Variations of glycogen: I. Following stimulation of Knollenorgan sensory cells,a lateral line electroreceptor of mormyrid fish

The metabolism of glycogen was studied in sensory cells of the mormyrid fish, Gnathonemus petersii. Knollenorgans, specific cutaneous electroreceptor organs of the lateral line system, have a spontancous electrical activity and their resting discharge in the absence of stimulation is about 0.04 kHz. Various types of stimulation can produce an increase in frequency; the highest frequency (1.30 kHz) is obtained by moving the Knollenorgan above water level. Glycogen was visualized in ultrathin sections after fixation in a solution of potassium ferricyanide and osmium tetroxide. The density of glycogen particles was determined morphometrically in sensory cells before stimulation, after high-frequency activity, and after reimmersion in water. An increase in the electrical activity of the Knollenorgan resulted in a decrease of the glycogen content of sensory cells. The glycogen store was replenished to about 85% of control within 40 min after stimulation and subsequent reimmersion. The results demonstrate that glycogen in the sensory cells of the Knollenorgan represents an energy source which can be catabolized during high electrical activity and replenished during rest.     

Soil humic substances affect transport properties of tonoplast vesicles isolated from oat roots

The effect of a low molecular size (<5 KDa) humic fraction, essentially fulvic acids, on microsomal and tonoplast ion-stimulated ATPase activity was studied. After 20 min of pre-incubation with microsomal vesicles from oat roots, humic substances at organic C concentration of up to 0.5 μg cm^-3 increased KCl-stimulated ATPase activity, while they inhibited enzyme activity at higher concentrations. Cl^--stimulated ATPase activity of tightly sealed tonoplast-enriched vesicles was similarly affected by <5 KDa humic substances. This behaviour was not observed when gramicidin D was added to the assay medium. Proton transport by vesicles incubated up to 5 min with <5 KDa humic molecules was affected in a concentration-dependent manner, strongly resembling that observed for ATP hydrolysis, whereas it was severely reduced when the assay conditions were close to those used for measuring ATP hydrolysis (20 min pre-incubation of vesicles with humic substances). The transmembrane electrical potential was negatively affected, irrespective of the concentration of humic molecules. Furthermore, a 15-min pre-incubation strongly reduced the formation of a potential gradient. The size and concentrations of humic substances employed make an interaction with the vacuolar membrane of root cells plausible. The results show that the main target of humic molecules is the electrical membrane potential and suggest a possible way of interference of these naturally occurring substances with the biochemical mechanisms involved in plant mineral nutrition.     

复合菌剂调控连作当归根围土壤养分及对产量的影响

【背景】连作可引起微生物群落结构失调,导致土壤环境恶化、养分循环不畅、当归[Angelica sinensis (Oliv.) Diels]产量降低,通过现代微生物技术改良土壤、消减连作障碍势在必行。【目的】于大田条件下,研究施用复合菌剂对当归根围土壤酶活、速效养分及产量的影响,明确增产机制,改进增产措施。【方法】利用溶磷圈法检测不同菌株溶磷活性、乙炔还原法检测固氮活性、试剂盒法检测过氧化物酶和硝化能力;复合菌剂T1[荧光假单胞菌(Pseudomonas fluorescens)CBS5、产碱假单胞菌(Pseudomonas alcaligenes) CBS7、嗜冷假单胞菌(Pseudomonas extremaustralis)CBSB、生枝动胶菌(Zoogloea ramigera) CBS4]和T2 (荧光假单胞菌CBS5、产碱假单胞菌CBS7、嗜冷假单胞菌CBSB)及对照CK (无菌马铃薯葡萄糖肉汤培养基)分别处理连作当归,分光光度法测定根围土壤及根中养分循环、转化相关酶活,氮、磷、钾速效养分含量;常规方法测产量;统计软件进行相关数据方差分析和主成分分析。【结果】产碱假单胞菌C...     

Mathematical Modeling of Heterogeneous Electrophysiological Responses in Human β-Cells

Electrical activity plays a pivotal role in glucose-stimulated insulin secretion from pancreatic -cells. Recent findings have shown that the electrophysiological characteristics of human -cells differ from their rodent counterparts. We show that the electrophysiological responses in human -cells to a range of ion channels antagonists are heterogeneous. In some cells, inhibition of small-conductance potassium currents has no effect on action potential firing, while it increases the firing frequency dramatically in other cells. Sodium channel block can sometimes reduce action potential amplitude, sometimes abolish electrical activity, and in some cells even change spiking electrical activity to rapid bursting. We show that, in contrast to L-type -channels, P/Q-type -currents are not necessary for action potential generation, and, surprisingly, a P/Q-type -channel antagonist even accelerates action potential firing. By including SK-channels and dynamics in a previous mathematical model of electrical activity in human -cells, we investigate the heterogeneous and nonintuitive electrophysiological responses to ion channel antagonists, and use our findings to obtain insight in previously published insulin secretion measurements. Using our model we also study paracrine signals, and simulate slow oscillations by adding a glycolytic oscillatory component to the electrophysiological model. The heterogenous electrophysiological responses in human -cells must be taken into account for a deeper understanding of the mechanisms underlying insulin secretion in health and disease, and as shown here, the interdisciplinary combination of experiments and modeling increases our understanding of human -cell physiology.     

The influence of plasma membrane ion permeability modulators on superoxide formation and bioelectrical characteristics of wheat root cells

Changes in superoxide radical formation and bioelectrical characteristics of excised wheat root cells under modification of plasma membrane ion permeability were studied. It was shown that a 2 h treatment of excised roots with valinomycin (Val, 20 microM), N, N'-dicyclohexylcarbodimide (DCCD, 100 microM), gramicidin S (Gr, 20 microM), chlorpromazine (CPZ, 100 microM) caused an increased loss of potassium by cells, lowering of membrane potential (MP) and electrical input resistance (Rin) of the cells. The superoxide formation by excised root cells diminished (under DCCD) or remained at the control level (under Val), which was accompanied by a minor decrease of MP and Rin of the cells, a small increase in potassium loss by excised roots, and in no change of pH of incubation medium. Significant depolarization of plasma membrane, dropping of Rin and essential loss of potassium ions by the cells correlated with a rise in the medium alkalinization and superoxide formation by excised roots (in the presence of Gr, CPZ). Ion channel blocker gadolinium (Gd3+, 200 microM) caused an increase of MP and Rin reduction of potassium loss by cells, and a decrease of pH of the incubation medium, and also enhancement of superoxide formation by excised root cells. It is suggested that upon plasma membrane ion permeability modification the activity of superoxide generating systems depends on the specificity and mechanisms of action of modulators, and is determined by their influence on redox state of plasma membrane as well as by peculiarities of ion transport disturbance.     

Responses of neuroblastoma cells to iontophoretically applied acetylcholine

Dissociated mouse neuroblastoma cells were studied in vitro by using intracellular microelectrodes for electrical stimulation and recording. Some, but not all cells, which exhibited well developed action potentials to electrical stimulation also showed changes in membrane potential to iontophoretically applied acetylcholine (ACh). The types of responses to ACh varied. Short latency depolarizing responses to pulses of ACh (similar to those obtained with skeletal muscle) as well as sustained depolarization to steady ACh application (D response) occurred. A longer latency prolonged hyperpolarizing response (H response) and bi- and triphasic combinations of H and D responses were also seen. Pairs of cells showing morphologic contact were tested for the occurrence of effective synaptic coupling by placing intracellular microelectrodes in each cell. In none of 95 cases tested did spike activity produced by direct electrical stimulation of one cell elicit a synaptic potential of 200 μv or more in the other.     

Characterization of chloroplast psbA transformants of Chlamydomonas reinhardtii with impaired processing of a precursor of a photosystem II reaction center protein,D1

One of the photosystem II reaction center proteins, D1, is encoded by the psbA gene and is synthesized as a precursor form with a carboxyl-terminal extension that is subsequently cleaved between Ala-344 and Ser-345. We have generated three psbA transformants of the green alga Chlamydomonas reinhardtii in which Ala-344 or Ser-345 have been substituted with Pro or Glu (A344P, S345E, and S345P) to understand the effects of the amino acid substitutions on the processing of the precursor D1. S345E grew photoautotrophically and showed PSII activity like the wild type. However, A344P and S345P were unable to grow photoautotrophically and were significantly photosensitive. A344P was deficient in the processing of precursor D1 and in oxygen-evolving activity, but assembled photosystem II complex capable of charge separation. In contrast, both precursor and mature forms of D1 accumulated in S345P cells from the logarithmic phase and the cells evolved oxygen at 18% of wild-type level. However, S345P cells from the stationary phase contained mostly the mature D1 and showed a twofold increase in oxygen-evolving activity. The rate of processing of the accumulated pD1 was estimated to be about 100 times slower than in the wild type. It is therefore concluded that the functional oxygen-evolving complex is assembled when the precursor D1 is processed, albeit at a very low rate. These results suggest the functional significance of the amino acid residues at the processing site of the precursor D1.     

Potassium Translocation into the Root Xylem

Abstract: Potassium is the most abundant cation in cells of higher plants and plays vital roles in plant growth and develop ment. Since the soil is the only source of potassium, plant roots are well adapted to exploit the soil for potassium and supply it to the leaves. Transport across the root can be divided into three stages: uptake into the root symplast, transport across the symplast and release into the xylem. Uptake kinetics of potassium have been studied extensively in the past and sug gested the presence of high and low affinity systems. Molecular and electrophysiological techniques have now confirmed the existence of discrete transporters encoded by a number of genes. Surprisingly, detailed characterisation of the transpor ters using reverse genetics and heterologous expression shows that a number of the transporters (AKT and AtKUP family) func tion both in the low (μM) and high (mM) K⁺ range. Electrophy siological studies indicate that K⁺ uptake by roots is coupled to H⁺, to drive uptake from micromolar K⁺. However, thus far only Na⁺ coupled K⁺ transport has been demonstrated (HKT1). Ion channels play a major role in the exchange of potassium be tween the symplast and the xylem. An outward rectifying chan nel (KORC) mediates potassium release. Cloning of the gene en coding this channel (SKOR) shows that it belongs to the Shaker super-family. Both electrophysiological and genetic studies demonstrate that K⁺ release through this channel is controlled by the stress hormone abscisic acid. Interestingly, xylem par enchyma cells of young barley roots also contain a number of in ward rectifying K⁺ channels that are controlled by G-proteins. The involvement of G-proteins emphasises once more that po tassium transport at the symplast/xylem boundary is under hor monal control. The role of the electrical potential difference across the symplastxylem boundary in controlling potassium release is discussed.     

Synthesis of water-soluble ruthenium porphyrins as DNA cleavers and potential cytotoxic agents

 Three new water-soluble ruthenium porphyrin complexes have been prepared and characterized, one with a cationic ligand, Ru(TMPyP), and two others with anionic ligands, Ru(p–COOH-PP) and Ru(TPPS). These different complexes and their manganese and iron analogues were tested in vivo as potential antitumor agents with mice bearing P388 leukemia cells, but these complexes have no significant antitumor activity. The nuclease activity of Ru(TMPyP) and Ru(p–COOH-PP) was evaluated on supercoiled plasmid DNA after activation by a reducing agent (ascorbate) in the presence of air or by potassium monopersulfate. No significant activity was evidenced for these ruthenium complexes, in contrast with the already known nuclease activity of the manganese and iron derivatives of TMPyP. Received: 15 November 1996 / Accepted: 7 April 1997     

An Electrophysiological Study of the Stomatal Complex of Tradescantia virginiana

Measurements of electrical potential difference (PD) and potassiumactivity were carried out on the intact leaf of Tradescantiavirginiana. PD gradients across the stomatal complex were observedwith both open and closed stomata. The guard cell PD appearedto be linearly related to stomatal aperture. With the stomataopen a gradient of potassium activity across the stomatal complexwas observed which became reversed on stomatal closure. Calculationof the driving forces on potassium suggested that it was distributedpassively between the vacuoles of the cells of the stomatalcomplex. The electrophysiological data obtained from this investigationenabled potassium activity in the apoplast to be calculated.The results showed that on stomatal closure there was a massiveincrease in the potassium activity in the guard cell wall. Key words: Stomata, Ionic gradients, Electrical potentials     

Modeling of Rhythmogenesis in an Ensemble of Cortical Neurons Connected with Electrical Synapses

Based on our own data on generation of spindle-like field electrical activity in neuronal barrels of the rat somatic cortex and also on the published data on the properties of voltage-dependent channels in the membranes of cortical cells, we developed a model of the ensemble (simple network) of neurons connected by electrical synapses. Such connections were found earlier in neurophysiological and ultramicroscopic studies. Model neurons with membranes having sodium, potassium, and calcium channels described in the literature were capable of generating bursting rhythmic impulse activity under conditions of switching off of synaptic connections between cells (isolation). With switching on of electrical synapses, spiking generated by separate neurons, which initially was nonsynchronous, became synchronized in time. Ipso facto, we demonstrated the ability of pacemaker oscillatory activity to be electrotonically synchronized in ensembles of neurons connected with electrical synapses.     

Mechanism‐based inactivation of cytochrome P450 2D6 by chelidonine

Chelidonine (CHE) is a major bioactive constituent of greater celandine, a plant used in traditional herbal medicines. CHE has widely been used as an analgesic in clinical settings. We evaluated the inhibitory effects of CHE on human cytochrome P450 enzymes. CHE produced time‐, concentration‐, and NADPH‐dependent inhibition of CYP2D6, with K _I and k _inact values of 20.49 μM and 11.05 min ^?1, respectively. Approximately 76% of CYP2D6 activity was suppressed after 9 minute incubation with CHE (50 μM). The loss of enzyme activity was not restored following dialysis. The estimated partition ratio of the inactivation was about 156. Quinidine, a competitive inhibitor of CYP2D6, attenuated the CHE‐mediated enzyme inactivation, while glutathione and catalase/superoxide dismutase did not markedly ameliorate the inhibitory effect. Upon oxidation using potassium ferricyanide, the 15.1% activity of CYP2D6 was restored. These findings indicate that CHE acted as a mechanism‐based inactivator of CYP2D6 and the observed effects may induce potential drug‐drug interactions.     

Mutations in the Kv1.5 channel gene KCNA5 in cardiac arrest patients

Mutations in one of the ion channels shaping the cardiac action potential can lead to action potential prolongation. However, only in a minority of cardiac arrest cases mutations in the known arrhythmia-related genes can be identified. In two patients with arrhythmia and cardiac arrest, we identified the point mutations P91L and E33V in the KCNA5 gene encoding the Kv1.5 potassium channel that has not previously been associated with arrhythmia. We functionally characterized the mutations in HEK293 cells. The mutated channels behaved similarly to the wild-type with respect to biophysical characteristics and drug sensitivity. Both patients also carried a D85N polymorphism in KCNE1, which was neither found to influence the Kv1.5 nor the Kv7.1 channel activity. We conclude that although the two N-terminal Kv1.5 mutations did not show any apparent electrophysiological phenotype, it is possible that they may influence other cellular mechanisms responsible for proper electrical behaviour of native cardiomyocytes.     

Growth and viability of mycorrhizal extraradical mycelia associated with three temperate orchid species

Growth and enzymatic activities of extraradical mycelia (ERM) of native mycorrhizal symbionts associated with three orchid species, Dactylorhiza fuchsii, D. majalis and Platanthera bifolia, were studied. ERM extracted from the mycorrhizosphere of these species showed features typical for fungi that form orchid mycorrhiza. In the first pot experiment, three different treatments were applied on tubers of D. fuchsii transplanted from a natural site: control (no specific treatment), reinoculated (surface-sterilized tubers reinoculated with mycorrhizal fungi-colonised roots), and benomyl (nonsterilized tubers treated with fungicide). However, no significant differences in ERM growth and intensity of root mycorrhizal colonisation at harvest were observed among these treatments. ERM associated with reinoculated D. fuchsii plants showed significantly higher alkaline phosphatase (ALP) enzymatic activity at week 36 than at week 24, but no differences were observed for NADH diaphorase activity. Benomyl application significantly reduced ALP activity in comparison with reinoculated plants at week 36. In the second experiment, plants of all three species were either untreated (control), or repeatedly treated with benomyl. Similarly to the results of the first experiment, benomyl application did not reduce the ERM growth of mycorrhizal symbionts associated with D. majalis and D. fuchsii. The low ERM growth associated with benomyl-treated P. bifolia was probably caused by poor root system development in this treatment. Significantly higher mycorrhizal colonisation was found for D. fuchsii compared to P. bifolia in control treatments at the end of cultivation. The ERM of native symbionts of the three orchid species studied seemed to have a different growth pattern over time and responded differently to fungicide application.     

Non-destructive measurement of acid phosphatase activity in the rhizosphere using nitrocellulose membranes and image analysis

We developed a method using nitrocellulose membranes and image analysis to localise and quantify acid phosphatase activity in the rhizosphere of two plant species, one with cluster roots (Dryandra sessilis (Knight) Domin) and another with ectomycorrhizal roots (Pinus taeda L.). Membranes were placed in contact with roots and then treated with a solution of x, α-naphthyl phosphate and Fast Red TR. Acid phosphatase activity was visualised as a red imprint on the membrane. We quantified acid phosphatase activity by image analysis of scanned imprints. The method was used to estimate the spatial distribution of acid phosphatase activity within particular root classes (lateral roots, mycorrhizal roots, root clusters). Over 95% of the acid phosphatase activity of the root system of D. sessilis was associated with cluster roots, and between 20 and 32% of the root surface active. About 26 % of the acid phosphatase activity of the root system of P. taeda was associated with mycorrhizal roots and unsuberised white root tips and less than 10% of the root surface was active, irrespective of root type. This non-destructive method can be used for rapid, semi-quantitative assessment of acid phosphatase activity in the laboratory and in situ. This revised version was published online in August 2006 with corrections to the Cover Date.     

Colchicine modulates calcium homeostasis and electrical property of HL‐1 cells

Colchicine is a microtubule disruptor that reduces the occurrence of atrial fibrillation (AF) after an operation or ablation. However, knowledge of the effects of colchicine on atrial myocytes is limited. The aim of this study was to determine if colchicine can regulate calcium (Ca²⁺) homeostasis and attenuate the electrical effects of the extracellular matrix on atrial myocytes. Whole‐cell clamp, confocal microscopy with fluorescence, and western blotting were used to evaluate the action potential and ionic currents of HL‐1 cells treated with and without (control) colchicine (3 nM) for 24 hrs. Compared with control cells, colchicine‐treated HL‐1 cells had a longer action potential duration with smaller intracellular Ca²⁺ transients and sarcoplasmic reticulum (SR) Ca²⁺ content by 10% and 47%, respectively. Colchicine‐treated HL‐1 cells showed a smaller L‐type Ca²⁺ current, reverse mode sodium–calcium exchanger (NCX) current and transient outward potassium current than control cells, but had a similar ultra‐rapid activating outward potassium current and apamin‐sensitive small‐conductance Ca²⁺‐activated potassium current compared with control cells. Colchicine‐treated HL‐1 cells expressed less SERCA2a, total, Thr17‐phosphorylated phospholamban, Cav1.2, CaMKII, NCX, Kv1.4 and Kv1.5, but they expressed similar levels of the ryanodine receptor, Ser16‐phosphorylated phospholamban and Kv4.2. Colchicine attenuated the shortening of the collagen‐induced action potential duration in HL‐1 cells. These findings suggest that colchicine modulates the atrial electrical activity and Ca²⁺ regulation and attenuates the electrical effects of collagen, which may contribute to its anti‐AF activity.     

Effects of hypothalamic peptides on electrical activity and membrane currents of 'patch perforated' clamped GH3 anterior pituitary cells.

'Perforated-patch' recordings of rat anterior pituitary GH3 cells allow long and stable monitoring of electrical activity and membrane currents. Under current clamp conditions, the biphasic effect of thryotropin releasing hormone (TRH) consisting of a transient hyperpolarization followed by a longer phase of increased action potential frequency is fully preserved. Somatostatin suppresses action potential activity and antagonizes the second phase of enhanced spiking caused by TRH. Voltage clamp records of isolated currents indicate that TRH affects calcium-dependent potassium currents, but does not alter either voltage-dependent potassium or calcium currents at times and concentrations at which the electrical activity is increased.     

Patch clamp analysis of the dominant plasma membrane K+ channel in root cell protoplasts of Plantago media L. Its significance for the P and K state

Ion channels in the plasma membrane of root cell protoplasts of Plantago media L. were studied with the patch clamp technique in the cell-attached patch and outside-out patch configuration. An outward rectifying potassium channel was dominantly present in the plasma membrane. It appears responsible for the diffusional part, dominated by the K⁺ diffusion potential, of the cell membrane potential, in vivo. This channel is activated at potentials near to and more positive than the K⁺ diffusion potential. The dependence of this ion channel on K⁺ activity and voltage has been characterized. The current-voltage relationships of the open channel at various K⁺ concentrations are described by a four-state model. The membrane potential of intact protoplasts appears either dominated by the K⁺ diffusion potential, the protoplast is then said to be in the K state, or by the pump potential generated by the plasma membrane-bound proton pump/H⁺ ATPase, the P state. An experimental procedure is described to determine in cell-attached patch mode the state of the protoplast, either K or P state.Institution paper no.: ECOTRANS publication no. 45.     

Electrogenic K+-basic amino-acid cotransport in the midgut of lepidopteran larvae

Summary Experiments performed on isolated midgut demonstrate that the model proposed for the absorption of neutral amino acids in the K⁺-transporting intestinal epithelium of lepidopteran larvae applies also to the transport of the basic amino acids histidine and lysine. The characteristics of these K⁺-basic amino-acid cotransports have been studied in brush-border membrane vesicles. Histidine and lysine are transported by different transport agencies, which share, to a different degree, a high sensitivity to transmembrane electrical potential difference. Kinetic analysis showed thatK _m for histidine and lysine increased 10-fold and three-fold, respectively, whereasV _max was only slightly modified when the electrical potential difference was abolished. The relationship between potassium concentration and histidine uptake indicates a cooperative binding of more than one potassium to the transporter. Countertransport experiments with glutamine as elicitor show that histidine and glutamine are transported through the same system.     

Measurements of Electrical Potential Differences on Single Nephrons of the Perfused Necturus Kidney

Stable electrical potential differences can be measured by means of conventional glass microelectrodes across the cell membrane of renal tubule cells and across the epithelial wall of single tubules in the doubly perfused kidney of Necturus. These measurements have been carried out with amphibian Ringer's solution, and with solutions of altered ionic composition. The proximal tubule cell has been found to be electrically asymmetrical inasmuch as a smaller potential difference is maintained across the luminal cell membrane than across the peritubular cell boundary. The tubule lumen is always electrically negative with respect to the peritubular extracellular medium. Observations on the effectiveness of potassium ions in depolarizing single tubule cells indicate that the transmembrane potential is essentially an inverse function of the logarithm of the external potassium concentration. The behavior of the peritubular transmembrane potential resembles more closely an ideal potassium electrode than that of the luminal transmembrane potential. From these results, and the effects of various ionic substitutions on the electrical profile of the renal tubular epithelium, a thesis concerning the origin of the observed potential differences is presented. A sodium extrusion mechanism is considered to be located at the peritubular cell boundary, and reasons are given for the hypothesis that the electrical asymmetry across the proximal renal tubule cell could arise as a consequence of differences in the relative sodium and potassium permeability at the luminal and peritubular cell boundaries.