THE ENERGETICS OF EXTRACELLULAR FE(III) REDUCTION BY IRON-LIMITED CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)

Fe-limited cells of the green alga Chlamydomonas reinhardtii (Fe-limited growth rate = 0.3 d⁻¹) reduced extracellular Fe(III) to Fe(II) when Fe(III) was supplied as ferricyanide or Fe(III)-EDTA; Fe(III) reduction was stimulated by light. In both darkness and during photosynthesis, ferricyanide reduction was accompanied by a decrease in cellular NADPH levels, with a concomitant increase in NADP⁺. NADH and NAD⁺ levels were not measurably altered during ferricyanide reduction. Furthermore, cellular hexose monophosphate levels declined and 6-phosphogluconate levels increased during ferricyanide reduction. Levels of most glycolytic and tricarboxylic acid cycle intermediates were mostly unaltered. Ferricyanide reduction was also associated with a decrease in cellular ATP levels, a concomitant increase in ADP and AMP, and increased extracellular acidification. The acidification was sensitive to inhibition by the H⁺-ATPase inhibitor N,N' -dicyclohexylcarbodiimide (DCCD). We conclude that the oxidative pentose phosphate pathway provides reducing equivalents for Fe(III) reduction in darkness and also contributes reducing equivalents to Fe(III) reduction during photosynthesis. The decline in ATP was likely due to activation of the plasma membrane H⁺-ATPase during ferricyanide reduction and was not directly associated with provision of reducing equivalents.     

Evidence for a pentose phosphate pathway in Helicobacter pylori

Abstract Evidence for the presence of enzymes of the pentose phosphate pathway in Helicobacter pylori was obtained using ³¹P nuclear magnetic resonance spectroscopy. Activities of enzymes which are part of the oxidative and non-oxidative phases of the pathway were observed directly in incubations of bacterial lysates with pathway intermediates. Generation of NADPH and 6-phosphogluconate from NADP⁺ and glucose 6-phosphate indicated the presence of glucose 6-phosphate dehydrogenase and 6-phosphogluconolactonase. Reduction of NADP⁺ with production of ribulose 5-phosphate from 6-phosphogluconate revealed 6-phosphogluconate dehydrogenase activity. Phosphopentose isomerase and transketolase activities were observed in incubations containing ribulose 5-phosphate and xylulose 5-phosphate, respectively. The formation of erythrose 4-phosphate from xylulose 5-phosphate and ribose 5-phosphate suggested the presence of transaldolase. The activities of this enzyme and triosephosphate isomerase were observed directly in incubations of bacterial lysates with dihydroxyacetone phosphate and sedoheptulose 7-phosphate. Glucose-6-phosphate isomerase activity was measured in incubations with fructos 6-phosphate. The presence of these enzymes in H. pylori suggested the existence of a pentose phosphate pathway in the bacterium, possibly as a mechanism to provide NADPH for reductive biosynthesis and ribose 5-phosphate for synthesis of nucleic acids.     

Nitric Oxide-Mediated Mitochondrial Damage in the Brain: Mechanisms and Implications for Neurodegenerative Diseases

Abstract: Within the CNS and under normal conditions, nitric oxide (^•NO) appears to be an important physiological signalling molecule. Its ability to increase cyclic GMP concentration suggests that ^•NO is implicated in the regulation of important metabolic pathways in the brain. Under certain circumstances ^•NO synthesis may be excessive and ^•NO may become neurotoxic. Excessive glutamate-receptor stimulation may lead to neuronal death through a mechanism implicating synthesis of both ^•NO and superoxide (O₂^•−) and hence peroxynitrite (ONOO⁻) formation. In response to lipopolysaccharide and cytokines, glial cells may also be induced to synthesize large amounts of ^•NO, which may be deleterious to the neighbouring neurones and oligodendrocytes. The precise mechanism of ^•NO neurotoxicity is not fully understood. One possibility is that it may involve neuronal energy deficiency. This may occur by ONOO⁻ interfering with key enzymes of the tricarboxylic acid cycle, the mitochondrial respiratory chain, mitochondrial calcium metabolism, or DNA damage with subsequent activation of the energy-consuming pathway involving poly(ADP-ribose) synthetase. Possible mechanisms whereby ONOO⁻ impairs the mitochondrial respiratory chain and the relevance for neurotoxicity are discussed. The intracellular content of reduced glutathione also appears important in determining the sensitivity of cells to ONOO⁻ production. It is concluded that neurotoxicity elicited by excessive ^•NO production may be mediated by mitochondrial dysfunction leading to an energy deficiency state.     

Ascorbate-glutathione cycle and H2O2 detoxification in elongating leaf bases of ryegrass: effect of inhibition of glutathione reductase activity on foliar regrowth

The role of the ascorbate-glutathione cycle and AOS detoxification was investigated during leaf growth of defoliated and undefoliated plants of ryegrass ( Lolium perenne L. cv. Bravo). Antioxidants and related enzymatic activities were located in elongating leaf bases (ELBs) of undefoliated plants, following a decreasing gradient from basal (meristem) to distal segments, inverse to H₂O₂ levels. In the meristematic zone, the intense activity of the ascorbate-glutathione cycle and the supply of reducing power by the oxidative pentose phosphate pathway allowed the maintenance of both antioxidant reduction and H₂O₂ detoxification. BCNU (1–3 bis(2-chloroethyl)- N -nitrosourea), a glutathione reductase inhibitor, induced an increase in the meristematic zone in both H₂O₂ and antioxidant levels and a decrease in reduced/oxidized ratios of glutathione and ascorbate. These changes were associated with a reduced foliar regrowth activity. In the absence of BCNU, defoliation did not modify the ratios of reduced/oxidized antioxidants, although it triggered a temporary increase in H₂O₂ level. The results are discussed on the basis of a possible control of leaf growth by glutathione and ascorbate.     

GRAMICIDIN INDUCED ION TRANSPORT IN BRAIN MITOCHONDRIA PREPARATIONS

Abstract— —(1) Gramicidin at low concentrations induces an uptake of K⁺ and Na⁺ in brain mitochondria in a manner similar to that observed with liver mitochondria.
(2) The cation uptake is energy dependent, and is accompanied by an ejection of H⁺ ions and a slight increase in respiration in the absence of added permeant anion.
(3) The cation uptake and hydrogen ion release are both inhibited by agents which inhibit electron transport. Barbiturates and chlorpromazine inhibit the transport phenomenon by inhibiting electron transport.
(4) In the presence of permeant anions (phosphate and acetate) respiration is stimulated quite significantly.
(5) At high gramicidin concentrations there is a release of Na⁺ and K⁺ from the mitochondria and uptake of H⁺. There is also a cyclic reduction-oxidation of the nicotinamide adenine dinucleotides, which is believed to be due to the release from the mitochondria of the reduced dinucleotides followed by their subsequent oxidation.
(6) The effect of high gramicidin on the mitochondrial nicotinamide-adenine dinucleotides and cation distribution is irreversible and is not blocked by individual inhibitors of respiration and of phosphorylation, but is prevented by prior addition of a mixture of these inhibitors.
(7) Gramicidin is therefore believed to have a bimodal function; one on the mitochondrial membrane per se , and the other on the energy dependent ion accumulation apparatus.
(8) A model of induced mitochondrial ion accumulation is presented.     

Cloning of sucrase genes from Streptococcus mutans in bacteriophage lambda

Abstract An extracellular peroxidase was purified by chromatofocusing column chromatography from the growth medium of ligninolytic cultures of the white-rot fungus Phanerochaete chrysosporium Burds BKM-1767. The enzyme was electrophoretically pure with an M _r of 45 000–47 000. It contained an easily dissociable heme, and required Mn²⁺ ions for activity. In the presence of hydrogen peroxide and Mn²⁺ it oxidized compounds such as vanillylacetone, 2,6-dimethyloxyphenol, curcumin, syringic acid, guaiacol, syringaldazine, divanillylacetone, and coniferyl alcohol. It did not oxidize veratryl alcohol. In reactions requiring Mn²⁺ and O₂, but not hydrogen peroxide, the enzyme oxidized glutathione, dithiothreitol, and NADPH with production of hydrogen peroxide. The hydrogen peroxide produced could be used as a co-substrate by ligninases such as those that oxidize veratryl alcohol, or by the peroxidase itself to oxidize lignin model compounds.     

Neurotrophic Factors Attenuate Glutamate-Induced Accumulation of Peroxides, Elevation of Intracellular Ca2+ Concentration, and Neurotoxicity and Increase Antioxidant Enzyme Activities in Hippocampal Neurons

Abstract: Exposure of cultured rat hippocampal neurons to glutamate resulted in accumulation of cellular peroxides (measured using the dye 2,7-dichlorofluorescein). Peroxide accumulation was prevented by an N -methyl- d -aspartate (NMDA) receptor antagonist and by removal of extracellular Ca²⁺, indicating the involvement of NMDA receptor-induced Ca²⁺ influx in peroxide accumulation. Glutamate-induced reactive oxygen species contributed to loss of Ca²⁺ homeostasis and excitotoxic injury because antioxidants (vitamin E, propyl gallate, and N-tert -butyl-α-phenylnitrone) suppressed glutamate-induced elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and cell death. Basic fibroblast growth factor (bFGF), nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF), but not ciliary neurotrophic factor, each suppressed accumulation of peroxides induced by glutamate and protected neurons against excitotoxicity. bFGF, NGF, and BDNF each increased (to varying degrees) activity levels of superoxide dismutases and glutathione reductase. NGF increased catalase activity, and BDNF increased glutathione peroxidase activity. The ability of the neurotrophic factors to suppress glutamate toxicity and glutamate-induced peroxide accumulation was attenuated by the tyrosine kinase inhibitor genistein, indicating the requirement for tyrosine phosphorylation in the neuroprotective signal transduction mechanism. The data suggest that glutamate toxicity involves peroxide production, which contributes to loss of Ca²⁺ homeostasis, and that induction of antioxidant defense systems is a mechanism underlying the [Ca²⁺]_i-stabilizing and excitoprotective actions of neurotrophic factors.     

Effects of Exogenous Spermidine on Antioxidant System Responses of Typha latifolia L. Under Cd^2＋ Stress

The effects of foliar spraying with spermidine (Spd), ranging in concentration from 0.25 to 0.50 mmol/L, on the antioxidant system under Cd^2 stress (range 0.1- 0.2 mmol/L Cd^2 ) in Typha latifolia L. grown hydroponically were investigated in order to offer a referenced evidence for an understanding of the mechanism by which polyamines (PAs) relieve the damage to plants by heavy metal and improve the phytoremediation efficiency of heavy metal-contaminated water. The results showed that Cd^2 stress induced oxidative injury, as evidenced by an increase in the generation of superoxide anion (O2), as well as the hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents in both leaves and caudices. With the exception of superoxide dismutase (SOD) activity in the leaves, an increase in the activities of catalase (CAT), guaiacol peroxidase (GPX), and glutathione reductase (GR) was observed in both leaves and caudices, SOD activity was increased in caudices, and ascorbate peroxidase (APX) activity was increased in leaves following Cd^2 treatment. The reduced glutathione (GSH) content in both leaves and caudices and the reductive ascorbate content in leaves was obviously increased, which were prompted by the application of exogenous Spd. Spraying with Spd increased the activity of GR and APX in both leaves and caudices, whereas the activity of SOD, CAT, and GPX was increased only in caudices following spraying with Spd. The generation of O2 and the H2O2 and MDA content in both leaves and caudices decreased after spraying with Spd. The decrease in MDA was more obvious following the application of 0.25 than 0.50 mmol/L Spd. It is supposed that exogenous Spd elevated the tolerance of T. latifolia under Cd^2 stress primarily by increasing GR activity and the GSH level.     

Mitochondrial Permeability Transition in the Central Nervous System: Induction by Calcium Cycling-Dependent and -Independent Pathways

Abstract: Isolated rat CNS mitochondria and cultured cortical astrocytes were examined for behavior indicative of a mitochondrial permeability transition (mPT). Exposure of isolated CNS mitochondria to elevated calcium or phosphate or both produced loss of absorbance indicative of mitochondrial swelling. The absorbance decreases were prevented by ADP and Mg²⁺ and reduced by cyclosporin A, dithiothreitol, and N -ethylmaleimide. Ruthenium red prevented calcium cycling-induced, but only attenuated phosphate-induced losses of absorbance. In cultured astrocytes permeabilized with digitonin or treated with the calcium ionophore, 4-bromo-A23187, elevations of external calcium altered mitochondrial morphology visualized with the dye, JC-1, from rod-like to rounded, swollen structures. Similar changes were observed in digitonin-permeabilized astrocytes exposed to phosphate. The incidence of calcium-induced changes in astrocyte mitochondria was prevented by Mg²⁺ and pretreatment with dithiothreitol and N -ethylmaleimide, and was reduced by cyclosporin A, ADP, and butacaine alone or in combinations. Ruthenium red and the Na⁺/Ca²⁺ exchange inhibitor CGP 37157 blocked calcium cycling and prevented mitochondrial shape changes in digitonin-treated, but not ionophore-treated astrocytes. Thus, the demonstrated induction conditions and pharmacological profile indicated the existence of an mPT in brain mitochondria. The mPT occurred consequent to activation of calcium cycling-dependent and -independent pathways. Induction of an mPT could contribute to neuronal injury following ischemia and reperfusion.     

Depolarization and Activation of Dihydropyridine-Sensitive Ca2+ Channels Stimulate Inositol Phosphate Accumulation in Photoreceptor-Enriched Chick Retinal Cell Cultures

Abstract: Elevated concentrations of extracellular K⁺ increased inositol phosphate accumulation in primary cultures of chick retinal photoreceptors and multipolar neurons. K⁺-evoked stimulation of inositol phosphate accumulation was greater in photoreceptor-enriched cell cultures than in cultures where multipolar neurons were the predominant cell type. Destroying multipolar neurons, but not photoreceptors, with kainic acid and N -methyl- d -aspartate did not reduce the K⁺-evoked stimulation of inositol phosphate accumulation. Both of these observations indicate that the observed effects occur in photoreceptor cells. The K⁺-evoked stimulation of inositol phosphate accumulation was blocked by omitting Ca²⁺ from the incubation medium or by adding the dihydropyridine-sensitive Ca²⁺-channel antagonists, nitrendipine and nifedipine. Bay K 8644, a dihydropyridine agonist, stimulated inositol phosphate accumulation and enhanced the effect of K⁺. ω-Conotoxin GVIA, an inhibitor of N-type Ca²⁺ channels, had no significant effect on K⁺-stimulated inositol phosphate accumulation. Pretreatment with pertussis toxin neither blocked K⁺-evoked inositol phosphate accumulation nor altered the inhibitory effect of nifedipine. K⁺-evoked inositol phosphate accumulation appears to reflect activation of phosphatidylinositol-specific phospholipase C, as it is inhibited by U-73122. These results indicate that Ca²⁺ influx through voltage-gated, dihydropyridine-sensitive channels activates phospholipase C in photoreceptor inner segments and/or synaptic terminals.     

Production of Reactive Oxygen Species and Release of l-Glutamate During Superoxide Anion-Induced Cell Death of Cerebellar Granule Neurons

Abstract: Enhanced production of superoxide anion (O₂⁻) is considered to play a pivotal role in the pathogenesis of CNS neurons. Here, we report that O₂⁻ generated by xanthine (XA) + xanthine oxidase (XO) triggered cell death associated with nuclear condensation and DNA fragmentation in cerebellar granule neuron. XA + XO induced significant increases in amounts of intracellular reactive oxygen species (ROS) before initiating loss of cell viability, as determined by measurement of 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) (C-DCDHF-DA) for O₂⁻ and other ROS and hydroethidine (HEt) specifically for O₂⁻ by using fluorescence microscopy and flow cytometry. Catalase, but not superoxide dismutase (SOD), significantly protected granule neurons from the XA + XO-induced cell death. Catalase effectively reduced C-DCDHF-DA but not HEt fluorescence, whereas SOD reduced HEt but not C-DCDHF-DA fluorescence, indicating that HEt and C-DCDHF-DA fluorescence correlated with O₂⁻ and hydrogen peroxide, respectively. The NMDA antagonist MK-801 prevented the death. XA + XO induced an increase in l -glutamate release from cerebellar granule neurons. These results indicate that elevation of O₂⁻ induces cell death associated with increasing ROS production in cerebellar granule neurons and that XA + XO enhanced release of l -glutamate.     

Resistance of Serratia marcescens to Hydrogen Peroxide

Irradiation with ultraviolet (u.v.) light (71 J/m²) reduced the viable count of suspenrsions of Serratia marcescens , grown in a glycerol-salts defined medium, to five in 10⁴ cells. Subsequent incubation of irradiated cells in hydrogen peroxide failed to decrease the survivors, but u.v. irradiation in the presence of hydrogen peroxide reduced the viable count to fewer than two in 10⁶ cells. Cells grown in defined medium with added iron had more measurable catalase activity and were more resistant to hydrogen peroxide alone and to simultaneous treatment with u.v. irradiation and hydrogen peroxide. Cells grown in a non-defined medium contained little iron and measurable catalase activity but were more resistant to hydrogen peroxide. Treatment with toluene, heat killing or sonication increased the catalase activity detected in all cell suspensions and showed that resistance to hydrogen peroxide and to u.v. irradiation in hydrogen peroxide was related to the total catalase activity within cells.     

Metabolism of hydrogen peroxide by the scavenging system in Chlamydomonas reinhardtii

The metabolism of hydrogen peroxide by the scavenging system was studied in Chlamydomonas grown in a selenium-lacking and a selenium-containing medium. In cells of the former, 40% of external hydrogen peroxide (H₂O₂) was scavenged by ascorbate peroxidase (AsAP; EC 1.11.1.11) and the residual H₂O₂ by catalase (EC 1.11.1.6). The enzymes involved in the ascorbate-glutathione cycle including AsAP. were localized in the chloroplast. In cells of the latter, glutathione peroxidase (GSHP; EC 1.11.1.9) functioned primarily in the removal of external H₂O₂. GSHP was located solely in the cytosol. The Chlamydomonas AsAP was relatively stable in ascorbate-depleted medium as compared with chloroplast AsAP of higher plants. No inactivation of the enzyme was found upon its incubation with hydroxyurea, an inhibitor of the chloroplast enzyme of higher plants. The enzyme showed higher specificity with pyrogallol than with ascorbate. The amino acid sequences in the N-terminal region of Chlamvdomonas AsAP showed no significant similarity to any other AsAP from higher plants and Euglena . The enzyme had a molecular mass of 34 kDa. The K_m values of the enzyme for ascorbate and H₂O₂ were 5.2±0.3 and 25±3.4 μ M , respectively. Hydrogen peroxide was generated at a rate of 6.1±0.8 μmol mg^-1 chlorophyll h^-1 in intact chloroplasts isolated from Chlamydomonas cells grown in the presence of Na-selenite, and it diffused from the organelles into the medium.     

Correlations between the ascorbate-glutathione pathway and effectiveness in legume root nodules

Legume root nodules use the ascorbate-glutathione pathway to remove harmful H₂O₂. In the present study. effective and ineffective nodules from soybean and alfalfa were compared with regard to this pathway. Effective nodules had higher activity of all 4 enzymes (ascorbate peroxidase, EC 1. 11. 1. 11: monodehydroascorbate reductase, EC 1. 6. 5. 4: dehydroascorbate reductase, EC 1. 8. 5. 1: and glutathione reductase, EC 1. 6. 4. 2). The concentration of thiol tripeptides (primarily homoglutathione) was about 1 m M in effective nodules – a level 3–4-fold higher than in ineffective nodules. Effective nodules contained higher levels of NAD⁺. NADP⁺ and NADPH. but not of NADH or ascorbate. The increased capacity for peroxide scavenging in effective nodules as compared to ineffective nodules emphasizes the important protective role that this pathway may play in processes related to nitrogen fixation.     

FREEZE-BLOWING: A NEW TECHNIQUE FOR THE STUDY OF BRAIN IN VIVO

Abstract— A new apparatus is described which removes and freezes brains of conscious rats more rapidly than was heretofore possible. The apparatus consists of two probes which are driven simultaneously into the cranial vault of the rat immobilized in a specially constructed restraining cage. When in position, air under pressure enters through one probe and blows the supratentorial portion of the brain tissue (situated between the olfactory bulbs and the superior colliculi) out the other probe and into a thin chamber previously cooled in liquid N₂. This method stops brain tissue metabolism more rapidly than the previously-described methods of microwave irradiation, decapitation into liquid N₂, or whole-animal immersion into liquid N₂, as evidenced by the measurement of labile metabolites and redox states. Thus, samples of freeze-blown brain had higher levels of a-oxoglutarate, creatine phosphate, pyruvate, glucose and glucose-6-phosphate and lower levels of lactate, malate and AMP than brain tissue obtained by the other methods. The free cytoplasmic [NAD⁺]/[NADH₂], [NADP⁺]/[NADPH₂] and [ATP]/[ADP] [HPO₄^2-] ratios were higher in freeze-blown samples. These data indicate that more extensive anoxic metabolism occurred when methods other than freeze-blowing were used. We conclude that the levels of metabolites measured in brain obtained with the freeze-blowing technique more closely resemble those which occur in vivo.     

Pivotal Role of Mitochondrial Calcium Uptake in Neural Cell Apoptosis and Necrosis

Abstract : Perturbed cellular calcium homeostasis has been implicated in both apoptosis and necrosis, but the role of altered mitochondrial calcium handling in the cell death process is unclear. The temporal ordering of changes in cytoplasmic ([Ca²⁺]C) and intramitochondrial ([Ca²⁺]M) calcium levels in relation to mitochondrial reactive oxygen species (ROS) accumulation and membrane depolarization (MD) was examined in cultured neural cells exposed to either an apoptotic (staurosporine ; STS) or a necrotic (the toxic aldehyde 4-hydroxynonenal ; HNE) insult. STS and HNE each induced an early increase of [Ca²⁺]C followed by delayed increase of [Ca²⁺]M. Overexpression of Bcl-2 blocked the elevation of [Ca²⁺]M and the MD in cells exposed to STS but not in cells exposed to HNE. The cytoplasmic calcium chelator BAPTA-AM and the inhibitor of mitochondrial calcium uptake ruthenium red prevented both apoptosis and necrosis. STS and HNE each induced mitochondrial ROS accumulation and MD, which followed the increase of [Ca²⁺]M. Cyclosporin A prevented both apoptosis and necrosis, indicating critical roles for MD in both forms of cell death. Caspase activation occurred only in cells undergoing apoptosis and preceded increased [Ca²⁺]M. Collectively, these findings suggest that mitochondrial calcium overload is a critical event in both apoptotic and necrotic cell death.     

Silencing of NtMPK4 impairs CO-induced stomatal closure, activation of anion channels and cytosolic Casignals in Nicotiana tabacum guard cells

Light-induced stomatal opening in C3 and C4 plants is mediated by two signalling pathways. One pathway is specific for blue light and involves phototropins, while the second pathway depends on photosyntheticaly active radiation (PAR). Here, the role of Nt MPK4 in light-induced stomatal opening was studied, as silencing of this MAP kinase stimulates stomatal opening. Stomata of Nt MPK4-silenced plants do not close in elevated atmospheric CO₂, and show a reduced response to PAR. However, stomatal closure can still be induced by abscisic acid. Measurements using multi-barrelled intracellular micro-electrodes showed that CO₂ activates plasma membrane anion channels in wild-type Nicotiana tabacum guard cells, but not in Nt MPK4-silenced cells. Anion channels were also activated in wild-type guard cells after switching off PAR. In approximately half of these cells, activation of anion channels was accompanied by an increase in the cytosolic free Ca²⁺ concentration. The activity of anion channels was higher in cells showing a parallel increase in cytosolic Ca²⁺ than in those with steady Ca²⁺ levels. Both the darkness-induced anion channel activation and Ca²⁺ signals were repressed in Nt MPK4-silenced guard cells. These data show that CO₂ and darkness can activate anion channels in a Ca²⁺-independent manner, but the anion channel activity is enhanced by parallel increases in the cytosolic Ca²⁺ concentration. Nt MPK4 plays an essential role in CO₂- and darkness-induced activation of guard-cell anion channels, through Ca²⁺-independent as well as Ca²⁺-dependent signalling pathways.     

Ca2+- and Mg2+-Modulated Lipolysis in Neonatal Rat Brain Slices Observed by One- and Two-Dimensional NMR

Abstract: Superfused cortical brain slices from neonatal rats demonstrated large increases in levels of NMR-detectable lipids after sample preparation and perfusion with standard artificial CSF. These increases were reduced by an average of 58% by perfusion with buffer with low (no added) Ca²⁺ or by perfusion in Ca²⁺-free buffer. Perfusion with buffer with elevated MgSO₄ (10 mmol/L) reduced the lipid changes by 47%. A reduction of 88% was observed in samples perfused in buffer with both low Ca²⁺ and high Mg²⁺, suggesting a role for Mg²⁺ in reducing lipolysis distinct from its known ability to block Ca²⁺ influx.     

Internalization of Voltage-Dependent Sodium Channels in Fetal Rat Brain Neurons: A Study of the Regulation of Endocytosis

Abstract: In fetal rat brain neurons, activation of voltage-dependent Na⁺ channels induced their own internalization, probably triggered by an increase in intracellular Na⁺ level. To investigate the role of phosphorylation in internalization, neurons were exposed to either activators or inhibitors of cyclic AMP- and cyclic GMP-dependent protein kinases, protein kinase C, and tyrosine kinase. None of the tested compounds mimicked or inhibited the effect of Na⁺ channel activation. An increase in intracellular Ca²⁺ concentration induced either by thapsigargin, a Ca²⁺-ATPase blocker, or by A23187, a Ca²⁺ ionophore, was unable to provoke Na⁺ channel internalization. However, Ca²⁺ seems to be necessary because both neurotoxin- and amphotericin B-induced Na⁺ channel internalizations were partially inhibited by BAPTA-AM. The selective inhibitor of Ca²⁺/calmodulin-dependent protein kinase II, KN-62, caused a dose-dependent inhibition of neurotoxin-induced internalization due to a blockade of channel activity but did not prevent amphotericin B-induced internalization. The rate of increase in Na⁺ channel density at the neuronal cell surface was similar before and after channel internalization, suggesting that recycling of internalized Na⁺ channels back to the cell surface was almost negligible. Pretreatment of the cells with an acidotropic agent such as chloroquine prevented Na⁺ channel internalization, indicating that an acidic endosomal/lysosomal compartment is involved in Na⁺ channel internalization in neurons.     

Influence of aluminium on phosphorus and calcium localization in roots of beech (Fagus sylvatica)

Beech plants ( Fagus sylvatica L. provenance Maramures) were grown in nutrient solution at low pH (4.2) and exposed to different concentrations of AlCl₃. Uptake and leakage of Ca²⁺(⁴⁵Ca²⁺) and H²PO₄-(³²P) were studied. A high external aluminium concentration (1.0m M ) reduced the uptake and export to the shoot of both calcium and phosphate, while 0.1 m M Al increased the phosphorus level in the roots. To determine the impact of aluminium on the localization of calcium and phosphate, leakage of the elements from both intact plants and plants frozen prior to the leakage experiment was studied. The leakage of Ca²⁺ from intact plants was not affected by prior exposure to 0.1 m M Al. Freezing of the beech plants before the leakage experiment increased leakage of calcium slightly more from roots of control plants than for roots exposed to 0.1 m M Al, indicating that even low concentrations of alminium may impede the influx of calcium across the plasma membrane in the roots. The patterns of Ca²⁺ leakage from roots previously exposed to 1.0 m M Al indicated that very little Ca²⁺ was located extracellularly. The extracellular fraction of phosphate increased with increasing Al concentration in the nutrient solution. Low Al concentration (0.1 m M ) only reduced the intracellular phosphate concentration to a minor extent, while 1.0 m M Al profoundly decreased it. It is concluded that 0.1 m M AlCl₃ has a limited effect upon the localization of Ca²⁺ and phosphate in the roots. At higher levels of Al, 0.1–1.0 m M , there is a more dramatic change in nutrient localization in the free space and uptake over the plasma membrane.