Rapid Membrane Depolarization Research Articles

Organometallic nucleosides induce non-classical leukemic cell death that is mitochondrial-ROS dependent and facilitated by TCL1-oncogene burden

BackgroundRedox stress is a hallmark of the rewired metabolic phenotype of cancer. The underlying dysregulation of reactive oxygen species (ROS) is interconnected with abnormal mitochondrial biogenesis and function. In chronic lymphocytic leukemia (CLL), elevated ROS are implicated in clonal outgrowth and drug resistance. The pro-survival oncogene T-cell leukemia 1 (TCL1) is causally linked to the high threshold towards classical apoptosis in CLL. We investigated how aberrant redox characteristics and bioenergetics of CLL are impacted by TCL1 and if this is therapeutically exploitable.MethodsBio-organometallic chemistry provided compounds containing a cytosine nucleobase, a metal core (ferrocene, ruthenocene, Fe(CO)3), and a 5’-CH2O-TDS substituent. Four of these metal-containing nucleoside analogues (MCNA) were tested for their efficacy and mode of action in CLL patient samples, gene-targeted cell lines, and murine TCL1-transgenic splenocytes.ResultsThe MCNA showed a marked and selective cytotoxicity towards CLL cells. MCNA activity was equally observed in high-risk disease groups, including those of del11q/del17p cytogenetics and of clinical fludarabine resistance. They overcame protective stromal cell interactions. MCNA-evoked PARP-mediated cell death was non-autophagic and non-necrotic as well as caspase- and P53-independent. This unconventional apoptosis involved early increases of ROS, which proved indispensible based on mitigation of MCNA-triggered death by various scavengers. MCNA exposure reduced mitochondrial respiration (oxygen consumption rate; OCR) and induced a rapid membrane depolarization (∆ΨM). These characteristics distinguished the MCNA from the alkylator bendamustine and from fludarabine. Higher cellular ROS and increased MCNA sensitivity were linked to TCL1 expression. The presence of TCL1 promoted a mitochondrial release of in part caspase-independent apoptotic factors (AIF, Smac, Cytochrome-c) in response to MCNA. Although basal mitochondrial respiration (OCR) and maximal respiratory capacity were not affected by TCL1 overexpression, it mediated a reduced aerobic glycolysis (lactate production) and a higher fraction of oxygen consumption coupled to ATP-synthesis.ConclusionsRedox-active substances such as organometallic nucleosides can confer specific cytotoxicity to ROS-stressed cancer cells. Their P53- and caspase-independent induction of non-classical apoptosis implicates that redox-based strategies can overcome resistance to conventional apoptotic triggers. The high TCL1-oncogenic burden of aggressive CLL cells instructs their particular dependence on mitochondrial energetic flux and renders them more susceptible towards agents interfering in mitochondrial homeostasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0378-1) contains supplementary material, which is available to authorized users.

Synthetic ciguatoxin CTX 3C induces a rapid imbalance in neuronal excitability.

Ciguatera is a human global disease caused by the consumption of contaminated fish that have accumulated ciguatoxins (CTXs), sodium channel activator toxins. Symptoms of ciguatera include neurological alterations such as paraesthesiae, dysaesthesiae, depression, and heightened nociperception, among others. An important issue to understand these long-term neurological alterations is to establish the role that changes in activity produced by CTX 3C represent to neurons. Here, the effects of synthetic ciguatoxin CTX 3C on membrane potential, spontaneous spiking, and properties of synaptic transmission in cultured cortical neurons of 11-18 days in vitro (DIV) were evaluated using electrophysiological approaches. CTX 3C induced a large depolarization that decreased neuronal firing and caused a rapid inward tonic current that was primarily GABAergic. Moreover, the toxin enhanced the amplitude of miniature postsynaptic inhibitory currents (mIPSCs), whereas it decreased the amplitude of miniature postsynaptic excitatory currents (mEPSCs). The frequency of mIPSCs increased, whereas the frequency of mEPSCs remained unaltered. We describe, for the first time, that a rapid membrane depolarization caused by CTX 3C in cortical neurons activates mechanisms that tend to suppress electrical activity by shifting the balance between excitatory and inhibitory synaptic transmission toward inhibition. Indeed, these results suggest that the acute effects of CTX on synaptic transmission could underlie some of the neurological symptoms caused by ciguatera in humans.

Membrane Disruption by Antimicrobial Fatty Acids Releases Low-Molecular-Weight Proteins from Staphylococcus aureus

The skin represents an important barrier for pathogens and is known to produce fatty acids that are toxic toward gram-positive bacteria. A screen of fatty acids as growth inhibitors of Staphylococcus aureus revealed structure-specific antibacterial activity. Fatty acids like oleate (18:1Δ9) were nontoxic, whereas palmitoleate (16:1Δ9) was a potent growth inhibitor. Cells treated with 16:1Δ9 exhibited rapid membrane depolarization, the disruption of all major branches of macromolecular synthesis, and the release of solutes and low-molecular-weight proteins into the medium. Other cytotoxic lipids, such as glycerol ethers, sphingosine, and acyl-amines blocked growth by the same mechanisms. Nontoxic 18:1Δ9 was used for phospholipid synthesis, whereas toxic 16:1Δ9 was not and required elongation to 18:1Δ11 prior to incorporation. However, blocking fatty acid metabolism using inhibitors to prevent acyl-acyl carrier protein formation or glycerol-phosphate acyltransferase activity did not increase the toxicity of 18:1Δ9, indicating that inefficient metabolism did not play a determinant role in fatty acid toxicity. Nontoxic 18:1Δ9 was as toxic as 16:1Δ9 in a strain lacking wall teichoic acids and led to growth arrest and enhanced release of intracellular contents. Thus, wall teichoic acids contribute to the structure-specific antimicrobial effects of unsaturated fatty acids. The ability of poorly metabolized 16:1 isomers to penetrate the cell wall defenses is a weakness that has been exploited by the innate immune system to combat S. aureus.

Antibacterial Properties of an Oligo-Acyl-Lysyl Hexamer Targeting Gram-Negative Species

Toward developing new tools for fighting resistance to antibiotics, we investigated the antibacterial properties of a new decanoyl-based oligo-acyl-lysyl (OAK) hexamer, aminododecanoyl-lysyl-[aminodecanoyl-lysyl](5) (α(12)-5α(10)). The OAK exhibited preferential activity against Gram-negative bacteria (GNB), as determined using 36 strains, including diverse species, with an MIC(90) of 6.2 μM. The OAK's bactericidal mode of action was associated with rapid membrane depolarization and cell permeabilization, suggesting that the inner membrane was the primary target, whereas the observed binding affinity to lipoteichoic acid suggested that inefficacy against Gram-positive species resulted from a cell wall interaction preventing α(12)-5α(10) from reaching internal targets. Interestingly, perturbation of the inner membrane structure and function was preserved at sub-MIC values. This prompted us to assess the OAK's effect on the proton motive force-dependent efflux pump AcrAB-TolC, implicated in the low sensitivity of GNB to various antibiotics, including erythromycin. We found that under sub-MIC conditions, wild-type Escherichia coli was significantly more sensitive to erythromycin (the MIC dropped by >10-fold), unlike its acr-deletion mutant. Collectively, the data suggest a useful approach for treating GNB infections through overcoming antibiotic efflux.

Multiple T-type Ca2+ current subtypes in electrophysiologically characterized hamster dorsal horn neurons: possible role in spinal sensory integration

Whole cell patch-clamp recordings were used to investigate the contribution of transient, low-threshold calcium currents (I(T)) to firing properties of hamster spinal dorsal horn neurons. I(T) was widely, though not uniformly, expressed by cells in Rexed's laminae I-IV and correlated with the pattern of action potential discharge evoked under current-clamp conditions: I(T) in neurons responding to constant membrane depolarization with one or two action potentials was nearly threefold larger than I(T) in cells responding to the same activation with continuous firing. I(T) was evoked by depolarizing voltage ramps exceeding 46 mV/s and increased with ramp slope (240-2,400 mV/s). Bath application of 200 μM Ni(2+) depressed ramp-activated I(T). Phasic firing recorded in current clamp could only be activated by membrane depolarizations exceeding ∼43-46 mV/s and was blocked by Ni(2+) and mibefradil, suggesting I(T) as an underlying mechanism. Two components of I(T), "fast" and "slow," were isolated based on a difference in time constant of inactivation (12 ms and 177 ms, respectively). The amplitude of the fast subtype depended on the slope of membrane depolarization and was twice as great in burst-firing cells than in cells having a tonic discharge. Post hoc single-cell RT-PCR analyses suggested that the fast component is associated with the Ca(V)3.1 channel subtype. I(T) may enhance responses of phasic-firing dorsal horn neurons to rapid membrane depolarizations and contribute to an ability to discriminate between afferent sensory inputs that encode high- and low-frequency stimulus information.

Effects of divalent cations on slow unblock of native NMDA receptors in mouse neocortical pyramidal neurons

The N-methyl-D-aspartate receptor (NMDAR) exhibits strong voltage-dependent block by extracellular Mg(2+) , which is relieved by sustained depolarization and glutamate binding, and which is central to the function of the NMDAR in synaptic plasticity. Rapid membrane depolarization during agonist application reveals a slow unblock of NMDARs, which has important functional implications, for example in the generation of NMDAR spikes, and in determining the narrow time window for spike-timing-dependent plasticity. However, its mechanism is still unclear. Here, we study unblock of divalent cations in native NMDARs in nucleated patches isolated from mouse cortical layer 2/3 pyramidal neurons. Comparing unblock kinetics of NMDARs in the presence of extracellular Mg(2+) or in nominally zero Mg(2+) , and with Mn(2+) or Co(2+) substituting for Mg(2+) , we found that the properties of slow unblock were determined by the identity of the blocking metal ion at the binding site, presumably by affecting the operation of a structural link to channel gating. The time course of slow unblock was not affected by zinc, or the zinc chelator TPEN [N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine], while the slower fraction of unblock was reduced by ifenprodil, an NR2B-selective antagonist. Slow unblock was only weakly temperature dependent, speeding up with rise in temperature with a Q(10) of ≈1.5. Finally, using action potential waveform voltage-clamp, we show that this slow relief from divalent cation block is a prominent feature in physiologically realistic patterns of changing membrane potential.

Sequential depolarization of root cortical and stelar cells induced by an acute salt shock – implications for Na+ and K+ transport into xylem vessels

Early events in NaCl-induced root ion and water transport were investigated in maize (Zea mays L) roots using a range of microelectrode and imaging techniques. Addition of 100 mm NaCl to the bath resulted in an exponential drop in root xylem pressure, rapid depolarization of trans-root potential and a transient drop in xylem K(+) activity (A(K+) ) within ∼1 min after stress onset. At this time, no detectable amounts of Na(+) were released into the xylem vessels. The observed drop in A(K+) was unexpected, given the fact that application of the physiologically relevant concentrations of Na(+) to isolated stele has caused rapid plasma membrane depolarization and a subsequent K(+) efflux from the stelar tissues. This controversy was explained by the difference in kinetics of NaCl-induced depolarization between cortical and stelar cells. As root cortical cells are first to be depolarized and lose K(+) to the environment, this is associated with some K(+) shift from the stelar symplast to the cortex, resulting in K(+) being transiently removed from the xylem. Once Na(+) is loaded into the xylem (between 1 and 5 min of root exposure to NaCl), stelar cells become more depolarized, and a gradual recovery in A(K+) occurs.

The Antioxidant Transcription Factor Nrf2 Negatively Regulates Autophagy and Growth Arrest Induced by the Anticancer Redox Agent Mitoquinone

Mitoquinone (MitoQ) is a synthetically modified, redox-active ubiquinone compound that accumulates predominantly in mitochondria. We found that MitoQ is 30-fold more cytotoxic to breast cancer cells than to healthy mammary cells. MitoQ treatment led to irreversible inhibition of clonogenic growth of breast cancer cells through a combination of autophagy and apoptotic cell death mechanisms. Relatively limited cytotoxicity was seen with the parent ubiquinone coenzyme Q(10.) Inhibition of cancer cell growth by MitoQ was associated with G(1)/S cell cycle arrest and phosphorylation of the checkpoint kinases Chk1 and Chk2. The possible role of oxidative stress in MitoQ activity was investigated by measuring the products of hydroethidine oxidation. Increases in ethidium and dihydroethidium levels, markers of one-electron oxidation of hydroethidine, were observed at cytotoxic concentrations of MitoQ. Keap1, an oxidative stress sensor protein that regulates the antioxidant transcription factor Nrf2, underwent oxidation, degradation, and dissociation from Nrf2 in MitoQ-treated cells. Nrf2 protein levels, nuclear localization, and transcriptional activity also increased following MitoQ treatment. Knockdown of Nrf2 caused a 2-fold increase in autophagy and an increase in G(1) cell cycle arrest in response to MitoQ but had no apparent effect on apoptosis. The Nrf2-regulated enzyme NQO1 is partly responsible for controlling the level of autophagy. Keap1 and Nrf2 act as redox sensors for oxidative perturbations that lead to autophagy. MitoQ and similar compounds should be further evaluated for novel anticancer activity.

Suppressing LPS-induced early signal transduction in macrophages by a polyphenol degradation product: a critical role of MKP-1

Macrophages represent the first defense line against bacterial infection and therefore, play a crucial role in early inflammatory response. In this study, we investigated the role of MAPKs and MKP-1 activation in regulation of an early inflammatory response in RAW 264.7 macrophage cells. We induced the inflammatory response by treating the macrophages with LPS and inhibited an early inflammatory response by using ferulaldehyde, a water-soluble end-product of dietary polyphenol degradation that we found previously to exert its beneficial anti-inflammatory effects during the early phase of in vivo inflammation. We found that LPS-induced ROS and nitrogen species formations were reduced by ferulaldehyde in a concentration-dependent manner, and ferulaldehyde protected mitochondria against LPS-induced rapid and massive membrane depolarization. LPS induced early suppression of MKP-1, which was accompanied by activation of JNK, ERK, and p38 MAPK. By reversing LPS-induced early suppression of MKP-1, ferulaldehyde diminished MAPK activation, thereby inhibiting NF-κB activation, mitochondrial depolarization, and ROS production. Taken together, our data suggest that ferulaldehyde exerts its early anti-inflammatory effect by preserving the mitochondrial membrane integrity and shifting the expression of MKP-1 forward in time in macrophages.

Modulatory action of acetylcholine on the Na+-dependent action potentials in Kenyon cells isolated from the mushroom body of the cricket brain

Kenyon cells, intrinsic neurons of the insect mushroom body, have been assumed to be a site of conditioning stimulus (CS) and unconditioned stimulus (US) association in olfactory learning and memory. Acetylcholine (ACh) has been implicated to be a neurotransmitter mediating CS reception in Kenyon cells, causing rapid membrane depolarization via nicotinic ACh receptors. However, the long-term effects of ACh on the membrane excitability of Kenyon cells are not fully understood. In this study, we examined the effects of ACh on Na+ dependent action potentials (Na+ spikes) elicited by depolarizing current injection and on net membrane currents under the voltage clamp condition in Kenyon cells isolated from the mushroom body of the cricket Gryllus bimaculatus. Current-clamp studies using amphotericin B perforated-patch recordings showed that freshly dispersed cricket Kenyon cells could produce repetitive Na+ spikes in response to prolonged depolarizing current injection. Bath application of ACh increased both the instantaneous frequency and the amplitudes of Na+ spikes. This excitatory action of ACh on Kenyon cells is attenuated by the pre-treatment of the cells with the muscarinic receptor antagonists, atropine and scopolamine, but not by the nicotinic receptor antagonist mecamylamine. Voltage-clamp studies further showed that bath application of ACh caused an increase in net inward currents that are sensitive to TTX, whereas outward currents were decreased by this treatment. These results indicate that in order to mediate CS, ACh may modulate the firing properties of Na+ spikes of Kenyon cells through muscarinic receptor activation, thus increasing Na conductance and decreasing K conductance.

Unique cellular effect of the herbicide bromoxynil revealed by electrophysiological studies using characean cells

In a previous paper, we proposed that the primary action of the herbicide bromoxynil (BX; 3,5-dibromo-4-hydroxybenzonitrile) is cytosol acidification, based on the fact that bromoxynil induced the inhibition of cytoplasmic streaming and cell death of Chara corallina in acidic external medium (Morimoto and Shimmen in J Plant Res 121:227-233, 2008). In the present study, electrophysiological analysis of the BX effect was carried out in internodal cells of C. corallina. Upon addition of BX, a large and rapid pH-dependent depolarization was induced, supporting our hypothesis. Ioxynil, which belongs to the same group as bromoxynil, also induced a large and rapid membrane depolarization in a pH-dependent manner. On the other hand, four herbicides belonging to other groups of herbicides did not induce such a membrane depolarization. Thus, BX has a unique cellular effect. The decrease in the electro-chemical potential gradient for H(+) across the plasma membrane appears to result in inhibition of cell growth and disturbance of intracellular homeostasis in the presence of BX.

Growth and functional responses of different cultivars of Lotus corniculatus to aluminum and low pH stress

Aluminum toxicity is an important stress factor in acid soils. Growth, respiration and permeability properties of root cells were studied in five cultivars of Lotus corniculatus subjected to aluminum (Al) or low pH stress. The cultivars showed significant differences in root elongation under stress conditions, which correlated with changes in membrane potential (E(M)) of root cortical cells. A pH drop from 5.5 to 4.0 resulted in significant membrane depolarization and root growth inhibition. The strongest inhibition was observed in cv. São Gabriel (33.6%) and least in cv. UFRGS (25.8%). Application of an extremely high Al concentration (2mM) stopped the root growth in cv. INIA Draco, while inhibition in cv. UFRGS reached only 75%. The E(M) values of cortical cells of Lotus roots varied between -115 and -144mV. Treatment with 250microM of AlCl(3) (pH 4) resulted in rapid membrane depolarization. The extent of the membrane depolarization ranged between 51mV (cv. UFGRS) and 16mV (cv. INIA Draco). The membrane depolarization was followed by a loss of K(+) from Al-treated roots (2mM Al) and resulted in a decrease of the diffusion potential (E(D)). The total amount of K(+) in Al-treated roots dropped from 31.4 to 16.8micromolg(-1) FW in sensitive cv. INIA Draco, or from 26.1 to 22.7micromolg(-1) FW in tolerant cv. UFGRS. The rate of root respiration under control conditions as well as under Al treatment was higher in cv. INIA Draco than in cv. UFRGS. Al-induced inhibition of root respiration was 21-34% of the control.

Chlorovirus-mediated membrane depolarization of Chlorella alters secondary active transport of solutes.

Paramecium bursaria chlorella virus 1 (PBCV-1) is the prototype of a family of large, double-stranded DNA, plaque-forming viruses that infect certain eukaryotic chlorella-like green algae from the genus Chlorovirus. PBCV-1 infection results in rapid host membrane depolarization and potassium ion release. One interesting feature of certain chloroviruses is that they code for functional potassium ion-selective channel proteins (Kcv) that are considered responsible for the host membrane depolarization and, as a consequence, the efflux of potassium ions. This report examines the relationship between cellular depolarization and solute uptake. Annotation of the virus host Chlorella strain NC64A genome revealed 482 putative transporter-encoding genes; 224 are secondary active transporters. Solute uptake experiments using seven radioactive compounds revealed that virus infection alters the transport of all the solutes. However, the degree of inhibition varied depending on the solute. Experiments with nystatin, a drug known to depolarize cell membranes, produced changes in solute uptake that are similar but not identical to those that occurred during virus infection. Therefore, these studies indicate that chlorovirus infection causes a rapid and sustained depolarization of the host plasma membrane and that this depolarization leads to the inhibition of secondary active transporters that changes solute uptake.

Ceragenins: Cholic Acid-Based Mimics of Antimicrobial Peptides

The prevalence of drug-resistant bacteria drives the quest for new antimicrobials, including those that are not expected to readily engender resistance. One option is to mimic Nature's most ubiquitous means of controlling bacterial growth, antimicrobial peptides, which have evolved over eons. In general, bacteria remain susceptible to these peptides. Human antimicrobial peptides play a central role in innate immunity, and deficiencies in these peptides have been tied to increased rates of infection. However, clinical use of antimicrobial peptides is hampered by issues of cost and stability. The development of nonpeptide mimics of antimicrobial peptides may provide the best of both worlds: a means of using the same mechanism chosen by Nature to control bacterial growth without the problems associated with peptide therapeutics. The ceragenins were developed to mimic the cationic, facially amphiphilic structures of most antimicrobial peptides. These compounds reproduce the required morphology using a bile-acid scaffolding and appended amine groups. The resulting compounds are actively bactericidal against both gram-positive and gram-negative organisms, including drug-resistant bacteria. This antimicrobial activity originates from selective association of the ceragenins with negatively charged bacterial membrane components. Association has been studied with synthetic models of bacterial membrane components, with bacterial lipopolysaccharide, with vesicles derived from bacterial phospholipids, and with whole cells. Comparisons of the antimicrobial activities of ceragenins and representative antimicrobial peptides suggest that these classes of compounds share a mechanism of action. Rapid membrane depolarization is caused by both classes as well as blebbing of bacterial membranes. Bacteria express the same genes in response to both classes of compounds. On the basis of the antibacterial activities of ceragenins and preliminary in vivo studies, we expect these compounds to find use in augmenting or replacing antimicrobial peptides in treating human disease.

An L-Tyrosine Derivative and PPAR Agonist, GW7845, Activates a Multifaceted Caspase Cascade in Bone Marrow B Cells

Apoptosis is a critical event in the deletion of B lymphocytes prior to their migration to the periphery. Synthetic peroxisome proliferator activated receptor gamma (PPARgamma) agonists, including the drug GW7845 and the environmental contaminant mono-(2-ethylhexyl) phthalate, as well as an endogenous ligand, 15-deoxy-Delta(12,14)-prostaglandin J(2), induce clonally unrestricted apoptosis in pro/pre-B cells. Considering that PPARgamma agonists are used clinically for the treatment of diabetes and postulated to be useful as chemotherapeutics, we used GW7845 as a model PPARgamma agonist to examine the mechanism of cell death that may contribute to tumor killing as well as normal bone marrow B lymphocyte toxicity. GW7845 induced rapid mitochondrial membrane depolarization and release of cytochrome c, along with nearly concurrent activation of capases-2, -3, -8, and -9 in primary pro-B cells and BU-11 cells, a nontransformed pro/pre-B cell line. GW7845-induced apoptosis was reduced significantly in Bax-deficient and Apaf-1 mutant primary pro-B cells, supporting the conclusion that GW7845-induced apoptosis is mitochondria- and apoptosome-dependent. Using benzyloxycarbonyl-VAD-fluoromethyl ketone (VAD-FMK) as a pan-caspase inhibitor, we demonstrated that an initial cytochrome c release occurred independently of caspase activation and that only caspase-9 activation was partially caspase independent. The attenuation of GW7845-induced apoptosis by multiple FMK-labeled peptide sequences suggests that multiple caspase pathways are responsible for initiating and executing apoptosis. The strong activation of Bid provides a mechanism by which caspases-2, -3, and -8 may amplify the apoptotic signal. These data support the hypothesis that pharmacologic concentrations of PPARgamma agonists induce an intrinsic apoptotic pathway that is driven in normal bone marrow B cells by multiple amplification loops.

Reactive oxidants regulate membrane repolarization and store-operated uptake of calcium by formyl peptide-activated human neutrophils

Although the rapid and considerable membrane depolarization response which accompanies activation of the phagocyte NADPH oxidase is due to transmembrane electron fluxes, little is known about the involvement of reactive oxidant species (ROS) in the subsequent repolarization response. In the current study, we have investigated the effects of superoxide dismutase (SOD), catalase, methionine, and the myeloperoxidase (MPO) inhibitors, sodium azide and 4-aminobenzoyl hydrazide (ABAH), as well as those of H 2O 2 and HOCl (both at 100 μM) on the alterations in membrane potential which accompany activation of human neutrophils with the chemoattractant, FMLP (1 μM), and on store-operated uptake of Ca 2+. The generation of ROS by FMLP-activated neutrophils was monitored according to the magnitude of oxygen consumption and autoiodination, while spectrofluorimetric procedures were used to measure alterations in membrane potential and influx of Ca 2+. Treatment of the cells with H 2O 2, and HOCl, significantly impeded membrane repolarization, while sodium azide, ABAH, methionine, and catalase exerted the opposite effects, potentiating both the rates and the magnitudes of membrane repolarization and store-operated uptake of Ca 2+. These observations demonstrate that NADPH oxidase regulates neutrophil membrane potential and Ca 2+ influx not only via its electrogenic activity, but also as a consequence of the generation of ROS.

Potassium Ion Channels of Chlorella Viruses Cause Rapid Depolarization of Host Cells during Infection

Previous studies have established that chlorella viruses encode K(+) channels with different structural and functional properties. In the current study, we exploit the different sensitivities of these channels to Cs(+) to determine if the membrane depolarization observed during virus infection is caused by the activities of these channels. Infection of Chlorella NC64A with four viruses caused rapid membrane depolarization of similar amplitudes, but with different kinetics. Depolarization was fastest after infection with virus SC-1A (half time [t(1/2)], about 9 min) and slowest with virus NY-2A (t(1/2), about 12 min). Cs(+) inhibited membrane depolarization only in viruses that encode a Cs(+)-sensitive K(+) channel. Collectively, the results indicate that membrane depolarization is an early event in chlorella virus-host interactions and that it is correlated with viral-channel activity. This suggestion was supported by investigations of thin sections of Chlorella cells, which show that channel blockers inhibit virus DNA release into the host cell. Together, the data indicate that the channel is probably packaged in the virion, presumably in its internal membrane. We hypothesize that fusion of the virus internal membrane with the host plasma membrane results in an increase in K(+) conductance and membrane depolarization; this depolarization lowers the energy barrier for DNA release into the host.

Mechanisms of nitric-oxide-induced increase of free cytosolic Ca 2+ concentration in Nicotiana plumbaginifolia cells

In this study, we investigated a role for nitric oxide (NO) in mediating the elevation of the free cytosolic Ca 2+ concentration ([Ca 2+] cyt) in plants using Nicotiana plumbaginifolia cells expressing the Ca 2+ reporter apoaequorin. Hyperosmotic stress induced a fast increase of [Ca 2+] cyt which was strongly reduced by pretreating cell suspensions with the NO scavenger carboxy PTIO, indicating that NO mediates [Ca 2+] cyt changes in plant cells challenged by abiotic stress. Accordingly, treatment of transgenic N. plumbaginifolia cells with the NO donor diethylamine NONOate was followed by a transient increase of [Ca 2+] cyt sensitive to plasma membrane Ca 2+ channel inhibitors and antagonist of cyclic ADP ribose. We provided evidence that NO might activate plasma membrane Ca 2+ channels by inducing a rapid and transient plasma membrane depolarization. Furthermore, NO-induced elevation of [Ca 2+] cyt was suppressed by the kinase inhibitor staurosporine, suggesting that NO enhances [Ca 2+] cyt by promoting phosphorylation-dependent events. This result was further supported by the demonstration that the NO donor induced the activation of a 42-kDa protein kinase which belongs to SnRK2 families and corresponds to Nicotiana tabacum osmotic-stress-activated protein kinase (NtOSAK). Interestingly, NtOSAK was activated in response to hyperosmotic stress through a NO-dependent process, supporting the hypothesis that NO also promotes protein kinase activation during physiological processes.

Estradiol inhibits the effects of extracellular ATP in human sperm by a non genomic mechanism of action

Steroid hormones, beside their classical genomic mechanism of action, exert rapid, non genomic effects in different cell types. These effects are mediated by still poorly characterized plasma membrane receptors that appear to be distinct from the classic intracellular receptors. In the present study we evaluated the non genomic effects of estradiol (17βE2) in human sperm and its effects on sperm stimulation by extracellular ATP, a potent activator of sperm acrosome reaction. In human sperm 17βE2 induced a rapid increase of intracellular calcium (Ca2+) concentrations dependent on an influx of Ca2+ from the extracellular medium. The monitoring of the plasma membrane potential variations induced by 17βE2 showed that this steroid induces a rapid plasma membrane hyperpolarization that was dependent on the presence of Ca2+ in the extracellular medium since it was absent in Ca2+ free-medium. When sperm were pre-incubated in the presence of the K+ channel inhibitor tetra-ethylammonium, the 17βE2 induced plasma membrane hyperpolarization was blunted suggesting the involvement of K+ channels in the hyperpolarizing effects of 17βE2. Extracellular ATP induced a rapid plasma membrane depolarization followed by acrosome reaction. Sperm pre-incubation with 17βE2 inhibited the effects of extracellular ATP on sperm plasma membrane potential variations and acrosome reaction. The effects of 17βE2 were specific since its inactive steroisomer 17αE2 was inactive. Furthermore the effects of 17βE2 were not inhibited by tamoxifen, an antagonist of the classic 17βE2 intracellular receptor.

Effect of p53 Silencing on Asenic Tioxide-Induced Apoptotic Pathways in a Myeloma Cell Line Expressing w.t.p53.

Mutations in the p53 gene are the most common genetic abnormality in human cancers. P53 is a tumor suppressor gene, which regulates a wide variety of target genes controlling cell cycle and apoptosis. Arsenic Trioxide (ATO) is an effective chemotherapeutic agent for the treatment of acute promyelocytic leukemia and is being tested in phase II studies in various types of hematological malignancies and solid tumors. We have previously shown that ATO is a potent inducer of apoptosis in multiple myeloma cells, engaging the intrinsic apoptotic pathway in cells expressing w.t. p53. In contrast, in cells expressing mutant p53, both the intrinsic and extrinsic apoptotic pathways are engaged. Our finding in this respect were further supported by our recent study using a p53 temperature sensitive (p53Ts) mutant cell line, BRK, expressing w.t. p53 at 32°C and mutant p53 phenotype at 37°C (Akay et al., AACR ; Abstract #5344, 2005). We hypothesized that knocking out of p53 or p21 mRNA will result in modifying ATO-induced apoptotic pathways as observed using the p53-Ts conditional mutant of BRK cells. Thus, IM9 cells (expressing w.t.p53) were transfected [AMAXA Nucleoporator, ~60% transfection by green fluorescence protein (GFP) tag] with a GFP-SiRNA expressing plasmid co-expressing anti-sense RNA sequences for p53 (GFP/Sip53) and p21 (GFP/Sip21). As a control, GFP expressing plasmid not containing SiRNA (GFP/cont) was used. We observed in GFP/cont-transfected cells a G1 arrest through activation of p21 and moderate apoptosis, primarily through activation of procaspase 9, with no activation of BID and procaspase 8. In cells transfected with GFP/Sip53 or GFP/Sip21 plasmids we observed rapid mitochondrial membrane depolarization and apoptosis primarily through activation of the extrinsic apoptotic pathway with downregulation of FLIP, activation of procaspase 8 and BID, enhanced release of AIF from mitochondria and rapid apoptosis. Depletion of glutathione was observed with or without silencing of p21 or p53. Furthermore, silencing of p53 or p21 resulted with a decrease in ATO-induced activation of c-jun and Erk and in a decrease in the activation of Akt. These results further substantiate the model we put forward for the role of p53 in ATO-induced apoptosis in myeloma and other cancer cell lines.