in the liver during the acute phase of the infection as expected, but of the tissues studied, the spleen was the main site of Fe binding. Zn tended to increase in all tissues, except in the spleen, where during the acute phase of the infection, the total content of Zn in groups L-1 and L-2 was lower than in group C. Cu increased mainly in the spleen and muscle. In general, each tissue presented its own pattern of redistribution related to its nature, functions, and number of parasites inoculated, and these patterns may have been altered by the tropism of the parasite.     

Blood-brain exchange routes and distribution of65Zn in rat brain

Zinc is essential for normal development and function of the CNS although much is to be learned about brain Zn homeostasis. In these experiments adult male Wistar rats within the weight range 500–600 g were used. Ventriculo-cisternal perfusion was performed to allow the measurement of⁶⁵Zn fluxes between blood and csf across the choroid plexuses. Blood-brain or blood-cerebrospinal fluid barrier permeability to⁶⁵Zn has been determined by graphical analysis in experiments that lasted between 5 and 180 minutes. Cerebral capillary permeability to⁶⁵Zn was found to be low with a K_in of about 5×10^–4ml/min/g. Choroid plexus permeability to⁶⁵Zn was about 12 fold greater, although Zn influx to brain via this route was <5% that across cerebral capillaries. The autoradiographic distribution of⁶⁵Zn in brain showed regional variation with lowest levels in white matter and high levels in the dentate gyrus and hippocampus.     

In silico QSAR studies of anilinoquinolines as EGFR inhibitors

Members of the epidermal growth factor receptor (EGFR) family of proteins are frequently overactive in solid tumors. A relatively new therapeutic approach to inhibit the kinase activity is the use of ATP-competitive small molecules. In silico techniques were employed to identify the key interactions between inhibitors and their protein receptors. A series of EGFR inhibitory anilinoquinolines was studied within the framework of hologram quantitative structure activity relationship (HQSAR), density functional theory (DFT)-based QSAR, and three-dimensional (3D) QSAR (CoMFA/CoMSIA). The HQSAR analysis implied that substitutions at certain sites on the inhibitors play an important role in EGFR inhibition. DFT-based QSAR results suggested that steric and electronic interactions contributed significantly to the activity. Ligand-based 3D-QSAR and receptor-guided 3D-QSAR analyses such as CoMFA and CoMSIA techniques were carried out, and the results corroborated the previous two approaches. The 3D QSAR models indicated that steric and hydrophobic interactions are dominant, and that substitution patterns are an important factor in determining activity. Molecular docking was helpful in identifying a bioactive conformer as well as a plausible binding mode. The docked geometry-based CoMFA model with steric and electrostatic fields effect gave q ² = 0.66, r ² = = 0.94 with r ² _predictive = 0.72. Similarly, CoMSIA with hydrophobic field gave q ² = 0.59, r ² = 0.85 with r ² _predictive = 0.63. Bulky groups around site 3 of ring “C”, and hydrophilic and bulky groups at position 6 of ring “A” are desirable, with a hydrophobic and electron-donating group at site 7 of ring “A” being helpful. Accordingly, potential EGFR inhibitors may be designed by modification of known inhibitors.     

The uptake of zinc by erythrocytes under near-physiological conditions

The in vitro uptake of zinc by erythrocytes was measured under near-physiological conditions, using⁶⁵Zn as a radioactive tracer. Because of the presence of serum albumin—a strong zinc ligand—a low concentration of medium free zinc was maintained. Under these conditions a high-affinity carrier for zinc transport over the cell membrane was identified. With human erythrocytes, a Michaelis constant (K _m ) of 0.2 nM with respect to free medium zinc was measured and aV _max of 4.5 nmoles Zn transported per h/g dry wt. TheK _m for medium Zn increases when the size of the internal erythrocytic Zn pool is augmented, whereasV _max remains virtually unchanged. A model to explain this phenomenon is proposed. It is suggested that this phenomenon could underlie observations, confirmed here, that the in vitro uptake of Zn by animal erythrocytes depends on the Zn status of the animal.     

The chemical fractionation of soil zinc and its specific and total adsorption by Egyptian alluvial soils

Summary The Zn contents of twenty-nine alluvial soils from Egypt were chemically fractionated into: water soluble+exchangeable, weakly bound to inorganic sites, organically bound, occluded as free oxide material, and residual mainly in the mineral structure. On the average these fractions constituted about 0.01, 1.20, 28.6, 21.5 and 45.5% of the total soil Zn respectively which averaged 76.25 ppm. Significant correlations were obtained between each individual Zn-fraction and some soil variable.Zinc adsorption isotherms were developed for seven soils suspended in dilute ZnCl₂ solution in the presence of either 0.05M CaCl₂ solution (Specific adsorption) or deionized water (Total adsorption). The Langmuir constants (adsorption maximum and bonding energy) were calculated. The average value of specific adsorption maximum was 1.94 mg Zn/g soil and of total adsorption maxima was 11.54 mg Zn/g soil. Correlation analysis showed that CEC, free Fe₂O₃ and clay content were the dominant soil variables contributing towards specific Zn adsorption. The (Zn) (OH)₂ ion concentration products in the solutions when Zn adsorption corresponded to the Langmuir adsorption maxima were 0.92×10^–17 in the specific adsorption treatment, and 1.35×10^–15 in the total adsorption treatment. These values are within the solubility range of Zn (OH)₂ and ZnCO₃. The values of Langmuir bonding energy constants showed that Zn was more strongly adsorbed by low carbonate or carbonate-free soils than by carbonate-rich soils.     

Physiological elevations in cytoplasmic free calcium by cold or ion injection result in transient closure of higher plant plasmodesmata

The concentration of cytoplasmic free calcium ([Ca²⁺]_cyt) required to close higher plant plasmodesmata was investigated using corn (Zea mays L. cv. Black Mexican Sweet) suspension-culture cells. Physiological elevations of [Ca²⁺]_cyt were applied by cold treatment, and ion injection was also used to increase [Ca²⁺]_cyt, by diffusion (for small increases) or by iontophoresis (for larger increases). The impact of such treatments on [Ca²⁺]_cyt was measured by ratiometric ion imaging. Intercellular communication during treatments was monitored using our recently developed electrophysiological technique that allows the electrical resistance of plasmodesmata and the plasma membranes of a sister-cell pair to be measured. A 4-fold increase in the calculated resistance of single plasmodesmata was observed in response to cold treatment that caused a 2-fold increase in average [Ca²⁺]_cyt (from 107 to 210 nM). In response to iontophoresis of Ca²⁺, plasmodesmata were observed to go from “open” (low resistance) to “shut” (high resistance) and then back “open” within 10 s. Our results thus indicate that higher plant plasmodesmata respond quickly to physiological changes in [Ca²⁺]_cyt. Received: 2 June 1999 / Accepted: 16 July 1999     

N6-(Δ2-Isopentenyl) adenine inhibits the alcohol dehydrogenase activity in cell-free extracts of Zymomonas mobilis

The plant growth substance N⁶-(²-isopentenyl) adenine (i⁶Ade) significantly inhibits the rates of ethanol oxidation and acetaldehyde reduction in vitro by cell-free extracts of Zymomonas mobilis and of an Escherichia coli recombinant strain, containing the Z. mobilis adhB gene. The two-substrate kinetics of ethanol oxidation (forward) is affected by increasing values of dissociation constants for coenzyme and coenzyme —enzyme complexes in the presence of i⁶Ade, whereas the reaction maximum velocity (V _m) remains unchanged and reflects the competitive type of inhibition. Changes of the kinetic constants of acetaldehyde reduction (back) are similar, except the increasing value of V _m and correspond to the CIS (competitive inhibition and stimulation) type of inhibition. The estimated values of inhibition constants of the forward and back reactions are 0.39 ± 0.05 mM and 0.19 ± 0.06 mM, respectively.     

Depolymerization of hyaluronan by sonication

High molecular weight hyaluronan (M _r 400 000) obtained from human umbilical cord was depolymerized by sonication for 10 h into small molecules and finally into molecules of constant size (M _r 11 000). The molecular size of the depolymerized hyaluronan was unaltered even under different conditions of sonication. After sonication, the main sugar residues at the reducing and non-reducing termini of depolymerized hyaluronan wereN-acetylglucosamine (86%) and glucuronic acid (98%), respectively. Hyaluronans derived from rooster comb (M _r 1×10⁶) andStreptococcus zooepidemicus (M _r 1.2×10⁶) were deploymerized into molecules of different but characteristic sizes by sonication. On the other hand, neither chondroitin sulfate nor glycogen was depolymerized by sonication. These results suggest that high molecular weight hyaluronan may have some weak linkages related toN-acetylglucosamine in the chain, which are extremely sensitive to sonication. At present, however, the nature of these linkages is still unclear.Abbreviations HA hyaluronan - PA 2-aminopyridine     

Determination of isotope ratios in human tissues enriched with zinc stable isotope tracers using inductively coupled plasma-mass spectrometry (ICP-MS)

Enriched fecal and urine samples were prepared using ion-exchange column chromatography for analysis by Inductively Coupled and Fast Atom Bombardment Mass spectrometry (ICP-MS, FAB-MS) to compare precision between methods. Unenriched samples of human milk, feces, and whole blood were prepared similarly to monitor instrumental precision and analytical error. A least squares fit of the ICP-MS results vs the FAB-MS for⁷⁰Zn/⁶⁴Zn gave a slope of 0.98, with a relative standard deviation (RSD) of only 0.7%. The results for⁶⁸Zn/⁶⁴Zn gave a slope of 0.82, with a RSD of 14%. For unenriched tissues, all potential interferences were removed by the preparation procedure with no significant differences between preparation for isotope ratios of⁷⁰Zn/⁶⁴Zn,⁶⁸Zn/⁶⁴Zn,⁶⁷Zn/⁶⁴Zn, and⁶⁶Zn/⁶⁴Zn. Poisson counting statistics are a major contribution to the total analytical error indicating the usefulness of this procedure for enrichment studies.     

High Precision Zinc Isotopic Measurements Applied to Mouse Organs

We present a procedure to measure with high precision zinc isotope ratios in mouse organs. Zinc is composed of 5 stable isotopes (⁶⁴Zn, ⁶⁶Zn, ⁶⁷Zn, ⁶⁸Zn and ⁷⁰Zn) which are naturally fractionated between mouse organs. We first show how to dissolve the different organs in order to free the Zn atoms; this step is realized by a mixture of HNO₃ and H₂O₂. We then purify the zinc atoms from all the other elements, in particular from isobaric interferences (e.g., Ni), by anion-exchange chromatography in a dilute HBr/HNO₃ medium. These first two steps are performed in a clean laboratory using high purity chemicals. Finally, the isotope ratios are measured by using a multi-collector inductively-coupled-plasma mass-spectrometer, in low resolution. The samples are injected using a spray chamber and the isotopic fractionation induced by the mass-spectrometer is corrected by comparing the ratio of the samples to the ratio of a standard (standard bracketing technique). This full typical procedure produces an isotope ratio with a 50 ppm (2 s.d.) reproducibility.     

Gender differences in selected zinc metabolism parameters in patients with mild primary arterial hypertension

The relationship between selected zinc (Zn) metabolism parameters, arterial blood pressure, age, and renin-angiotensin-aldosterone system in subjects of both sexes with mild primary arterial hypertension is presented in this study. The following parameters were measured: systolic and diastolic arterial blood pressure, total and ouabain-dependent efflux rate constants of Zn from lymphocytes, serum and lymphocyte Zn concentrations, serum aldosterone, angiotensin-converting enzyme, sodium and potassium concentrations, body mass index, and plasma rennin activity. When all subjects are taken into account, no significant age-related differences were found for serum Zn. If divided into men and women, negative (r=−0.39) and positive (r=0.34) correlations are observed, respectively. Lymphocyte Zn correlated negatively with age in the entire group (r=−0.55) and also for men (r=−0.54) and women (r=−0.57). The renin-agiotensin-aldosterone system parameters correlated with those of Zn metabolism only for women: plasma rennin activity with total Zn efflux from lymphocytes (r=−0.33) and with lymphocyte Zn (r=0.71); the angiotensin-converting enzyme with total Zn efflux from lymphocytes (r=−0.35), with the oubain-dependent Zn efflux from lymphocytes (r=−0.33) and with lymphocyte Zn (r=0.57); serum aldosterone with oubain-dependent Zn efflux from lymphocytes (r=−0.44) and with lymphocyte Zn (r=0.59). For the men, the only positive correlation was that of serum Zn and aldosterone (r=0.45). In all cases (men and women), there was no negative correlation between serum Zn and angiotensin-converting enzyme. In women, the diastolic blood pressure correlated negatively with total Zn efflux from lymphocytes (r=−0.39), oubain-dependent Zn efflux from lymphocytes (r=−0.49), and serum Zn (r=−0.46); systolic blood pressure correlated negatively with lymphocyte zinc (r=−0.38). In men, the systolic blood pressure had a negative correlation with lymphocyte zinc (r=−0.32), which was also true for the entire group (r=−0.34). These results clearly show gender-related differences in Zn metabolism and indicate the need for further research to elucidate the possible causes of this phenomenon not only for Zn but for other elements as well.     

Carbon fixation and carbonic anhydrase activity in Haslea ostrearia (Bacillariophyceae) in relation to growth irradiance

The metabolic pathway of primary carbon fixation was studied in a peculiar pennate marine diatom, Haslea ostrearia (Bory) Simonsen, which synthesizes and accumulates a blue pigment known as “marennine”. Cells were cultured in a semi-continuous mode under saturating [350 μmol(photon) m⁻² s⁻¹] or non-saturating [25 μmol(photon) m⁻² s⁻¹] irradiance producing “blue” (BC) and “green” (GC) cells, characterized by high and low marennine accumulation, respectively. Growth, pigment contents (chlorophyll a and marennine), ¹⁴C accumulation in the metabolites, and the carbonic anhydrase (CA) activity of the cells were determined during the exponential growth phase. Growth rate and marennine content were closely linked to irradiance during growth: higher irradiance increased both growth rate and marennine content. On the other hand, the Chl a concentration was lower under saturating irradiance. The distribution between the Calvin-Benson (C₃) and β-carboxylation (C₄) pathways was very different depending on the irradiance during growth. Metabolites of the C₃ cycle contained about 70 % of the total fixed radioactivity after 60 s of incorporation into cells cultured under the non-saturating irradiance (GC), but only 47 % under saturating irradiance (BC). At the same time, carbon fixation by β-carboxylation was 24 % in GC versus about 41 % in BC, becoming equal to that in the C₃ fixation pathway in the latter. Internal CA activity remained constant, but the periplasmic CA activity was higher under low than high irradiance.     

Concentrations of cadmium,lead, selenium,and zinc in human blood and seminal plasma

The concentrations of cadmium, lead, selenium, and zinc in blood and seminal plasma were determined in 76 Singapore males. Except for zinc, the concentrations were generally higher in blood than in seminal plasma (cadmium, 1.31 μg/L vs 0.61 μg/L; lead, 82.6 μg/L vs 12.4 μg/L, and selenium, 163.6 μg/L vs 71.5 μg/L). The mean concentration of zinc in seminal plasma was more than 30 times higher than in blood (202 mg/L vs 6.2 mg/L). Significant positive correlations were found between the concentrations in blood and seminal plasma for the two essential trace elements: selenium (r=0.45,p<0.001) and zinc (r=0.25,p<0.05). However, no relationships were found between the concentrations in blood and seminal plasma for two toxic metals (cadmium and lead). Significant inverse correlations were observed between Cd and Zn (r=−0.40,p<0.01), and Pb and Se (r=−0.32,p<0.05) in blood, whereas significant positive correlations were noted between Cd and Se (r=0.45,p<0.01), Cd and Zn (r=0.35,p<0.05), and Se and Zn (r=0.57,p<0.001) in seminal plasma. The physiological significance of these relationships are also discussed in this paper.     

Palatine fenestrae,the orangutan and hominoid evolution

Although most mammals develop relatively large double anterior palatine fenestrae that patently communicate with the nasal cavity, four extant primates—Homo sapiens, Pongo, Pan andGorilla—do not. While these four have closed-down these foramenal structures,Homo sapiens andPongo are unique in forming a single foramen palatally. Among fossil taxa,Homo, Australopithecus, Sivapithecus (=Ramapithecus) andRudapithecus also develop a single foramen palatally. Dryopithecines, the presumed fossil apes, preserve the two patent fenestrae. In light of dental features that are considered diagnostically “hominid,” which are also found in the orangutan, it is suggested that this “ape,” rather thanPan, is phylogenetically closer toHomo.     

Low voltage-activated Ca2+ conductances in thalamic neurons

Low voltage-activated (LVA) Ca²⁺ conductances were characterized in the neurons of the associative laterodorsal (LD) thalamic nucleus in rat brain slices and in enzymatically isolated thalamic units using electrophysiological techniques. Voltage dependence, kinetics of inactivation, pharmacology, and selectivity of the LVA current in the thalamic neurons from animals older than 14 postnatal days were consistent with the existence of two, “fast” and “slow,” subtypes of LVA Ca²⁺ channels. “Slow” LVA current in enzymatically isolated thalamic neurons was much less prominent, compared with that in slice neurons, suggesting that respective channels are predominatly located on the distal dendrites. “Fast” Ca²⁺ channels were sensitive to nifedipine (K _d−2.6 μM) and La³⁺ (K _d−1.0 mM), whereas “slow” Ca²⁺ channels were sensitive to Ni²⁺ (25 μM). Selectivity of the “fast” Ca²⁺ channels was similar to that found for the LVA Ca²⁺ channels in other preparations (I _Ca:I _Sr:I _Ba−1.0: 1.23: 0.94), while selectivity of the “slow” Ca²⁺ channels more resembled selectivity of the HVA Ca²⁺ channels (I _Ca:I _Sr:I _Ba−1.0: 2.5: 3.4).     

Fast activation of a time-dependent outward current in protoplasts derived from coats of developing Phaseolus vulgaris seeds

An outward current that appeared to activate instantaneously in response to depolarising voltage pulses at low sampling frequencies predominated in the plasma membrane of ground-parenchyma protoplasts derived from coats of developing Phaseolus vulgaris L. (cv. Redland Pioneer) seeds. However, the outward current showed time-dependent activation when higher sampling frequencies were used to measure the current. Activation of the current was best described as a double-exponential time course with the fast and slow time constants being 1 and 20 ms, respectively. The current also exhibited a rapid deactivation that followed a double-exponential time course with time constants of approximately 2 and 30 ms, respectively. “Tail-current” analysis allowed us to show that this current exhibited a low selectivity between K⁺ and Cl⁻ (P _K:Cl=1.8). Such a fast-activating current may account for some of the reports of time-independent, instantaneous currents that have been observed in plasma membranes of plant cells digitised at low sampling frequencies. Therefore, when “instantaneous” currents appear it is advisable to characterise these currents using higher sampling frequencies with correspondingly higher filtering frequency cut-offs. Received: 12 May 2000 / Accepted: 26 June 2000     

Computer simulation of Zn(II) speciation and effect of Gd(III) on Zn(II) speciation in human blood plasma

The speciation and distribution of Zn(II) and the effect of Gd(III) on Zn(II) speciation in human blood plasma were studied by computer simulation. The results show that, in normal blood plasma, the most predominant species of Zn(II) are [Zn(HSA)] (58.2%), [Zn(IgG)](20.1%), [Zn(Tf)] (10.4%), ternary complexes of [Zn(Cit)(Cys)] (6.6%) and of [Zn(Cys)(His)H] (1.6%), and the binary complex of [Zn(Cys)₂H] (1.2%). When zinc is deficient, the distribution of Zn(II) species is similar to that in normal blood plasma. Then, the distribution changes with increasing zinc(II) total concentration. Overloading Zn(II) is initially mainly bound to human serum albumin (HSA). As the available amount of HSA is exceeded, phosphate metal and carbonate metal species are established. Gd(III) entering human blood plasma predominantly competes for phosphate and carbonate to form precipitate species. However, Zn(II) complexes with phosphate and carbonate are negligible in normal blood plasma, so Gd(III) only have a little effect on zinc(II) species in human blood plasma at a concentration above 1.0×10⁻⁴ M.     

Geochemical evidence for the formation of a large miocene “travertine” mound at a sublacustrine spring in a soda lake (Wallerstein castle rock, nördlinger ries, Germany)

Summary “Travertines” (tufa pinnacles) of the Miocene Riescrater basin have been investigated to test whether carbon, oxygen and strontium isotopes can be used for the recognition of fossil subaquatic spring deposits in high-alkalinity settings. The Ries basin “travertines” have so far been interpreted as a product of subaerial to sublacustrine artesian springs discharging calcareous groundwater into a freshwater or slightly saline lake. However, recent studies on microfacies and fabric development propose a formation at Ca²⁺-supplying sublacustrine springs of a soda lake. Geochemical analysis of “travertines” of the castle rock Wallerstein, including “sickle-cell” limestones, thrombolites, non-skeletal stromatolites, and speleothems, now support the latter interpretation. High Sr contents surpassing that of the contemporaneously formed dolomitic algal biocherms of the lake shore point to an aragonitic composition of primary precipitates. the δ¹³C values of diagenetically moderately to weakly altered “travertine” facies types are in the same range of the impact-brecciated Upper Jurassic limestones, thus, are inconsistent with a mixture of soil-derived CO₂ and CO₃ ²⁻ from the Jurassic limestones. In addition, the δ¹⁸O values are too high to support a significant contribution of CO₃ ²⁻ by meteoric waters seeping through marine Jurassic limestones. Instead the δ¹³C and δ¹⁸O values indicate an origin of the CO₃ ²⁻ from a lake water body characterized by evaporation. This is consistent with a sodium-rich lake water as indicated by high sodium contents of aragonitic algal bioherms of the lake shore. The⁸⁷Sr/⁸⁶Sr isotope ratio of the “travertine” mound carbonates are consistent with calculated mixing of spring waters discharging from the crystalline basement and lake water high in dissolved inorganic carbon. This points to an origin of the divalent cations from sublacustrine spring waters. In turn,⁸⁷Sr/⁸⁶Sr isotope ratios of green algal reef carbonates of the lake shore are closer to that of the Upper Jurassic carbonates, due to surface run-off from surrounding limestone uplands.     

A polarographic, oxygen-selective, vibrating-microelectrode system for the spatial and temporal characterisation of transmembrane oxygen fluxes in plants

 A simple procedure is described for the fabrication of micrometer to nanometer-scale platinum electrodes to be used in a vibrating oxygen-selective system. The electrode was prepared by etching a fine platinum wire and insulating it with an electrophoretic paint. The dimensions allowed this electrode to be used with the “vibrating probe technique” in exploratory studies aimed at mapping and measuring the patterns of net influxes as well as effluxes of oxygen in Olea europaea L. leaves and roots with spatial and temporal resolutions of a few microns and a few seconds, respectively. The magnitude and spatial localisation of O₂ influxes in roots was characterised by two distinct peaks. The first, in the division zone, averaged 38 ± 5 nmol m⁻² s⁻¹; the second, in the elongation region, averaged 68 ± 6 nmol m⁻² s⁻¹. Long-term records of oxygen influx in the elongation region of the root showed an oscillatory regime characterised by a fast oscillation with periods of about 8–9 min. In leaves, the system allowed the measurement of real-time changes in O₂ evolution following changes in light. Furthermore, it was possible to obtain “topographical” images of the photosynthetically generated oxygen diffusing through different stomata from a region of the leaf of 120 μm × 120 μm. The combination of topographic and electrochemical information at the micrometer scale makes the system an efficient tool for studying biological phenomena involving oxygen diffusion. Received: 12 November 1999 / Accepted: 1 February 2000