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.

Succinate, iron chelation, and monovalent cations affect the transformation efficiency of Acinetobacter baylyi ATCC 33305 during growth in complex media.

Natural transformation is the acquisition of new genetic material via the uptake of exogenous DNA by competent bacteria. Acinetobacter baylyi is model for natural transformation. Here we focus on the natural transformation of A. baylyi ATCC 33305 grown in complex media and seek environmental conditions that appreciably affect transformation efficiency. We find that the transformation efficiency for A. baylyi is a resilient characteristic that remains high under most conditions tested. We do find several distinct conditions that alter natural transformation efficiency including addition of succinate, Fe2+ (ferrous) iron chelation, and substitution of sodium ions with potassium ones. These distinct conditions could be useful to fine tune transformation efficiency for researchers using A. baylyi as a model organism to study natural transformation.

A novel copper(I) complex induces ER-stress-mediated apoptosis and sensitizes B-acute lymphoblastic leukemia cells to chemotherapeutic agents.

A phosphine copper(I) complex [Cu(thp)4][PF6] (CP) was recently identified as an efficient in vitro antitumor agent. In this study, we evaluated the antiproliferative activity of CP in leukemia cell lines finding a significant efficacy, especially against SEM and RS4;11 cells. Immunoblot analysis showed the activation of caspase-12 and caspase-9 and of the two effector caspase-3 and -7, suggesting that cell death occurred in a caspase-dependent manner. Interestingly we did not observe mitochondrial involvement in the process of cell death. Measures on semipurified proteasome from RS4;11 and SEM cell extracts demonstrated that chymotrypsin-, trypsin- and caspase-like activity decreased in the presence of CP. Moreover, we found an accumulation of ubiquitinated proteins and a remarkable increase of ER stress markers: GRP78, CHOP, and the spliced form of XBP1. Accordingly, the protein synthesis inhibitor cycloheximide significantly protected cancer cells from CP-induced cell death, suggesting that protein synthesis machinery was involved. In well agreement with results obtained on stabilized cell lines, CP induced ER-stress and apoptosis also in primary cells from B-acute lymphoblastic leukemia patients. Importantly, we showed that the combination of CP with some chemotherapeutic drugs displayed a good synergy that strongly affected the survival of both RS4;11 and SEM cells.

pH dependent transfer of nano-pores into membrane of cancer cells to induce apoptosis.

Proper balance of ions in intracellular and extracellular space is the key for normal cell functioning. Changes in the conductance of membranes for ions will lead to cell death. One of the main differences between normal and cancerous cells is the low extracellular pHe and the reverse pH gradient: intracellular pHi is higher than extracellular pHe. We report here pH-selective transfer of nano-pores to cancer cells for the dis-regulation of balance of monovalent cations to induce cell death at mildly acidic pHe as it is in most solid tumors. Our approach is based on the pH-sensitive fusion of cellular membrane with the liposomes containing gramicidin A forming cation-conductive ß-helix in the membrane. Fusion is promoted only at low extracellular pH by the pH (Low) Insertion Peptide (pHLIP®) attached to the liposomes. Gramicidin channels inserted into the cancer cells open flux of protons into the cytoplasm and disrupt balance of other monovalent cations, which induces cell apoptosis.

[Influence of metal ions and specific chemical reagents on activity of alpha-L-rhamnosidase of Eupenicillium erubescens].

The effect of cations, anions and specific chemical reagents: 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide methiodide, EDTA, o-phenanthroline, dithiotreitol, L-cysteine, beta-mercaptoethanol, p-chlormercurybenzoate (p-ChMB), N-ethylmaleimide on the activity of alpha-L-rhamnosidase of Eupenicillium erubescens has been investigated. The essential role of Ag+ and Hg2+ which inhibit the alpha-L-rhamnosidase activity by 47-73% has been shown. Whereas L-cysteine exhibits the protective effect, rhamnose in concentration of 1-5 mM does not protect the enzyme from negative effect of Ag+ and Hg2+. Basing on the inhibitory and kinetic analysis it was supposed that the carboxyl group of C-terminal aminoacid and imidazole group of histidine take part in the catalytic action of alpha-L-rhamnosidase. It was assumed that sulphydryl groups took part in catalysis, carried out by alpha-L-rhamnosidase of E. erubescens, since the activity of alpha-L-rhamnosidase inhibited by p-ChMB and thiol reagents such as dithiothreitol, L-cysteine, beta-mercaptoethanol did not remove its inhibitory action.

Single molecular observation of self-regulated kinesin motility.

Kinesin-1 is an ATP-driven molecular motor that transports various cargoes in cells, a process that can be regulated by the kinesin tail domain. Here, kinesin ATPase activity and motility were inhibited in vitro by interacting the kinesin heavy chain C-terminal tail domain with the kinesin N-terminal motor domain. Though the tail domain can directly interact with microtubules, we found 70% of tail domains failed to bind in the presence of >100 mM (high) KCl, which also modulated the ATPase inhibition manner. These observations suggest that self-inhibition of kinesin depends on electrostatic interactions between the motor domain, the tail domain, and a microtubule. Furthermore, we observed self-regulated behavior of kinesin at the single molecule level. The tail domain did not affect motility velocity, but it did lower the binding affinity of the motor domain to the microtubule. The decrement in binding was coupled to ATPase inhibition. Meanwhile, the tail domain transfected into living cells not only failed to bind to microtubules but also inhibited the motor domain and microtubule interaction, in agreement with our in vitro results. Furthermore, at high potassium concentrations, the self-regulation of kinesin observed in cells was like that in vitro. The results favor a way tail inhibition mechanism where the tail domain masks the microtubule binding site of the motor domain in high potassium concentration.

Functional expression of purinergic P2X7 receptors in pregnant rat myometrium.

ATP has been reported to enhance the membrane conductance of myometrial cells and uterine contractility. Purinergic P2 receptor expression has been reported in the myometrium, using molecular biology, but the functional identity of the receptor subtype has not been determined. In this study, ATP-induced currents were recorded and characterized in single myometrial cells from pregnant rats using whole cell patch clamping. Extracellular ATP was applied in the range of 10 muM-1 mM and induced currents with an EC(50) of 74 muM, with no desensitization, time dependency, or voltage dependency. The currents induced carried multiple monovalent cations, with conductances ranked as K(+) > Cs(+) > Li(+) > Na(+). They were activated by P2X receptor agonists, with their effectiveness ranked as 2',3'-O-(4-benzoylbenzoyl)-ATP >> ATP > alphabeta-methylene-ATP > 2-methylthio ATP > or = UTP > or = GTP > ADP. These currents were blocked by the selective P2X7 receptor antagonist 3-[5-(2,3-dichlorophenyl)-1 H-tetrazol-1-yl]methyl pyridine (A-438079). We therefore concluded that ATP-induced currents in rat myometrial cells crossed cell membranes via P2X7 receptors. We further showed that the ATP-induced currents were blocked by extracellular Mg(2+) (IC(50) = 0.26 mM). Clinically, administering extracellular Mg(2+) is known to inhibit uterine contraction. It therefore seems likely that uterine contraction may be induced by raised extracellular ATP and suppressed via Mg(2+) inhibiting P2X7 receptors. Further research is needed into the P2X7 receptor as a therapeutic target in abnormal uterine contraction, as a possible treatment for premature labor.

Influence of salts and natural organic matter on the stability of bacteriophage MS2.

The stability of functionalized nanoparticles generally results from both steric and electrostatic interactions. Viruses like bacteriophage MS2 have adopted similar strategies for stability against aggregation, including a net negative charge under natural water conditions and using polypeptides that form loops extending from the surface of the protein capsid for stabilization. In natural systems, dissolved organic matter can adsorb to and effectively functionalize nanoparticle surfaces, affecting the fate and transport of these nanoparticles. We used time-resolved dynamic light scattering to measure the aggregation kinetics of a model virus, bacteriophage MS2, across a range of solution chemistries to determine what factors might destabilize viruses in aquatic systems. In monovalent electrolytes (LiCl, NaCl, and KCl), aggregation of MS2 could not be induced within a reasonable kinetic time frame, and MS2 was stable even at salt concentrations greater than 1.0 M. Aggregation of MS2 could be induced in divalent electrolytes when we employed Ca(2+). This trend was also observed in solutions containing 10 mg/L Suwannee River organic matter (SROM) reference material. Even at Ca(2+) concentrations as high 200 mM, diffusion-controlled aggregation was never achieved, demonstrating an additional barrier to aggregation. These results were confirmed by small-angle X-ray scattering experiments, which indicate a transition from repulsive to attractive interactions between MS2 virus particles as monovalent salts are replaced by divalent salts.

Characterization of ATPase activity of class II chaperonin from the hyperthermophilic archaeon Pyrococcus furiosus.

To understand how molecular damage under harsh environmental conditions can be controlled, we investigated the properties of ATPase activity of the chaperonin molecular machinery from the hyperthermophilic archaeon Pyrococcus furiosus (PfCPN). PfCPN ATPase activity depended on K(+) and Mg(2+) and its optimal pH was 7.5. PfCPN had almost no ADPase activity. ADP strongly competitively inhibited PfCPN ATPase activity. Inhibition of PfCPN ATPase decreased its chaperonin activity in protecting lysozyme from heat-induced inactivation.

Effect of monovalent cations and G-quadruplex structures on the outcome of intramolecular homologous recombination.

Homologous recombination is a very important cellular process, as it provides a major pathway for the repair of DNA double-strand breaks. This complex process is affected by many factors within cells. Here, we have studied the effect of monovalent cations (K+, Na+, and NH4+) on the outcome of recombination events, as their presence affects the biochemical activities of the proteins involved in recombination as well as the structure of DNA. For this purpose, we used an in vitro recombination system that includes a protein nuclear extract, as a source of recombination machinery, and two plasmids as substrates for intramolecular homologous recombination, each with two copies of different alleles of the human minisatellite MsH43. We found that the presence of monovalent cations induced a decrease in the recombination frequency, accompanied by an increase in the fidelity of the recombination. Moreover, there is an emerging consensus that secondary structures of DNA have the potential to induce genomic instability. Therefore, we analyzed the effect of the sequences capable of forming G-quadruplex on the production of recombinant molecules, taking advantage of the capacity of some MsH43 alleles to generate these kinds of structure in the presence of K+. We observed that the MsH43 recombinants containing duplications, generated in the presence of K+, did not include the repeats located towards the 5'-side of the G-quadruplex motif, suggesting that this structure may be involved in the recombination events leading to duplications. Our results provide new insights into the molecular mechanisms underlying the recombination of repetitive sequences.

Mechanism of direct conversion between C8 adducts and N7 adducts in carcinogenic reactions of arylnitrenium ions with purine nucleosides: a theoretical study.

To validate the mechanism of direct formation of C8 adducts for a series of complicated and controversial carcinogenic reactions, in this study, we examine the key direct conversion process between N7 adducts and C8 adducts in some theoretical models. First, the mechanism of direct conversions between N7 adducts and C8 adducts suggested by some experiments is explored. For the first time, it is approved that direct conversion between N7 and C8 adducts does not proceed because of very high activation energy. Second, the relative stabilities of the N7 adducts and C8 adducts for these model reactions are evaluated by high-level calculations. Our results indicate that C8 adducts are more stable in aqueous solution. Third, on the basis of these findings, experimental phenomenon of rich C8 adducts and rare N7 adducts is well explained theoretically, and the correctness of the three different existing experimental schemes of these reactions is discussed. Finally, the mechanism of formation of C8 adducts by additions of arylnitrenium ions directly to C8 positions of nucleotide bases in DNA is supported.

Influence of extracellular monovalent cations on pore and gating properties of P2X7 receptor-operated single-channel currents.

Using the patch-clamp method, we studied the influence of external alkali and organic monovalent cations on the single-channel properties of the adenosine triphosphate (ATP)-activated recombinant human P2X(7) receptor. The slope conductance of the hP2X(7) channel decreased and the reversal potential was shifted to more negative values as the ionic diameter of the organic test cations increased. From the relationship between single-channel conductance and the dimensions of the inward current carrier, the narrowest portion of the pore was estimated to have a mean diameter of approximately 8.5 A. Single-channel kinetics and permeation properties remained unchanged during receptor activation by up to 1 mM ATP(4-) for >1 min, arguing against a molecular correlate of pore dilation at the single P2X(7) channel level. Substitution of extracellular Na(+) by any other alkali or organic cation drastically increased the open probability of the channels by prolonging the mean open time. This effect seems to be mediated allosterically through an extracellular voltage-dependent Na(+) binding site with a K(d) of approximately 5 mM Na(+) at a membrane potential of -120 mV. The modulation of the ATP-induced hP2X(7) receptor gating by extracellular Na(+) could be well described by altering the rate constant from the open to the neighboring closed state in a C-C-C-O kinetic receptor model. We suggest that P2X(7) receptor-induced depolarization and associated K(+)-efflux may reduce Na(+) occupancy of the regulatory Na(+) binding site and thus increase the efficacy of ATP(4-) in a feed-forward manner in P2X(7) receptor-expressing cells.

Molecular cloning and characterization of ouabain-insensitive Na(+)-ATPase in the parasitic protist, Trypanosoma cruzi.

Maintaining low intracellular sodium concentrations is vital for almost all organisms. Na(+) efflux is generally governed by P-type ATPases, Na(+)/K(+)-ATPase in animals and Na(+)-ATPase, called ENA, in fungi and plants. Trypanosoma cruzi, which parasitizes mammalian cells, must undergo drastic adaptations to high Na(+) concentrations outside and low Na(+) concentrations inside host cells. However, T. cruzi Na(+) efflux pumps have not been identified. We report here the cloning and characterization of the gene encoding Na(+)-ATPase in T cruzi, which resembled fungal and plant ENAs, termed TcENA. TcENA was a plasma membrane protein expressed throughout the parasite life cycle. The transcription level of TcENA was higher in insect stage epimastigotes and blood stream trypomastigotes than in intracellular amastigotes, probably reflecting the high Na(+) concentration outside the host cells. Biochemical analysis of TcENA expressed heterologously in mammalian cells demonstrated, for the fist time, that the ATPase activity of TcENA is stimulated by both Na(+) and K(+) and is insensitive to ouabain, a specific inhibitor of Na(+)/K(+)-ATPases. Furthermore, epimastigotes overproducing TcENA showed increased tolerance to high Na(+) stress. Our findings suggest that TcENA acts as a sodium pump and provide insights into the regulation of ion homeostasis in the parasitic protist.

Effect of different salts and detergents on luciferin-luciferase luminescence of the enchytraeid Fridericia heliota.

The study addresses the effect produced by different inorganic salts and detergents (SDS, Triton X-100, the Tween series) on the ATP-dependent bioluminescent reaction catalyzed by the luciferase of the new earthworm species Fridericia heliota (Annelida: Clitellata: Oligochaeta: Enchytraeidae). It has been shown that the effect of divalent metal salts on luminescence is determined by the action of cations. Three of them - Mg(2+), Mn(2+) and Ca(2+) - can stimulate luciferase activity at concentrations varying within a wide range, and Mn(2+) can act as a 100%-effective substitute for Mg(2+) in F. heliota luminescence reaction in vitro. The inhibitory effect of monovalent metal salts on luminescence is largely determined by the action of the anion part of the molecule. The effectiveness of the inhibitory effect of anions increases in the following order: Cl(-)

[Effect of separate effectors on activity of extracellular fructosobisphosphatase of Acholeplasma laidlawii var. Granulum, strain 118].

The influence of 16 substances-effectors on the extracellular mollicute fructosobisphosphatase (FBPhase) was studied for the first time. These effectors are used, as a rule, when studying properties of this enzyme biopreparations newly isolated from the cells of animals, plants and cells of microorganisms. It was established that optimum pH for FBPhase of mollicutes is whithin 7.3-7.5 and on the basis of this index it is attributed to the group of the "neutral" of these enzymes. Cations of K, Mn, Mg, NH4, Tl and phosphoenolpyruvate (PEP) increase its activity. Cations of Li and adenosin 5'-monophosphate (AMP) proved to be the inhibitors of mollicute FBPhase activity. Chelators (EDTA, citrate, imidasol pyruvate, L-histidine) activated it inconsiderably (by 10-20%). Cations of Zn, in contrast to those of other tested metals, in low concentrations (0.1 - 0.2 microM) inhibited FBPhase activity, but when increasing their concentration (6 microM and above) activated the enzyme even better than it was observed for Mn and Mg cations, the necessary components of the reacting mixtures. Thus, when determining the components of the reacting mixtures with the purpose to study the properties of mollicute FBPhase and to regulate its activity in the in vitro systems under pH values optimal for the enzyme, the monovalent (NH4, K, Tl) and bivalent (Mg, Mn, Zn) cations may be introduced in their compositions as activators, AMP and Li cations should be used as inhibitors. Other substances which were studied when making their work proved to be inessential effectors is respect of mollicute FBPhase.

Characterization of the norovirus 3C-like protease.

The recombinant 3C-like protease of Chiba virus, a Norovirus, expressed in Escherichia coli cells was purified and characterized as to effects of pH, temperature, salt contents, and SH reagents on its proteolytic activity. The optimal pH and temperature of the 3C-like protease for the proteolytic activity were 8.6 and 37 degrees C, respectively. Increased concentration (approximately 100 mM) of monovalent cations such as Na+ and K+ was inhibitory to the activity. Hg2+ and Zn2+ remarkably inhibited the protease activity, while Mg2+ and Ca2+ had no virtual effect. Several sulfhydryl reagents such as p-chloromercuribenzoic acid, methyl methanethiosulfonate, N-ethylmaleimide and N-phenylmaleimide also blocked the activity, confirming the previous result that cysteine residue(s) were responsible for the proteolysis.

Interaction between muscle aldolase and muscle fructose 1,6-bisphosphatase results in the substrate channeling.

Fructose 1,6-bisphosphatase (FBPase) is known to form a supramolecular complex with alpha-actinin and aldolase on both sides of the Z-line in skeletal muscle cells. It has been proposed that association of aldolase with FBPase not only desensitizes muscle FBPase toward AMP inhibition but it also might enable the channeling of intermediates between the enzymes [Rakus et al. (2003) FEBS Lett. 547, 11-14]. In the present paper, we tested the possibility of fructose 1,6-bisphosphate (F1,6-P(2)) channeling between aldolase and FBPase using the approach in which an inactive form of FBPase competed with active FBPase for binding to aldolase and thus decreased the rate of aldolase-FBPase reaction. The results showed that F1,6-P(2) is transferred directly from aldolase to FBPase without mixing with the bulk phase. Further evidence that F1,6-P(2) is channeled from aldolase to FBPase comes from the experiments investigating the inhibitory effect of a high concentration of magnesium ions on aldolase-FBPase activity. FBPase in a complex with aldolase, contrary to free muscle FBPase, was not inhibited by high Mg(2+) concentrations, which suggests that free F1,6-P(2) was not present in the assay mixture during the reaction. A real-time interaction analysis between aldolase and FBPase revealed a dual role of Mg(2+) in the regulation of the aldolase-FBPase complex stability. A physiological concentration of Mg(2+) increased the affinity of muscle FBPase to muscle aldolase, whereas higher concentrations of the cation decreased the concentration of the complex. We hypothesized that the presence of Mg(2+) stabilizes a positively charged cavity within FBPase and that it might enable an interaction with aldolase. Because magnesium decreased the binding constant (K(a)) between aldolase and FBPase in a manner similar to the decrease of K(a) caused by monovalent cations, it is postulated that electrostatic attraction might be a driving force for the complex formation. It is presumed that the biological relevance of F1,6-P(2) channeling between aldolase and FBPase is protection of this glyconeogenic, as well as glycolytic, intermediate against degradation by cytosolic aldolase, which is one of the most abundant enzyme of glycolysis.

Effects of potassium and chloride on ribosome association with the RNA of foot-and-mouth disease virus.

Foot-and-mouth disease virus (FMDV) and other picornaviruses initiate translation of their polyprotein cap-independently at an internal site of the positive-strand viral RNA. This process is mediated by the internal ribosome entry site (IRES), a highly structured cis-acting RNA element that binds translation initiation factors and ribosomal subunits. During their life cycle, picornaviruses induce proliferation of membrane structures involved in viral replication and an increase in membrane permeability probably facilitating virus progeny release. Here, I analyze the efficiency of association of the ribosomal subunits with the FMDV IRES RNA at elevated salt concentrations. Potassium stimulates FMDV translation, whereas sodium chloride concentrations up to 150 mM neither stimulate nor interfere with FMDV translation. Even high potassium concentrations allow binding of the viral RNA to ribosomes. Chloride stimulates binding of ribosomes to the viral RNA at the stage of 48S initiation complex formation and FMDV translation at concentrations up to 150 mM. Only at elevated concentrations, binding of ribosomal subunits and translation are inhibited by chloride. However, FMDV start site selection is not influenced by potassium salts. These results indicate that the association of the viral RNA with ribosomal subunits is well adapted to high salt conditions that are induced during picornavirus infection.