The inhibition of ATP-dependent shape change of human erythrocyte ghosts correlates with an inhibition of Mg(2+)-ATPase activity by fluoride and aluminofluoride complexes.

The vanadate-sensitive Mg(2+)-dependent ATPase activity of the human erythrocyte ghost is believed to be involved in the shape change events that convert echinocytic ghosts to smoothed forms (biconcave discs and stomatocytes). At physiological salt concentration, pH 7.4, 2 mM ATP, 5 mM Mg2+ and 1 mM EGTA, the Mg(2+)-ATPase activity of ghosts was inhibited strongly by millimolar concentrations of sodium fluoride: I50 = 1.31 +/- 0.23 mM (mean +/- S.D.; n = 12). The addition of aluminium chloride to 15 microM reduced the concentration of NaF required for 50% inhibition to 0.76 +/- 0.21 mM (n = 10). Aluminium alone had only a small inhibitory effect on the ATPase activity (13 +/- 9%; n = 10). Desferrioxamine, a strong chelator of tervalent aluminium ion, failed to reverse the inhibition by fluoride and reversed the inhibition in the presence of aluminium and fluoride back to those values obtained with fluoride alone. Of several metal salts tested only beryllium sulfate was able to replace aluminium as an effective inhibitor in the presence of fluoride. Inhibition of the Mg(2+)-ATPase activity by fluoride and the aluminofluoride complexes correlated with an inhibition of the rate of MgATP-dependent change in red cell ghost shape from echinocytes to smoothed forms. All gross morphological changes of the smoothing process were affected, including the production of discocytes, stomatocytes and endocyctic vesicles.     

Inhibition of nitrifiers and methanotrophs from an agricultural humisol by allylsulfide and its implications for environmental studies.

Allylsulfide, an inhibitor of ammonia monooxygenase, was tested to determine its ability to inhibit nitrification and methane oxidation in pure cultures, in agricultural humisol enrichment cultures, and in humisol slurries. We confirmed that allylsulfide is a differential inhibitor of cultures of nitrifiers and methanotrophs at concentrations of 1 and 200 microM, respectively, which result in 50% inhibition. However, although a nitrifying enrichment culture added to sterilized humisol was inhibited 50% by 4 microM allylsulfide, 500 microM allylsulfide was necessary for 50% inhibition of the endogenous nitrifying activity in nonsterile humisol. We concluded that native nitrifiers were protected, possibly by being in colonial aggregates or sheltered microenvironments.     

Effects of docosahexaenoic acid on the survival and neurite outgrowth of rat cortical neurons in primary cultures

Effects of docosahexaenoic acid (DHA) on survival and neurite outgrowth were investigated in primary cultures of rat cortical neurons. Cell cultures were prepared from cortex on embryonic day 18 (E-18) for treatment with a series of DHA concentrations (12.5, 25, 50, 75, 100 and 200 microM). Docosahexaenoic acid (25-50 microM) significantly enhanced neuronal viability, but lower concentration of DHA (12.5 microM) did not show an obvious effect. In contrast, higher concentrations of DHA (100-200 microM) exerted the significant opposite effects by decreasing neuronal viability. Furthermore, treatment with 25 microM DHA significantly prevented the neurons from death after different culture days in vitro (DIV). Moreover, measurements from the cultures exposed to 25 microM DHA immediately after plating showed significant increases in the percentage of cells with neurites, the mean number of neurite branches, the total neuritic length per cell and the length of the longest neurite in each cell after 24 and 48 h in vitro (HIV). The DHA-treated neurons had greater growth-associated protein-43 (GAP-43) immunoactivity and higher phosphatidylserine (PS) and phosphatidylethanolamine (PE) contents, but lower phosphatidylcholine (PC) content than control neurons. The significant increased DHA contents were also observed in both PE and PS in the treated neurons. These findings suggest that optimal DHA (25 microM) may have positive effects on the survival and the neurite outgrowth of the cultured fetal rat cortical neurons, and the effects probably are related to DHA-stimulating neuron-specific protein synthesis and its enhancing the discrete phospholipid (PL) content through enrichment of DHA in the PL species.     

Embryogenic callus formation,growth and regeneration in callus and suspension cultures of Miscanthus x ogiformis Honda Giganteus' as affected by proline

The effects of proline additions to culture systems of Miscanthus x ogiformis Honda Giganteus' were investigated. Proline was added in concentrations of 0, 12.5, 25, 50, 100 or 300 mM to the callus induction and suspension culture media containing either Murashige and Skoog or N6 basal salts and 22.6 μM 2,4-dichlorophenoxyacetic acid. Shoot apices and leaves from in vitro-propagated shoots, and immature inflorescences from greenhouse-grown plants were used as explants for callus induction and formation. Suspension cultures initiated from embryogenic callus of immature inflorescences were used to test the effect of proline in suspension cultures. The proline additions affected the formation of embryogenic callus and the growth of suspension cultures. Improvements depended on the proline concentration and the basal salts of the medium. Addition of 12.5 to 50 mM proline to callus induction medium with Murashige and Skoog salts increased embryogenic callus formation on shoot apices and leaf explants while proline had no effect on embryogenic callus formation in medium with N6 salts. Increased growth with increasing proline concentration was obtained in suspension aggregates grown in medium with N6 salts, whereas proline only increased growth of suspension aggregates grown in medium with Murashige and Skoog salts at concentrations of 12.5 or 25 mM. A stimulating effect of proline on plant regeneration was observed in short-term cultures of callus as well as in long-term cultures of suspension aggregates. An optimum proline concentration for plant regeneration was found at 12.5 mM. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effect of aluminium on the heam biosynthesis (in vitro) in bone marrow cells in rats.

The study has been carried out on male Wistar rats. The aim of the present study was to trace the effect of aluminium chloride and aluminium nitrate at concentration 10 microM and 20 microM on haem biosynthesis in vitro in bone marrow cell culture. The ability of haem biosynthesis in bone marrow cell culture after 48 h of experiments with aluminium chloride and aluminium nitrate significantly decreased in relation with the control value.     

Model studies of the precipitation of silica in the presence of aluminium; implications for biology and industry

The unique chemical affinity between the oxides of silicon and aluminium has been cited as a potential route for the amelioration of the detrimental effects of aluminium in the environment and in biological systems. A greater understanding of silicon-aluminium interactions may assist in this endeavour and also provide a means of overcoming silica fouling problems encountered by industry which are exacerbated by the presence of aluminium. It is also conceivable that this increased knowledge may demonstrate a positive use for aluminium in the processing of the silicon dioxide phase. In this study we report the effect of aluminium ions, derived from aluminium chloride, on silicic acid species obtained from potassium catecholato complexes of silicon at circumneutral pH at the molar ratios 1000Si:Al, 100Si:Al and 50Si:Al. Silica and low levels of aluminium-rich silica materials were formed with Si:Al ratios of about 3.5:1 comparable with the element ratios detected in senile plaques and aluminium-rich scale. A kinetic study showed that aluminium in the reaction medium slowed down the rate of formation of one of the silica species formed early in the condensation process, e.g. trimers, but increased the rate at which silicic acid was removed from sub 1 nm diameter particles. The materials precipitated in the presence of aluminium were composed of smaller particles and aggregates with smaller pores (Si100:Al and Si50:Al systems) or larger pores (Si1000:Al) compared to the control. The nature of the interactions responsible for these differences is discussed. The effects described here demonstrate the ability of silica and aluminium to interact under conditions such as those found in biological systems. That silica reacts with aluminium in the presence of catechol supports the protective role assigned to silicon.     

Development of a colorimetric method for functional chloride channel assay

Anion channels play significant physiological roles in humans and animals. However, the effort of screening for anion channel modulators was limited by the available assay technologies. This report discusses the development of a cell-based functional chloride channel assay using iodine as the chloride channel functional indicator. Iodine concentrations were measured with modified Sandell-Kolthoff reaction using colorimetric detection. The assay was rapid and quantitative. When WSS-1 cells were activated by gamma-aminobutyric acid (GABA) in the condition that gamma-aminobutyric acid type A receptor (GABAA receptor) conducted outwardly rectifying chloride channel function, the EC50 of GABA was 7.69 microM. IC50s were 0.53 microM for bicuculline and 3.1 microM for picrotoxin, respectively, in the presence of 10 microM GABA. When Capan-1 cells were activated by forskolin, the EC50 was 0.14 microM. The assay can also be applied to inwardly rectifying anion channels as exemplified by GABAA channel with an EC50 of 294 microM. Thus, the assay is universal and reliable and can be used for anion channel high-throughput screening.     

Metallothionein-III antagonizes the neurotoxic and neurotrophic effects of amyloid beta peptides

Metallothionein-III (MT-III) protects cerebral cortical neurons in established culture from the toxic effect of amyloid beta peptides (Abetas). Protection is concentration dependent and approaches 100% at 0.1 microM. The EC(50) value estimated at 5 microM Abeta(1-40) is 2 nM. At higher concentrations (>0.1 microM), MT-III also antagonizes the trophic effect of Abeta(1-40) on cerebral cortical neurons in early cultures. Because only the fibrillar, SDS-resistant form of Abeta aggregates are thought to be neurotoxic, we analyzed and compared Abeta(1-40) aggregates formed in the presence and absence of MT-III using SDS-PAGE. Results show that aggregates formed in the absence of MT-III are predominantly SDS-resistant whereas those formed in its presence are mostly SDS-soluble. Neither MT-I nor -II exhibits any of the effects of MT-III. On the basis of these results, we propose that MT-III alleviates Abetas' neurotoxic effect by abolishing the formation of toxic aggregates of Abetas and that it may play a specific and important role in protecting the brain from the deleterious effects of Abetas.     

Membrane Transport in Isolated Vesicles from Sugarbeet Taproot: III. Effects of Fluoride on ATPase Activity and Transport

The effects of fluoride on the tonoplast type ATPase and transport activities associated with sealed membrane vesicles isolated from sugarbeet (Beta vulgaris L.) storage tissue were examined. This anion had two distinct effects upon the proton-pumping vesicles. When ATP hydrolysis was measured in the presence of gramicidin D, significant inhibition (approximately 50%) only occurred when the fluoride concentration approached 50 millimolar. In contrast, the same degree of inhibition of proton transport occurred when the fluoride concentration was about 24 millimolar. Effects on proton pumping at this concentration of fluoride could be attributed to an inhibition of chloride movement which serves to dissipate the vesicle membrane potential. Valinomycin could partially restore ATPase activity in sealed vesicles which were inhibited by fluoride and this restoration occurred with a reduction in the membrane potential. Fluoride demonstrated a competitive interaction with chloride-stimulation of proton transport and inhibited the uptake of radioactive chloride into sealed vesicles. When the vesicles were allowed to develop a pH gradient in the absence of KCl, and KCl was subsequently added, fluoride reduced enhancement of the existing pH gradient by KCl. The results are consistent with a chloride carrier that is inhibited by fluoride.     

Kinetics of succinic dehydrogenase inhibition by methyl parathion and its recovery by oximes and L-cysteine in hepatopancreas of Lamellidens marginalis

Effects in vitro of methyl parathion on some kinetic constants of succinic dehydrogenase (SDH) in hepatopancreas of freshwater mussel, L. marginalis were studied. Altered pH vs. specific activity curves for SDH demonstrated significant inhibition by methyl parathion in buffered acidic, neutral and alkaline ranges. At high pH ranges IC50 (12.5 microM) of methyl parathion did not cause 50% inhibition enzyme as it did at neutral and acidic pHs. Activation energies (delta E) were found to be increased suggesting decreased efficiency of enzyme in presence of methyl parathion. Non-competitive inhibition with respect to activation by succinate was indicated by decreased maximal velocity (V) without change in Michaelis Menten constant (Km). Pyridine-2-aldoxime (25 microM), pyridine-4-aldoxime (15 microM) and L-cysteine (40 microM) neutralized the inhibition of SDH by methyl parathion (12.5 microM). The kinetic data suggests that inhibition of SDH by methyl parathion was pH and temperature independent.     

Passive loss of proteoglycan from articular cartilage explants

The addition of proteinase inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide, 0.25 mM benzamidine hydrochloride, 6.25 mM EDTA, 12.5 mM 6-aminohexanoic acid and 2 mM iodoacetic acid) to explant cultures of adult bovine articular cartilage inhibits proteoglycan synthesis as well as the loss of the macromolecule from the tissue. Those proteoglycans lost to the medium of explant cultures treated with proteinase inhibitors were either aggregates or monomers with functional hyaluronic acid-binding regions, whereas proteoglycans lost from metabolically active tissue also included a population of monomers that were unable to aggregate with hyaluronate. Analysis of the core protein from proteoglycans lost into the medium of inhibitor-treated cultures showed the same size distribution as the core proteins of proteoglycans present in the extracellular matrix of metabolically active cultures. The core proteins of proteoglycans appearing in the medium of metabolically active cultures showed that proteolytic cleavage of these macromolecules occurred as a result of their loss from the tissue. Explant cultures of articular cartilage maintained in medium with proteinase inhibitors were used to investigate the passive loss of proteoglycan from the tissue. The rate of passive loss of proteoglycan from the tissue was dependent on surface area, but no difference in the proportion of proteoglycan aggregate to monomer appearing in the medium was observed. Furthermore, proteoglycans were lost at the same rate from the articular and cut surfaces of cartilage. Proteoglycan aggregates and monomer were lost from articular cartilage over a period of time, which indicates that proteoglycans are free to move through the extracellular matrix of cartilage. The movement of proteoglycans out of the tissue was shown to be temperature dependent, but was different from the change of the viscosity of water with temperature, which indicates that the loss of proteoglycan was not solely due to diffusion. The activation energy for the loss of proteoglycans from articular cartilage was found to be similar to the binding energies for electrostatic and hydrogen bonds.     

Anti-curare action of stannous ion in the frog neuromuscular junction

For the purpose of elucidating the mechanism of action of stannous ion (Sn2+), we investigated effects of stannous chloride (SnCl2) on the twitch and on the electrical phenomena in the muscle fiber. Sciatic nerve-sartorius muscle preparations from the bullfrog were used as the material. Effect of SnCl2 was examined on the twitch partially inhibited by pretreatment with d-tubocurarine. SnCl2 (1-100 microM) antagonized d-tubocurarine and enhanced the twitch dose-dependently. Tartaric acid, which is the solvent used for SnCl2 solution, had no augmentative effect on the twitch, even at a concentration as high as 250 microM. SnCl2 (1-50 microM) increased the amplitude of the endplate potential; that is, it exerted an anti-curare action. The resting potential and the membrane resistance of the muscle fiber were not altered by 30 microM SnCl2. These findings lead to the conclusion that Sn2+ enhances the twitch by increasing the endplate potential of the muscle fibers.     

Phenylarsine oxide evokes intracellular calcium increases and amylase secretion in isolated rat pancreatic acinar cells

The effects of the thiol reagent, phenylarsine oxide (PAO, 10(-5)-10(-3) M ), a membrane-permeable trivalent arsenical compound that specifically complexes vicinal sulfhydryl groups of proteins to form stable ring structures, were studied by monitoring intracellular free calcium concentration ([Ca2+]i) and amylase secretion in collagenase dispersed rat pancreatic acinar cells. PAO increased [Ca2+]i by mobilizing calcium from intracellular stores, since this increase was observed in the absence of extracellular calcium. PAO also prevented the CCK-8-induced signal of [Ca2+]i and inhibited the oscillatory pattern initiated by aluminium fluoride (AlF-4). In addition to the effects of PAO on calcium mobilization, it caused a significant increase in amylase secretion and reduced the secretory response to either CCK-8 or AlF-4. The effects of PAO on both [Ca2+]i and amylase release were reversed by the sulfhydryl reducing agent, dithiothreitol (2 mM). Pretreatment of acinar cells with high concentration of ryanodine (50 microM) reduced the PAO-evoked calcium release. However, PAO was still able to release a small fraction of Ca2+ from acinar cells in which agonist-releasable Ca2+ pools had been previously depleted by thapsigargin (0.5 microM) and ryanodine receptors were blocked by 50 microM ryanodine. We conclude that, in pancreatic acinar cells, PAO mainly releases Ca2+ from the ryanodine-sensitive calcium pool and consequently induces amylase secretion. These effects are likely to be due to the oxidizing effects of this compound.     

Prevention of thermal inactivation and aggregation of lysozyme by polyamines.

Proteins tend to form inactive aggregates at high temperatures. We show that polyamines, which have a relatively simple structure as oligoamids, effectively prevent thermal inactivation and aggregation of hen egg lysozyme. In the presence of additives, including arginine and guanidine (100 microM), more than 30% of 0.2 mg x mL(-1) lysozyme in sodium phosphate buffer (pH 6.5) formed insoluble aggregates by heat treatment (98 degrees C for 30 min). However, in the presence of 50 mm spermine or spermidine, no aggregates were observed after the same heat treatment. The residual activity of lysozyme after this heat treatment was very low (< 5%), even in the presence of 100 microM arginine and guanidine, while it was maintained at approximately 50% in the presence of 100 microM spermine and spermidine. These results imply that polyamines are new candidates as molecular additives for preventing the thermal aggregation and inactivation of heat-labile proteins.     

Concanavalin A stimulates neuron-substratum adhesion and nerve fiber outgrowth in culture

Dorsal root ganglion neurons in culture proceed through a series of shape changes before growing nerve fibers. These shape changes involve: attachment to the substratum, extension of filopodia, and spreading of part of the cell to form broad lamellipodia. With the formation of lamellipodia, neurons adhere firmly to the substratum and retrogradely transport lectins (concanavalin A, wheat germ agglutinin) on their surfaces. In unspread neurons concanavalin A, but not wheat germ agglutinin, rapidly stimulates lamellipodium formation and neuron-substratum adhesion. Neurons treated with concanavalin A also have more, branched nerve fibers than untreated neurons, but otherwise appear similar. These effects of concanavalin A are concentration dependent, blocked by alpha-methyl-D-mannoside (100 mM), and are accompanied by receptor redistribution. Stimulation of lamellipodium extension by concanavalin A is inhibited by low temperature (4 degrees C), 2,4-dinitrophenol (0.2 mM), cytochalasin D (4 microM), or trifluoperazine (10 microM), but not by cycloheximide (360 microM) or colchicine (12.5 microM). Attachment of neurons to the culture substratum was affected little by these treatments. These results indicate differences in the neuron's metabolic requirements for simple attachment to the substratum and the early phases of nerve fiber growth. Moreover, they suggest a convenient system in which to study the cellular and biochemical events of rapid nerve fiber outgrowth in primary neuronal cultures.     

Effect of quinolinic acid on neurons in dissociated cultures of cells of different structures of the brain

Using neurohistological and cytochemical methods in the living cells, the peculiarities of the action of endogenous neurotoxin, quinolinic acid (QUIN), on the neurons developing in the cell cultures of the hippocamp, neocortex and septum have been investigated in 17-19-day-old mouse embryos. The addition of 500 microM of QUIN on the 21st--22nd day into the nutrition medium in vitro resulted in the rapid destruction of neurons localized in glioneuronal aggregates, while the isolated nervous cells as well as septal cholinergic neurons remained intact. At earlier stages of cultivation (up to 2 weeks) QUIN did not provoke degenerative changes in the cultivated neurons. The comparison of our results with the literary data suggests that in nervous cell cultures QUIN, having mature synaptic connections with afferent nervous fibers, causes destruction of neurons.     

Alpha-adrenergic stimulation activates a calcium-sensitive chloride current in brown fat cells

The first response of brown adipocytes to adrenergic stimulation is a rapid depolarizing conductance increase mediated by alpha-adrenergic receptors. We used patch recording techniques on cultured brown fat cells from neonatal rats to characterize this conductance. Measurements in perforated patch clamped cells showed that fast depolarizing responses were frequent in cells maintained in culture for 1 d or less, but were seen less often in cells cultured for longer periods. Ion substitution showed that the depolarization was due to a selective increase in membrane chloride permeability. The reversal potential for the depolarizing current in perforated patch clamped cells indicated that intracellular chloride concentrations were significantly higher than expected if chloride were passively distributed. The chloride conductance could be activated by increases in intracellular calcium, either by exposing intact cells to the ionophore A23187 or by using pipette solutions with free calcium levels of 0.2-1.0 microM in whole- cell configuration. The chloride conductance did not increase monotonically with increases in intracellular calcium, and going whole cell with pipette-free calcium concentrations > or = 10 microM rapidly inactivated the current. The chloride currents ran down in whole-cell recordings using intracellular solutions of various compositions, and were absent in excised patches. These findings imply that cytoplasmic factors in addition to intracellular calcium are involved in regulation of the chloride conductance. The chloride currents could be blocked by niflumic acid or flufenamic acid with IC50s of 3 and 7 microM, or by higher concentrations of SITS (IC50 = 170 microM), DIDS (IC50 = 50 microM), or 9-anthracene carboxylic acid (IC50 = 80 microM). The chloride conductance activated in whole cell by intracellular calcium had the permeability sequence PNOS > PI > PBr > PCl >> Paspartate, measured from either reversal potentials or conductances. Instantaneous current-voltage relations for the calcium-activated chloride currents were linear in symmetric chloride solutions. Much of the current was time and voltage independent and active at all membrane potentials between -100 and +100 mV, but an additional component of variable amplitude showed time-dependent activation with depolarization. Volume- sensitive chloride currents were also present in brown fat cells, but differed from the calcium-activated currents in that they responded to cell swelling, required intracellular ATP in whole-cell recordings, showed no sensitivity to intracellular or extracellular calcium levels, and were relatively resistant to block by niflumic and flufenamic acids. (ABSTRACT TRUNCATED AT 400 WORDS)     

Formation and characterization of a transition state complex of Azotobacter vinelandii nitrogenase

A stable complex is formed between the nitrogenase proteins of Azotobacter vinelandii, aluminium fluoride and MgADP. All nitrogenase activities are inhibited. The complex formation was found to be reversible. An incubation at 50°C recovers nitrogenase activity. The complex has been characterized with respect to protein and nucleotide composition and redox state of the metal-sulphur clusters. Based on the inhibition by aluminium fluoride together with MgADP, it is proposed that a stable transition state complex of nitrogenase is isolated.     

Cadmium chloride-induced oxidative stress in skeletal muscle cells in vitro

The effects of cadmium chloride (CdCl(2)) on oxidative stress in the skeletal muscle cell line C(2)C(12) were investigated. Myoblast cells that differentiated into myotubes were treated with CdCl(2) (1, 3, 5, 7.5, 10, and 12.5 microM) for 24, 48, and 72 h. Subsequent assay of cell homogenates for MTT (3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide) reduction, neutral red uptake and nucleic acid content showed that cadmium was toxic to C(2)C(12) cells in a concentration-dependent manner. Glutathione-S-transferase activity (nmol microg of protein(-1) min(-1)) was increased with 1 and 3 microM CdCl(2) (36.9 +/- 5.6 and 32.1 +/- 6.0, respectively) compared to control cells (21.8 +/- 1.5), but decreased at higher concentrations (7.5 microM = 15.9 +/- 3.3, 10 microM = 15.9 +/- 4.6, and 12.5 microM = 10.5 +/- 2.8). An increase in malondialdehyde content (nmol microg of protein(-1)), especially at high CdCl(2) concentrations (control = 7.3 +/- 0.5; CdCl(2): 7.5 microM = 11.2 +/- 3.1, 10 microM = 14.6 +/- 3.8, and 12.5 microM = 20.5 +/- 6.5) indicated that there was enhanced lipid peroxidation. Light and scanning electron microscopy showed that there was a concentration-dependent loss of adherent cells and the formation of vesicles indicative of cell death. These results indicated that CdCl(2) increased oxidative stress in C(2)C(12) cells, and this stress probably compromised cell adhesion and the cellular antioxidant defense mechanisms.