Cytotoxicity of Exogenous Acetoacetate in Lithium Salt Form Is Mediated by Lithium and Not Acetoacetate.

BACKGROUND/AIM: The ketogenic diet has recently gained interest as potential adjuvant therapy for cancer. Many researchers have endeavored to support this claim in vitro. One common model utilizes treatment with exogenous acetoacetate in lithium salt form (LiAcAc). We aimed to determine whether the effects of treatment with LiAcAc on cell viability, as reported in the literature, accurately reflect the influence of acetoacetate. MATERIALS AND METHODS: Breast cancer and normal cell lines were treated with acetoacetate, in lithium and sodium salt forms, and cell viability was assessed. RESULTS: The effect of LiAcAc on cells was mediated by Li ions. Our results showed that the cytotoxic effects of LiAcAc treatment were significantly similar to those caused by LiCl, and also treatment with NaAcAc did not cause any significant cytotoxic effect. CONCLUSION: Treatment of cells with LiAcAc is not a convincing in vitro model for studying ketogenic diet. These findings are highly important for interpreting previously published results, and for designing new experiments to study the ketogenic diet in vitro.

Deposition and aggregation kinetics of rotavirus in divalent cation solutions.

Aggregation kinetics of rotavirus in aqueous solutions and its deposition kinetics on silica surface in the presence of divalent (Ca(2+), Mg(2+)) cations were studied using complementary techniques of time-resolved dynamic light scattering (TR-DLS) and quartz crystal microbalance (QCM). Within a reasonable temporal window of 4 h, aggregation could be observed at levels as low as 10 mM of Ca(2+) and 20 mM of Mg(2+). Attachment efficiencies were always greater in Ca(2+) solutions of the same concentration, and the critical coagulation concentration (CCC) for rotavirus in Ca(2+) solutions was slightly smaller than that in Mg(2+) solutions. No aggregation was detected in Na(+) solution within the temporal window of 4 h. Deposition experiments showed higher attachment coefficients in solutions containing Ca(2+) compared to those obtained in Mg(2+) solution. The classic Derjaguin-Landau-Verwey-Overbeek (DLVO) theory failed to predict both the aggregation behavior of rotavirus and its deposition on silica surface. Besides electrostatic interactions, steric repulsions and specific interactions with divalent cations were important mechanisms in controlling rotavirus deposition and aggregation. Experimental results presented here suggest that rotavirus is not expected to aggregate in groundwater with typical hardness (up to 6 mM Ca(2+)) and rotavirus deposition on silica soil would be more favorable in the presence of Ca(2+) than Mg(2+).

Effect of proline and K+ on the stimulation of cellular activities in Escherichia coli K-12 under high salinity.

Growth of Escherichia coli K-12 in a modified Davis minimal medium was inhibited under high osmolarity, but it recovered remarkably with the addition of 1 mM proline. The co-existence of K+ with proline enhanced the recovery of growth under high osmolarity more than that in the presence of proline alone. The same was true for the activities of respiration and glucose uptake. A similar supplementary effect of K+ was observed for the activities of proline uptake under high osmolarity. These results suggest that K+ and proline support not only growth but respiration and uptake of the respiratory substrate glucose in the cell cytoplasm when exposed to high osmolarity. External K+ almost disappeared with 1 h of incubation at low osmolarity, indicating that active accumulation of K+ in the cells occurred. On the other hand, a gradual accumulation of K+ was recognized at high osmolarity in the presence of 1 M NaCl, especially at > 2 h of incubation. This study of L-[5-3H]proline uptake in the cell cytoplasm indicates that proline was incorporated as a substrate of protein synthesis in the absence of NaCl, but was efficiently utilized as a compatible solute in the presence of high concentrations of NaCl.

Evaluation of potassium ion as the endothelium-derived hyperpolarizing factor (EDHF) in the bovine coronary artery.

1. This study explored the role of the potassium ion in endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilatation in the bovine coronary artery. 2. Bradykinin-induced, EDHF-mediated vasodilatation was blocked by the Na(+)-K(+) ATPase inhibitor, ouabain (1 micro M), in a time-dependent manner, with maximal blockade seen after 90 min. In contrast, the K(IR) channel inhibitor, Ba(2+) (30 micro M), had no effect. 3. When the potassium content of the bathing solution was increased in a single step from 5.9 to 7-19 mM, powerful vasodilatation (max. 75.9+/-3.6%) was observed. Vasodilatation was transient and, consequently, cumulative addition of potassium produced little vasodilatation, with vasoconstriction predominating at the higher concentrations. 4. The magnitude of potassium-induced vasodilatation was similar in endothelium-containing and endothelium-denuded rings, and was unaffected by Ba(2+) (30 micro M), but abolished by ouabain (1 micro M). 5. Ouabain (1 micro M, 90 min) powerfully blocked bradykinin-induced, nitric oxide-mediated vasodilatation as well as that induced by the nitrovasodilator, glyceryl trinitrate, but that induced by the K(ATP) channel opener, levcromakalim, was hardly affected. 6. Thus, activation of Na(+)-K(+) ATPase is likely to be involved in the vasodilator responses of the bovine coronary artery to both nitric oxide and EDHF. These findings, together with the ability of potassium to induce powerful, ouabain- but not Ba(2+)-sensitive, endothelium-independent vasodilatation, are consistent with this ion contributing to the EDHF response in this tissue.

Characteristic changes of the S2/S1 difference FTIR spectrum induced by Ca2+ depletion and metal cation substitution in the photosynthetic oxygen-evolving complex.

Effects of Ca2+ depletion and substitution with other metal cations on the structure of the protein matrices of the oxygen-evolving complex (OEC) and their corresponding changes upon the S1 to S2 transition were examined using Fourier transform infrared (FTIR) spectroscopy. Ca2+ depletion and further supplementation with Li+, Na+, Mg2+, Ca2+, or Sr2+ did not significantly affect the typical vibrational features in the double difference S2/S1 spectrum, including the symmetric [1365(+)/1404(-) cm(-1)] and the asymmetric [1587(+)/1566(-) cm(-1)] stretching modes of the carboxylate ligand and the amide I and II modes of the backbone polypeptides. On the other hand, supplementation with K+, Rb+, Cs+, or Ba2+ significantly modified the S2/S1 spectrum, in which the carboxylate modes disappeared and the amide I and II modes were modified. Results indicate that the binding of metal cations that have ionic radii larger than that of Ca2+ to the Ca2+ site induces perturbations in the protein matrices in the vicinity of the Mn cluster to interrupt the characteristic structural and/or conformational changes upon the oxidation of the Mn cluster accompanied with the S1 to S2 transition. The spectrum was also altered by the supplementation of Cd2+, which has an ionic radius comparable to that of Ca2+. A single-pulse-induced S2/S1 difference spectrum revealed that bands that have been assigned to the vibrational modes for the Y(Z) tyrosine and the histidine ligand for the Mn cluster were not induced in the K+-supplemented membranes, although the histidine band is likely to be preserved in the Ca2+-depleted membranes. The Y(Z) band was considerably small in the double difference S2/S1 spectrum in the Ca2+-depleted and the cation-substituted membranes but distinctively present in the Sr2+- or Ca2+-replenished membranes. Furthermore, cation supplementation induced several new bands that disappeared following the Ca2+ replenishment. These results suggest that the proper organization of the hydrogen bond network within OEC for the water oxidation chemistry requires the Ca2+ ion and indicate that the role of Ca2+ is not purely structurally defined by the physical properties of the ion, such as valence and ionic radius. On the basis of these and other findings, we propose that Ca2+ is necessary for the formation of the hydrogen bond network that is involved in the reaction step of water oxidation.

Characterization of rabbit kidney and brain pancreatic polypeptide-binding neuropeptide Y receptors: differences with Y1 and Y2 sites in sensitivity to amiloride derivatives affecting sodium transport.

Sites sensitive to human and rat pancreatic polypeptides (hPP and rPP) accounted for more than 30% of the specific binding of [125I](Leu31,Pro34) human peptide YY (LP-PYY) in particulates from rabbit kidney cortex, and about 10% of the specific binding in membranes from rabbit hypothalamus. The binding of [125I]hPP or [125I]rPP showed a high-affinity displacement with either hPP, rPP, LP-PYY, neuropeptide Y or peptide YY (Ki below 50 pM for all), while being quite insensitive to Y2-selective ligands. The PP binding had a high sensitivity to alkali cations and inhibitors of phospholipase C, very similar to that of LP-PYY binding 'masked' by excess cold hPP. However, as different from the Y1-like LP-PYY binding, but similar to the binding of the Y2-selective ligand [125I]human peptide YY(3-36) (hPYY(3-36)), the PP binding showed a low sensitivity to guanosine polyphosphates. The PP binding was much more sensitive to N5-substituted amiloride inhibitors of Na+ transport than the binding of LP-PYY, or that of hPYY(3-36). The inhibition of PP binding by N5-substituted amilorides was not enhanced by guanine nucleotides or by phospholipase C blockers. However, pairing of N5-substituted amilorides disproportionately increased the inhibition of hPP binding. Thus, in rabbit kidney or hypothalamus, the high-affinity PP-responding sites share some of the basic properties of the Y1 and Y2 sites, but are distinguished from both by a high sensitivity to compounds affecting sodium transport. These PP/NPY receptors could associate with membrane structures involved in the control of ion balance and osmotic responses.

Influence of altered plasma membrane fatty acid composition on cesium transport characteristics and toxicity in Saccharomyces cerevisiae.

The influence of altered plasma membrane fatty acid composition on cesium uptake and toxicity was investigated in Saccharomyces cerevisiae. Detailed kinetic studies revealed that both the Vmax and Km values for Cs+ transport increased (by approximately twofold in the latter case) when S. cerevisiae was grown in medium supplemented with the polyunsaturated fatty acid linoleate. In addition, Cs+ uptake by linoleate-enriched cells was considerably less sensitive to the competitive effects of other monovalent cations (K+, Rb+, and NH4+) than that by unsupplemented cells. Stimulation of Cs+ uptake in the presence of certain K+ and Rb+ concentrations was only evident in linoleate-enriched S. cerevisiae. At 100 mM CsCl, the initial rate of Cs+ uptake was greater in linoleate-supplemented cells than in unsupplemented cells and this was reflected in a more rapid displacement of cellular K+. However, little difference in net Cs+ accumulation between linoleate-supplemented and unsupplemented cells was evident during prolonged incubation in buffer or during growth. Thus, Cs+ toxicity was similar in linoleate-supplemented and unsupplemented cells. The results were consistent with the Cs+ (K+) transport mechanism adopting an altered conformational state in linoleate-enriched S. cerevisiae.

Characterization of vacuolar arginine uptake and amino acid efflux in Neurospora crassa using cupric ion to permeabilize the plasma membrane.

Treatment of Neurospora crassa mycelia with cupric ion has been shown to permeabilize the plasma and mitochondrial membranes. Permeabilized mycelia were shown to take up arginine into the vacuoles. Uptake was ATP-independent and appeared to be driven by an existing K+-gradient. The kinetic characteristics of the observed uptake were similar to those observed using vacuolar membrane vesicles: the Km for arginine uptake was found to be 4.2-4.5 mM. Permeabilized mycelia were used to study the regulation of arginine uptake into vacuoles. The results suggest that uptake is relatively indifferent to the contents of the vacuoles and is not affected by growth of mycelia in amino acid-supplemented medium. Efflux of arginine, lysine, and ornithine from vacuoles was also measured using mycelia permeabilized with cupric ion. Arginine release was shown to be specifically enhanced by cytosolic ornithine and/or increases in the vacuolar pool of arginine or ornithine. Lysine efflux was shown be indifferent to the presence of other amino acids. These observations emphasize the importance of vacuolar compartmentation in controlling arginine and ornithine metabolism and suggest that vacuolar compartmentation may play an important role in nitrogen homeostasis of filamentous fungi.

Differences in cation sensitivity of ligand binding to Y1 and Y2 subtype of neuropeptide Y receptor of rat brain.

The binding of selective ligands to the Y1 subtype of neuropeptide Y receptor in rat brain particulates was promoted by Ca2+ and also stimulated by Sr2+, but reversibly reduced by Ba2+, Mg2+, Mn2+, by the organic polycations neomycin and spermidine, and by chelating agents. The alkali monovalent cations inhibited the Ca(2+)-enabled Y1 subtype binding with some selectivity (Cs+ > or = NH4+ > Li+ > Na+, K+), with half-inhibition between 70-120 mM. The specific Y2 subtype binding was enhanced by all alkaline-earth divalent cations, Mn2+, neomycin and spermidine in the range of 0.1-10 mM, and by alkali cations at up to 100 mM, and also by Na+ salts of the chelators EGTA and EDTA. The large disparity in cation sensitivity indicates substantial differences in the structure of the binding sites of the Y1 and Y2 receptors, predictable from known distinct features of ligand epitopes and of primary structure of the receptors.

Active recombinant human cytosolic phospholipase A2 is expressed in Escherichia coli.

The cDNA encoding human cytosolic phospholipase A2 (cPLA2) has been subcloned into a prokaryotic pET16b expression vector which also encodes an amino-terminal deca-histidine affinity tag to facilitate purification of the recombinant enzyme. Soluble, active fusion protein, designated His-cPLA2, has been obtained reproducibly from this expression system using the E. coli strain BL21 (DE3). The protein has been purified to homogeneity in four steps and the mass confirmed by electrospray mass spectrometry. His-cPLA2 was characterized by kinetic analysis which demonstrated that the enzyme is similar to native cPLA2 in all respects investigated. Specifically, the enzyme binds to anionic vesicles containing substrate, and acts processively on these vesicles. Enzymatic activity is supported by the presence of Ca2+ and several other divalent metal ions, and is inhibited by several transition metal ions. Finally, the enzyme demonstrates lysophospholipase activity and exhibits a high selectivity for sn-2 arachidonyl esters. This prokaryotic expression system yields moderate amounts of unmodified recombinant His-cPLA2 and is advantageous for rapid production of protein and mutational analyses.

Ammonium ions stimulate in vitro the activity of phosphoenolpyruvate carboxylase from leaves of Amaranthus hypochondriacus, a C4 plant: evidence for allosteric activation.

Ammonium ions stimulated in vitro the activity of PEP carboxylase (PEPC) extracted from dark-adapted leaves of Amaranthus hypochondriacus. Maximum stimulation of 80 to 85% occurred at 50 microM ammonium chloride. There was a marginal inhibition of PEPC at 5 mM ammonium chloride. Among several ions tested, potassium ions stimulated PEPC to a limited extent of about 30%. In presence of ammonium, there was no change either in the sensitivity of enzyme to malate or in the affinity for substrate, PEP. On the other hand, glucose-6-phosphate, an allosteric activator, which stimulated the enzyme by two-fold, could enhance PEPC activity by < 20% in the presence of ammonium. The light-activated form of PEPC from leaves of Amaranthus hypochondriacus was not stimulated, but was inhibited in the presence of ammonium. Our results demonstrate that ammonium ions stimulate PEPC by acting at the allosteric site. Ammonium ion being a component of plant metabolism could be an important regulator of PEPC, particularly in C4 plants.

Calcium release-activated calcium current in rat mast cells.

1. Whole-cell patch clamp recordings of membrane currents and fura-2 measurements of free intracellular calcium concentration ([Ca2+]i) were used to study the biophysical properties of a calcium current activated by depletion of intracellular calcium stores in rat peritoneal mast cells. 2. Calcium influx through an inward calcium release-activated calcium current (ICRAC) was induced by three independent mechanisms that result in store depletion: intracellular infusion of inositol 1,4,5-trisphosphate (InsP3) or extracellular application of ionomycin (active depletion), and intracellular infusion of calcium chelators (ethylene glycol bis-N,N,N',N'-tetraacetic acid (EGTA) or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)) to prevent reuptake of leaked-out calcium into the stores (passive depletion). 3. The activation of ICRAC induced by active store depletion has a short delay (4-14 s) following intracellular infusion of InsP3 or extracellular application of ionomycin. It has a monoexponential time course with a time constant of 20-30 s and, depending on the complementary Ca2+ buffer, a mean normalized amplitude (at 0 mV) of 0.6 pA pF-1 (with EGTA) and 1.1 pA pF-1 (with BAPTA). 4. After full activation of ICRAC by InsP3 in the presence of EGTA (10 mM), hyperpolarizing pulses to -100 mV induced an instantaneous inward current that decayed by 64% within 50 ms. This inactivation is probably mediated by [Ca2+]i, since the decrease of inward current in the presence of the fast Ca2+ buffer BAPTA (10 mM) was only 30%. 5. The amplitude of ICRAC was dependent on the extracellular Ca2+ concentration with an apparent dissociation constant (KD) of 3.3 mM. Inward currents were nonsaturating up to -200 mV. 6. The selectivity of ICRAC for Ca2+ was assessed by using fura-2 as the dominant intracellular buffer (at a concentration of 2 mM) and relating the absolute changes in the calcium-sensitive fluorescence (390 nm excitation) with the calcium current integral. This relationship was almost identical to the one determined for Ca2+ influx through voltage-activated calcium currents in chromaffin cells, suggesting a similar selectivity. Replacing Na+ and K+ by N-methyl-D-glucamine (with Ca2+ ions as exclusive charge carriers) reduced the amplitude of ICRAC by only 9% further suggesting a high specificity for Ca2+ ions. 7. The current amplitude was not greatly affected by variations of external Mg2+ in the range of 0-12 mM. Even at 12 mM Mg2+ the current amplitude was reduced by only 23%. 8. ICRAC was dose-dependently inhibited by Cd2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonselective cation channel activated by patch excision from lobster olfactory receptor neurons.

A nonselective cation channel activated by patch excision was characterized in inside-out patches from spiny lobster olfactory receptor neurons. The channel, which was permeable to Na+, K+ and Cs+, had a conductance of 320 pS and was weakly voltage dependent in the presence of micromolar divalent cations. Millimolar internal divalent cations caused a voltage- and concentration-dependent block of Na+ permeation. Analysis of the voltage dependence indicated that the proportion of the membrane's electric field sensed by Mg2+ was greater than 1, suggesting that the channel contains a multi-ion pore. Internal divalent cations also reduced the frequency of channel opening in a concentration-dependent, but not voltage-dependent, manner, indicating that different cation binding sites affect gating and conductance. While block of gating prevented determining if internal divalent cations permeate the channel, a channel highly permeable to external divalent cations was observed upon patch excision to the inside-out configuration. The monovalent and divalent cation conductances shared activation by patch excision, weak voltage dependence, and steady-state activity, suggesting that they are the same channel. These data extend our understanding of this type of channel by demonstrating permeation by monovalent cations, detailing Mg2+ block of Na+ permeation, and demonstrating the channel's presence in arthropods.

Effects of monovalent cations on red cell shape and size.

Human erythrocytes were incubated in isotonic solutions of different monovalent cations. The apparent size of the red cells measured on scanning electron microscopic pictures decreases in the order Li+ greater than Na+ = K+ greater than Rb+. These differences in size are abolished after pretreatment with trypsin, which removes a large part of the charges associated with membrane glycoproteins. Shape alterations are also observed. Normal biconcave shapes are visible after Na+ or K+ incubation, whereas Li+ leads to flabby, flattened cells with a certain tendency to crenation, and Rb+ causes more pronounced biconcavity with a certain tendency to cupping. The overall effects of pretreatment with trypsin are similar to those of Li+. Our results provide evidence that the electrostatic repulsion of glycoproteins and other charged membrane components may play an essential role in maintaining red cell shape.

Calcium ion binding to lobster nerve membranes.

The binding of 45Ca2+ to membrane material isolated from lobster walking leg nerves was studied using a rapid filtration technique. In solutions of high ionic strength (450 mM), the amount of 45Ca2+ bound to this membrane material was found to be highly dependent on the monovalent cation used in the incubating solution. The amount of 45Ca2+ bound was larger when the membranes were incubated in a KCl solution compared to when they were incubated in a NaCl solution. This difference was attributed to the ability of these closed membrane vesicles to accumulate Ca2+ into the vesicle when incubating in a KCl solution but not in a NaCl solution. This accumulation of Ca2+ was found to be independent of metabolic energy and depended primarily on the absence of Na+ from the incubation medium. At low ionic strength, the membranes formed open fragments and the amount of Ca2+ bound was no longer sensitive to the monovalent cation species in the incubation solution. The 45Ca2+ bound under these low ionic strength conditions was considered to be bound to anionic sites on the membranes.

Inhibition of dihydrostreptomycin action on Mycobacterium smegmatis by monovalent and divalent cation salts.

We have examined and compared the effects of monovalent and divalent cation salts on dihydrostreptomycin (DSM) action against Mycobacterium smegmatis. The Sauton synthetic liquid medium used was supplemented with test salts on the basis of ionic strength (mu). Turbidimetric growth experiments showed that 0.02 M MgSO(4) (mu = 0.08) prevented growth inhibition by 0.1 mug of dihydrostreptomycin per ml, but 0.02 M NaCl (mu = 0.02) did not. However, at molarities equivalent to mu = 0.08, four monovalent cation salts, including NaCl, Na(2)SO(4), NH(4)Cl, and (NH(4))(2)SO(4), all prevented inhibition by dihydrostreptomycin. When magnesium and sodium salts were compared at mu = 0.02, 0.04, and 0.05, two distinct growth protective patterns were seen. These data were indicative of two different mechanisms of dihydrostreptomycin antagnosim by salts; the first being divalent cation and concentration dependent, and the second being nonspecific and ionic strength dependent. Viability studies supported the existence of two mechanisms.