Antiviral Activity of the Glucan Lichenan (Poly-β(l→3, 1→4)D-anhydroglucose)

Lichenan is a linear glucan of (1→3, 1→4)-β-glycosidic bonds in a ratio cf 1:2 that originates from the lichen Cetraria islandica . Lichenan at 5–1000 μg/ml exhibited antiviral activity against mechanically-transmitted viruses of different taxonomic groups. The antiviral activity was demonstrated by the inhibition of symptom development and virus accumulation in greenhouse-grown Nicotiana tabacum (four cultivars), N. benthamiana and N. glutinosa . In plants of N. tabacum cv. "Xanthi-nc", which react hypersensitively in response to TMV infection, lichenan reduced the number, but not the size, of necrctic lesions both, when added to purified virions or naked viral RNA. Partly hydrolized lichenan representing different degrees of polymerization (DP) of 6–30, 30–60, 60–190, and > 190, reduced the number of local and systemic infections to the same degree as the unchanged glucan. The antiviral effect of lichenan was restricted to the leaf-area of application. Depending on the time of application, the strongest effect was achieved when lichenan was added directly to the inoculum. Post-infectionally, it was inhibitory only up to 3 h post-inoculation, indicating that early events of virus replication were affected. Viral inhibition caused by lichenan results frotn a plant-specific protection, which does not function in N. rustica .
Although less inhibitory than lichenan, there were other mixed-linkage β-glticans (schizophylian, pacbytnan, laminaran, barley-β-glucan) causing antiviral effects. In contrast, pustulan as well as glucans of the a-coniiguration (amyiose, dextran, nigeran, puUulan) were ineffective.     

A β-(1→4)-xylosyltransferase involved in the synthesis of arabinoxylans in developing barley endosperms

A β-(1→4)-xylosyltransferase (XylTase; EC 2.4.2.24) participating in the synthesis of arabinoxylans was investigated using microsomal membranes prepared from developing barley ( Hordeum vulgare L.) endosperms. The microsomal fraction transferred Xyl from uridine 5'-diphosphoxylose (UDP-Xyl) into exogenous β-(1→4)-xylooligosaccharides derivatized at their reducing ends with 2-aminopyridine. HPLC analysis showed chain elongation of pyridylaminated β-(1→4)-xylotriose (Xyl₃-PA) by repeated attachment of one to five single xylosyl residues depending on the reaction time, leading to the formation of Xyl₄₋₈-PA. Methylation analysis and enzymatic digestions with β-xylosidase (EC 3.2.1.37) and endo -β-(1→4)-xylanase (EC 3.2.1.8) confirmed that the transfer of xylosyl residues into the newly synthesized products occurred through β-(1→4)-linkages. The activity of the XylTase was maximal at pH 6.8 and 20°C and most enhanced in the presence of 0.5% Triton X-100 and 5 m M MnCl₂. The apparent Michaelis constant and maximal velocity of the enzyme for Xyl₃-PA were 2.1 m M and 25 400 pmol min⁻¹ mg protein⁻¹, respectively. The enzyme also transferred [¹⁴C]Xyl from UDP-[¹⁴C]Xyl into higher β-(1→4)-xylooligosaccharides and birchwood xylans through β-(1→4)-linkages. The enzyme activity varied according to the stage of development (7–35 days after flowering) of the endosperms. Maximal activity occurred at 13–16 days; no activity was detectable in mature seeds. A comparison of endosperms from 10 different cultivars of barley harvested 11–22 days after flowering showed no correlation between enzyme activity and the amount of Xyl in the cell walls.     

The phytotoxin syringomycin elicits Ca2+-dependent callose synthesis in suspension-cultured cells of Catharanthus roseus

Deposition of the 1,3-β-glucan callose onto the cell wall represents one of the defence reactions of plants against pathogens. This process can be induced in suspension cells of Catharanthus roseus by subtoxic concentrations of the bacterial phytotoxin syringomycin and is associated with a slight increase in Ca²⁺ uptake and some K⁺ release. Under these conditions callose formation can be prevented by complexing external Ca²⁺, indicating that some Ca²⁺ uptake is essential as a signal. However, higher syringomycin concentrations elicit increased Ca²⁺ uptake without increasing callose formation, although the potential for callose synthesis is not exhausted – as shown using digitonin as an additional elicitor. These results suggest that, superimposed on Ca²⁺, another, yet unknown signal is also involved in the regulation of callose synthesis.     

Endo-(1 → 3)β-D-glucanase activity secreted by Bacillus sp. no. 215

The production of an extracellular endo-(1 → 3)-β-D-glucanase by Bacillus sp. no. 215 was induced during growth with (1 → 3)-β-D-glucan (curdlan) from Cellulomonas flavigena strain KU as carbon and energy source. Maximum levels of activity (0.26 U ml^-1 resp. 1.40 U mg^-1) were detected in cell-free culture supernatant fluid after 25 h of aerobic growth at 55°C. The cells secreted an endo-(1 → 3)-β-D-glucanase with low electrophoretic mobility that used curdlan from C. flavigena strain KU and from Agrobacterium sp. (formerly Alcaligenes faecalis var. myxogenes ) as substrates. The enzyme activity was highest at pH 7.0 and 55°C. It exhibited a remarkably low thermal stability with a half-life of 14 min at 55°C in the presence of substrate. Divalent metal cations were required for enzyme activity.     

Paradoxical effect of ethanol on potassium channel currents and cell survival in cerebellar granule neurons

Transient exposure to ethanol (EtOH) results in a massive neurodegeneration in the developing brain leading to behavioral and cognitive deficits observed in fetal alcohol syndrome. There is now compelling evidence that K⁺ channels play an important role in the control of programmed cell death. The aim of the present work was to investigate the involvement of K⁺ channels in the EtOH-induced cerebellar granule cell death and/or survival. At low and high concentrations, EtOH evoked membrane depolarization and hyperpolarization, respectively. Bath perfusion of EtOH (10 mM) depressed the I _A (transient K⁺ current) potassium current whereas EtOH (400 mM) provoked a marked potentiation of the specific I _K (delayed rectifier K⁺ current) current. Pipette dialysis with GTPγS or GDPβS did not modify the effects of EtOH (400 mM) on both membrane potential and I _K current. In contrast, the reversible depolarization and slowly recovering inhibition of I _A induced by EtOH (10 mM) became irreversible in the presence of GTPγS. EtOH (400 mM) induced prodeath responses whereas EtOH (10 mM) and K⁺ channel blockers promoted cell survival. Altogether, these results indicate that in cerebellar granule cells, EtOH mediates a dual effect on K⁺ currents partly involved in the control of granule cell death.     

Potassium Fluxes on Hyperosmotic Shock and the Effect of Phenol and Bronopol (2-bromo-2-nitropropan-1,3-diol) on Deplasmolysis of Pseudomonas aeruginosa

Potassium fluxes and the effect of phenol and bronopol on deplasmolysis of Pseudomonas aeruginosa were followed in sucrose and glycerol plasmolysing systems.
In sucrose, K⁺ uptake related to the solute concentration. Proline increased the rate and overall K⁺ uptake, the latter by a factor of three. It was concluded that there was no rigid maximum in the accumulation of intracellular K⁺ as long as intracellular neutrality in electrical charges was maintained.
In glycerol, K⁺ uptake was parallel with glycerol penetration. The process was reversed, however, on equilibration of glycerol. This suggested that glycerol inhibited K⁺ retention against a concentration gradient rather than that K⁺ was excluded as a consequence of the osmotic established steady state. This view was enforced by the fact that the reversal of K⁺ uptake occurred in 20 and 30% glycerol but not in 10%.
Phenol and bronopol did not affect deplasmolysis in glycerol significantly, although some effect on K⁺ uptake and glycerol permeability could be seen. In the sucrose system, phenol acted according to its mode of action generally accepted, i.e. inhibiting deplasmolysis at low and allowing solute penetration at higher concentrations, whereas very high concentrations caused coagulation of the cytoplasm. Bronopol inhibited deplasmolysis, except at very low concentrations. Proline did not prevent the inhibition of deplasmolysis in either of the solute systems, except at the very low bronopol concentrations where the deplasmolysis rate only was affected.     

Axonal Contact Regulates Expression of α2 and β2 Isoforms of Na+,K+-ATPase in Schwann Cells: Adhesion Molecules and Nerve Regeneration

Abstract: Three isoforms of catalytic α subunits and two isoforms of β subunits of Na⁺,K⁺-ATPase were detected in rat sciatic nerves by western blotting. Unlike the enzyme in brain, sciatic nerve Na⁺,K⁺-ATPase was highly resistant to ouabain. The ouabain-resistant α1 isoform was demonstrated to be the predominant form in rat intact sciatic nerve by quantitative densitometric analysis and is mainly responsible for sciatic nerve Na⁺,K⁺-ATPase activity. After sciatic nerve injury, the α3 and β1 isoforms completely disappeared from the distal segment owing to Wallerian degeneration. In contrast, α2 and β2 isoform expression and Na⁺,K⁺-ATPase activity sensitive to pyrithiamine (a specific inhibitor of the α2 isoform) were markedly increased in Schwann cells in the distal segment of the injured sciatic nerve. These latter levels returned to baseline with nerve regeneration. Our results suggest that α3 and β1 isoforms are exclusive for the axon and α2 and β2 isoforms are exclusive for the Schwann cell, although axonal contact regulates α2 and β2 isoform expressions. Because the β2 isoform of Na⁺,K⁺-ATPase is known as an adhesion molecule on glia (AMOG), increased expression of AMOG/β2 on Schwann cells in the segment distal to sciatic nerve injury suggests that AMOG/β2 may act as an adhesion molecule in peripheral nerve regeneration.     

Interaction of potassium ions and gibberellin in the control of hypocotyl growth in Amaranthus caudatus

Potassium promotes growth in several plant tissues. Elongation growth of the hypocotyls of Amaranthus caudatus L. ev. Lalsag is mainly controlled by gibberellins, but K⁺ also promotes growth. In the present study the interaction of K⁺ with gibberellin (GA₃) and chlorocholine chloride (CCC) has been investigated. When K⁺ was applied externally in the dark, hypocotyl growth was promoted in the seedlings. External application of GA₃ did not promote growth in the dark. GA₃ was effective in the light and K⁺ was synergistic with GA₃ in promoting elongation. Application of CCC in the dark makes the seedlings sensitive to GA₃. The inhibition of growth by CCC was also reversed by K⁺. The results indicate a possible role of K⁺ in GA₃ induced elongation of hypocotyls.     

The effect of xanthoxylin (2-oxy, 4-6-dimethoxyacetophenone) on potassium-dependent acid: extrusion in wheat and maize root segments

Abstract. Xanthoxylin is a cytotoxic and fungicidal compound with the characteristics of a typical phytoalexin. At a concentration of 0.3 mol m⁻³ it inhibits K⁺-dependent acid extrusion and K⁺ net uptake (or uptake of equivalent alkaline cations such as Rb⁺ and CS⁺) by up to about 80% and hyperpolarizes by about 20% the membrane electrical potential. Its inhibition of the acid extrusion does not depend on altered ion exchange involving the anions in the media, a reduction of the metabolic energy available, or detectable changes in the permeability of the cell membrane to H⁺ ions. The drop in K⁺ net uptake depends on a decrease in the influx of K⁺ into the cell. In functional terms, xanthoxylin is an inhibitor of the K⁺ permeation mechanism and does not appear to interact with the mechanisms creating the electrochemical energy gradient.     

Net Glutamate Release from Astrocytes Is Not Induced by Extracellular Potassium Concentrations Attainable in Brain

Abstract: Elevated extracellular potassium concentration ([K⁺]_e) has been shown to induce reversal of glial Na⁺-dependent glutamate uptake in whole-cell patch clamp preparations. It is uncertain, however, whether elevated [K⁺]_e similarly induces a net glutamate efflux from intact cells with a physiological intracellular milieu. To answer this question, astrocyte cultures prepared from rat and mouse cortices were incubated in medium with elevated [K⁺]_e (by equimolar substitution of K⁺ for Na⁺), and glutamate accumulation was measured by HPLC. With [K⁺]_e elevations to 60 m M , medium glutamate concentrations did not increase during incubation periods of 5–120 min. By contrast, 45 min of combined inhibition of glycolytic and oxidative ATP production increased medium glutamate concentrations 50–100-fold. Similar results were obtained in both rat and mouse cultures. Studies were also performed using astrocytes loaded with the nonmetabolized glutamate tracer d -aspartate, and parallel results were obtained; no increase in medium d -aspartate content resulted from [K⁺]_e elevation up to 90 m M , whereas a large increase occurred during inhibition of energy metabolism. These results suggest that a net efflux of glutamate from intact astrocytes is not induced by any [K⁺]_e attainable in brain.     

The influences of calcium and magnesium ions on the exchange of monovalent cations in Neurospora crassa

Low-K⁺, high-Na⁺ cells of strain RL21a of Neurospora crassa , in steady state with 25 m M Na⁺, were used to study K⁺/Na⁺ exchanges in the presence or absence of Ca²⁺ and Mg²⁺. In the presence of Ca²⁺ and Mg²⁺, a low concentration of K⁺ (0.3 m M ) triggered a rapid exchange, but in the absence of the divalents, a high K⁺ concentration (30 m M ) was required to initiate the exchange at a rapid rate. In the absence of Ca²⁺ and Mg²⁺, K⁺ uptake did not occur at low K⁺ concentration, internal K⁺ did not regulate Na⁺ influx in the presence of external K⁺, and the efflux of Na⁺ proceeded at maximum activity at very low-K⁺ contents.     

Effects of desiccation on the 77°K fluorescence properties of the liverwort Porella navicularis and the isolated lichen green alga Trebouxia pyriformis

The uptake of the auxin type herbicide 2,4-D into rice seedlings ( Oryza sativa L. cv. Dunghan Shali) and its effects on the K⁺, NH⁺₄ and NO₃ ion uptake and the K⁺ content were investigated at different pH values. A short incubation of the roots in 0.01 m M 2,4-D caused a marked ion uptake inhibition only at low pH. The non-auxin type herbicide benthiocarb did not produce such an inhibitory effect. Lowering of the pH in the external medium led to an increased 2,4-D uptake by the roots. These results can be explained by the increased H⁺ permeability of the membranes, allowing a more rapid entrance of 2,4-D into the root cells, thereby inhibiting the active ion uptake. Rice roots not subjected to 2,4-D treatment responded to H⁺ stress with an increased anomalous K⁺ uptake and a decreased K⁺ content. With reference to the effects of pH changes on the ion and 2,4-D uptake, possible transport mechanism of NH⁺₄ and 2,4-D are briefly discussed.     

Potassium transport in wheat seedlings grown with different potassium supplies.

The K⁺(⁸⁶Rb) uptake into the roots and the translocation to the shoots of 11-day-old intact wheat seedlings ( Triticum aestivum L. cv. Martonvásári 8) were investigated using plants grown with different K⁺ supplies. The effects of environmental conditions (darkness, humidity) and of metabolic and transport inhibitors (oligomycin, disalicylidene-propanediamine, 2,4-dinitriphenol, diethylstilbestrol, colchicine) were also studied. Plants with K content of about 0.2 mmol/g dry weight in the root and 0.5 mmol/g dry weight in the shoot (low K status) showed high K⁺ uptake into the roots and high translocation rates to the shoots. Both transport processes were very low in plants with K content of more than 1.5 and 2.2 mmol/g dry weight in the root and shoot, respectively (high K status).
Darkness and a relative humidity of the air of 100% did not influence K⁺ uptake by roots, but did inhibit upward translocation and water transport. Inhibition of photosynthesis and treatments with diethylstilbestrol (10⁻⁵ mol/dm³), as well as with colchicine resulted in inhibition of translocation in plants of low K status, but these inhibitors had little effect on K⁺ uptake by the roots. Oligomycin, 2,4-dinitrophenol and diethylstilbestrol (10⁻⁴ mol/dm³), however, inhibited K⁺ uptake by the roots. In general, K⁺ transport processes were almost unchanged in plants of high K status. It is concluded that only plants of low K status operating with active K⁺ transport mechanisms are responsive to environmental factors. In high K⁺ plants the transport processes are passive and are uncoupled from the metabolic energy flow.     

Effects of aluminium on growth and kinetics of K+(86Rb) uptake in two cultivars of wheat (Triticum aestivum) with different sensitivity to aluminium

Two cultivars of wheat (Triticum aestivum L. cvs Kadett and WW 20299) were grown for 9 days with 20% relative increase in nutrient supply per day at pH 4.1. Aluminium at 50 μ M retarded the growth of roots more than that of shoots in both cultivars, thus decreasing the root/shoot ratio. The inhibition was largest in WW 20299. With long term Al treatment (9 days), K_m for K⁺(⁸⁶Rb) influx increased five times in both cultivars and V_max decreased in WW 20299. Efflux of K⁺(⁸⁶Rb) was little affected. When the roots were treated with aluminium for two days, only relative growth rate of roots was retarded, while growth of shoots was unaffected and influx of K⁺(⁸⁶Rb) adjusted to the actual K⁺ demand of the plants. It is concluded that the effects of aluminium on K⁺ uptake in these wheat cultivars are not primary factors contributing to aluminium sensitivity. However, in soil with Al the demand for a comparatively high concentration of K⁺ to maintain an adequate K⁺ uptake rate, in combination with a slow growth rate of the roots, may secondarily lead to K⁺ deficiency in the plants.     

Isoform-Specific Up-Regulation by Ouabain of Na+,K+-ATPase in Cultured Rat Astrocytes

Abstract: There are two α-subunit isoforms (α1 and α2) and two β-subunit isoforms (β1 and β2) of Na⁺,K⁺-ATPase in astrocytes, but the functional heterodimer composition is not known. Ouabain (0.5–1.0 m M ) increased the levels of α1 and β1 mRNAs, whereas it decreased those of α2 and β2 mRNAs in cultured rat astrocytes. The increases in α1 and β1 mRNAs were observed at 6–48 h after addition of the inhibitor. Immunochemical analyses showed that ouabain increased α1 and β1, but not α2 and β2, proteins, and that the isoforms in control and ouabain-treated cultures were of glial origin. Low extracellular K⁺ and monensin (20 µ M ) mimicked the effect of ouabain on α1 mRNA. The ouabain-induced increase in α1 mRNA was blocked by the protein synthesis inhibitor cycloheximide (10 µ M ), the intracellular Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane- N,N,N',N' -tetraacetic acid tetraacetoxymethyl ester (30 µ M ), and the calcineurin inhibitor FK506 (1 n M ). These findings indicate that chronic inhibition of Na⁺,K⁺-ATPase up-regulates the α1 and β1, but not α2 and β2, isoforms in astrocytes, suggesting a functional coupling of α1β1 complex. They also suggest that intracellular Na⁺, Ca²⁺, and calcineurin may be involved in ouabain-induced up-regulation of the enzyme in astrocytes.     

Sodium ion transport in Neurospora crassa

Na⁺ influx and efflux in Neurospora crassa RL21a can be studied separately to calculate net Na⁺ movements. In the absence of external K⁺, Na⁺ influx was independent of the K⁺ content of the cells, but when K⁺ was present, the inhibition of Na⁺ influx by external K⁺ was higher the higher the K⁺ content. Efflux depended on the K⁺ and Na⁺ content, and on the history of the cells. Efflux was higher the higher the Na⁺ and K⁺ contents, and, in low-K⁺ cells, the efflux was also higher in cells grown in the presence of Na⁺ than when Na⁺ was given to cells grown in the absence of Na⁺. Addition of K⁺ to cells in steady state with external Na⁺ resulted in a net Na⁺-loss. In cells grown without Na⁺ this loss was a consequence of the inhibition of Na⁺ influx. In Na⁺-grown cells, addition of K⁺ inhibited Na⁺ influx and increased Na⁺ efflux.     

ɛ-(γ-Glutamyl)lysine cross-links of spore coat proteins and transglutaminase activity in Bacillus subtilis

Abstract The capacity to transport potassium and to discriminate between the different alkali cations has been found to affect sodium tolerance in Saccharomyces cerevisiae . Mutants with a defective capacity to transport K⁺ were more sensitive to high concentrations of Na⁺ because they accumulated more Na⁺ and less K⁺ than wild-type cells which showed high discrimination between K⁺ and Na⁺.     

ENERGETICS OF AMINO ACID TRANSPORT INTO BRAIN SLICES: EFFECTS OF K+ DEPLETION AND Rb+ OR Cs+ SUBSTITUTION ON AMINO ACID UPTAKE

Abstract— Mouse brain slices were depleted of K⁺ by three 10-min incubations-in oxygenated HEPES-buffered medium lacking glucose and K⁺. Addition of K⁺ or Rb⁺ (or Cs⁺, to a smaller degree) with glucose, or with succinate, malate, and pyruvate (SMP) before incubation at 37°C with ¹⁴C-amino acids restored active low-affinity transport of d -Glu, α-aminoisobutyrate (AIB), GABA, Gly, His, Val, Leu, Lys, and Orn. Ouabain at 1–2μ m with Rb⁺ was more inhibitory with SMP than with glucose, suggesting that the glycoside may affect specific energy coupling to transport. Valinomycin, in contrast, showed no specificity of inhibition of amino acid uptake with glucose or SMP and K⁺ or Rb⁺. Cs⁺ partially restored amino acid uptake, but Li⁺ was less effective than Cs ⁺. NaF at 10 m m with SMP + Rb⁺, or SMP + K⁺ did not inhibit amino acid uptake. Therefore, it was possible to dissociate glycolysis and Na⁺, K ⁺ -ATPase activity from amino acid transport. The ion replacements for K ⁺ that supported active amino acid transport indicate that the specificity of ions in possible ionic gradients for transport energetics should be reexamined.     

Passive uptake of K+(86Rb+) in sunflower roots at low external K+ concentrations

Passive fluxes of K⁺ (⁸⁶Rb) into roots of sunflower ( Helianthus annuus L. cv. Uniflorus) were determined at low K⁺ concentration (0.1 and 1.0 mM K⁺) in the ambient solution. Metabolic uptake of K⁺ was inhibited by 10⁻⁴M 2,4-dinitrophenol (DNP). K⁺ (⁸⁶Rb) fluxes were studied both continuously and by time differentiation of uptake. In high K⁺ roots passive uptake was directly proportional to the K⁺ concentration of the uptake solution, indicating free diffusion. This assumption was supported by the fact that passive Rb⁺ uptake was not affected by high K⁺ concentrations. In low K⁺ roots the passive uptake of K⁺ was higher than in high K⁺ roots. The increase was possibly due to carrier-mediated K⁺ transport. As K⁺ effluxes were quantitatively similar to influxes, it is suggested that passive K⁺ fluxes represent exchange diffusion without relation to net K⁺ transport.     

Effect of K+ and H+ stress and role of Ca2+ in the regulation of intracellular K+ concentration in mung bean roots

The effects of external K⁺, H⁺ and Ca2⁺ concentrations on the intracellular K+ concentration, [K⁺]i, and the K⁺-ATPase activity in 2-day-old mung bean roots [ Vigna mungo (L.) Hepper] were investigated. [K⁺]i, in mung bean roots was markedly decreased by external K⁺ or H⁺ stress and did not recover the initial value even after the stress was removed. This decrease in [K⁺]i, gradually disappeared with the addition of (Ca2⁺. Ca2⁺ may offset the harmful effects of ion stress. Ca2⁺ seems to have two effects on K+ transport; control of K⁺ permeability and activation of K⁺ uptake, although K⁺-ATPase activity was inhibited by Ca2⁺ concentrations higher than 10–4 M. We suggest that Ca2⁺ activates K⁺ uptake indirectly through the acidification of the cytoplasm.