Mechanism Of Potentiation Research Articles

Shortening speed dependent force potentiation is attenuated but not eliminated in skeletal muscles without myosin phosphorylation.

We investigated the influence of shortening speed on concentric force potentiation at different frequencies in muscles devoid of skeletal myosin light chain kinase (skMLCK-/-) and unable to phosphorylate myosin. EDL muscles from skMLCK-/- mice were activated in vitro (25 °C) across a range of stimulation frequencies (10-100Hz) during shortening ramps at 0.10, 0.30, or 0.50 of maximum shortening velocity (Vmax) before and after a potentiating stimulus (PS). When collapsed across all frequencies, the PS increased relative (post/pre) concentric force to 1.27 ± 0.02 and 1.17 ± 0.02 of pre-PS values at 0.50 and 0.30 Vmax, respectively (n = 4, P < 0.05 for all speeds). In addition, potentiation was significantly greater at low and intermediate-than at high stimulus frequencies at both speeds. In contrast, during shortening at 0.10 Vmax, a posttetanic depression was observed as mean concentric forces were reduced to 0.85 ± 0.02 of pre-PS values. Thus, although reduced compared to published values for wildtype muscles (Gittings et al., J Muscle Res Cell Motil 33:359-368, 2012), skMLCK-/- muscles displayed a speed dependent potentiation of concentric force during moderate and fast shortening speed at all frequencies tested. Our data support the presence of a myosin phosphorylation-independent mechanism(s) for concentric force potentiation at moderate speeds of shortening, and also suggests that myosin phosphorylation may be necessary to prevent the concentric force depression that may be present at slow speeds of shortening. Although additive in nature, further work is needed to parse out the relative influence of myosin phosphorylation-independent and dependent potentiation mechanisms on wildtype contractile function during dynamic conditions.

Binding Properties of the Transforming Growth Factor-β Coreceptor Betaglycan: Proposed Mechanism for Potentiation of Receptor Complex Assembly and Signaling.

Transforming growth factor (TGF) β1, β2, and β3 (TGF-β1–TGF-β3, respectively) are small secreted signaling proteins that each signal through the TGF-β type I and type II receptors (TβRI and TβRII, respectively). However, TGF-β2, which is well-known to bind TβRII several hundred-fold more weakly than TGF-β1 and TGF-β3, has an additional requirement for betaglycan, a membrane-anchored nonsignaling receptor. Betaglycan has two domains that bind TGF-β2 at independent sites, but how it binds TGF-β2 to potentiate TβRII binding and how the complex with TGF-β, TβRII, and betaglycan undergoes the transition to the signaling complex with TGF-β, TβRII, and TβRI are not understood. To investigate the mechanism, the binding of the TGF-βs to the betaglycan extracellular domain, as well as its two independent binding domains, either directly or in combination with the TβRI and TβRII ectodomains, was studied using surface plasmon resonance, isothermal titration calorimetry, and size-exclusion chromatography. These studies show that betaglycan binds TGF-β homodimers with a 1:1 stoichiometry in a manner that allows one molecule of TβRII to bind. These studies further show that betaglycan modestly potentiates the binding of TβRII and must be displaced to allow TβRI to bind. These findings suggest that betaglycan functions to bind and concentrate TGF-β2 on the cell surface and thus promote the binding of TβRII by both membrane-localization effects and allostery. These studies further suggest that the transition to the signaling complex is mediated by the recruitment of TβRI, which simultaneously displaces betaglycan and stabilizes the bound TβRII by direct receptor–receptor contact.

Wnt signaling inhibitor FH535 selectively inhibits cell proliferation and potentiates imatinib-induced apoptosis in myeloid leukemia cell lines.

Wnt signaling pathway plays a major role in leukemogenesis of myeloid leukemia. Aberrancy in its regulation results in hyperactivity of the pathway contributing to leukemia propagation and maintenance. To investigate effects of Wnt pathway inhibition in leukemia, we used human leukemia cell lines (i.e., K562, HL60, THP1, and Jurkat) and several Wnt inhibitors, including XAV939, IWP2 and FH535. Our results showed that leukemia cell lines (>95% cells) had increased endogenous levels of β-catenin as compared to mononuclear cells from healthy donors (0%). Among the tested inhibitors, FH535 demonstrated a markedly suppressive effect (IC50=358nM) on mRNA levels of β-catenin target genes (LEF1, CCND1, and cMYC). In addition, FH535 significantly potentiated imatinib-induced apoptosis. Evaluation of erythrocyte and megakaryocyte lineage using flow cytometry demonstrated that the potentiation mechanism is independent of the developmental stage, and is more likely due to crosstalk between other pathways and β-catenin. FH535 also displayed antiproliferative properties in other cell lines used in this study. In summary, FH535 showed significantly high antiproliferative effects at submicromolar dosages, and additionally enhanced imatinib-induced apoptosis in human leukemia cell lines. Our results highlight its potential antileukemic promise when used in conjunction with other conventional therapeutic regimens.

Natural Killer Cells and Their Interaction with Dendritic Cells in Hepatitis C Infection

Background: Hepatitis C is a viral infection of the liver that has affected around 200 million people globally. The immune response against HCV infection includes both the innate and adaptive arms of immunity, with crosstalk between liver inhabitant and infiltrating cells. In the current study, we aimed to investigate the natural killer cells activation and inhibition status, and their role in interaction with DCs utilizing different combinations between NK cells and DCs in the presence of HCV peptides in a ratio of 5 NK: 1DC.Results: HCV NK cells upregulated both activation and inhibition markers. This could be attributed to HCV infection and their interaction with DCs especially healthy DCs. Moreover, apoptosis of DCs and NK cells occurred more in HCV NK cultures due to their higher frequency of NKp30 and KLRG1. The death of NK cells was more than DCs despite DCs maturation defect due to HCV infection, suggesting that the inhibitory marker KLRG1 took the upper hand over the upregulated activation markers leading to impaired cytotoxic activity and apoptosis of NK cells. Conclusion: The bidirectional crosstalk between NK cells and DCs is important in both potentiating mechanisms of the innate immune responses and the subsequent adaptive immune responses in the immune surveillance of cancer and infections. HCV infection impairs this crosstalk which may be a leading cause in viral persistence and chronicity.

Ubiquitin-Conjugating Enzyme 9 Phosphorylation as a Novel Mechanism for Potentiation of the Inflammatory Response

Lipopolysaccharide (LPS), a bacterial endotoxin, induces inflammation in macrophages via activation of NF-κB signaling. Sumoylation is a post-translational modification mediated by the small ubiquitin-like modifier, SUMO. Ubiquitin-conjugating enzyme 9 (UBC9) is the only known SUMO conjugating enzyme. LPS treatment lowers SUMO-1 and UBC9 mRNA levels in primary astrocytes. UBC9 can degrade NF-κB inhibitor α (Ikbα) via a SUMO2/3-ubiquitin pathway. However, UBC9 may also promote Ikbα stability by SUMO-1 conjugation that further regulates NF-κB signaling. The role of UBC9 in liver inflammation is unknown. We reported that CDK1-mediated phosphorylation of UBC9 enhanced its stability. Herein, we describe an anti-inflammatory role of UBC9 that is lost when it is phosphorylated during inflammation. LPS exposure caused induction in UBC9 phosphorylation and CDK1 activation specifically in Kupffer cells invivo and in RAW264.7 macrophages invitro. Silencing or overexpression experiments invitro and invivo showed that UBC9 was required to blunt the proinflammatory response elicited by LPS. LPS stimulation raised the binding of phospho-UBC9 but not the unphosphorylated counterpart, to Ikbα in RAW264.7 macrophages. Hence, phospho-UBC9 may promote NF-κB signaling by regulating Ikbα andthis may be a novel mechanism that deregulates liver inflammatory signaling.

Long-term potentiation-like cortical plasticity is disrupted in Alzheimer's disease patients independently from age of onset.

Alzheimer's disease (AD) is considered an age-related disorder. However, it is unclear whether AD induces the same pathological and neurophysiological modifications in synaptic functions independently from age of disease onset. We used transcranial magnetic stimulation tools to investigate the mechanisms of cortical plasticity and sensory-motor integration in AD patients with a wide range of disease onset. We evaluated newly diagnosed sporadic AD (n = 54) in comparison with healthy age-matched controls (HS; n = 24). Cortical plasticity mechanisms of long-term potentiation (LTP) or of long-term depression (LTD) were assessed using respectively intermittent (iTBS) or continuous theta burst stimulation (cTBS) protocols. Sensory-motor integration was evaluated by means of short afferent inhibition (SAI) protocol. AD patients show after iTBS an impairment of LTP-like cortical plasticity forming a paradoxical LTD in comparison to HS. LTD-like cortical plasticity is similar between AD and HS. LTP-like cortical plasticity is not associated with age, but AD patients presenting with more altered LTP-like cortical plasticity have more-severe cognitive decline at 18 months. SAI is impaired in AD and shows a strong association with the individual age of subjects rather than with disease age of onset. Cortical LTP disruption is a central mechanism of AD that is independent from age of onset. AD can be described primarily as a disorder of LTP-like cortical plasticity not influenced by physiological aging and associated with a more-severe cognitive decline. Ann Neurol 2016;80:202-210.

Two-ligand priming mechanism for potentiated phosphoinositide synthesis is an evolutionarily conserved feature of Sec14-like phosphatidylinositol and phosphatidylcholine exchange proteins

Lipid signaling, particularly phosphoinositide signaling, plays a key role in regulating the extreme polarized membrane growth that drives root hair development in plants. The Arabidopsis AtSFH1 gene encodes a two-domain protein with an amino-terminal Sec14-like phosphatidylinositol transfer protein (PITP) domain linked to a carboxy-terminal nodulin domain. AtSfh1 is critical for promoting the spatially highly organized phosphatidylinositol-4,5-bisphosphate signaling program required for establishment and maintenance of polarized root hair growth. Here we demonstrate that, like the yeast Sec14, the AtSfh1 PITP domain requires both its phosphatidylinositol (PtdIns)- and phosphatidylcholine (PtdCho)-binding properties to stimulate PtdIns-4-phosphate [PtdIns(4)P] synthesis. Moreover, we show that both phospholipid-binding activities are essential for AtSfh1 activity in supporting polarized root hair growth. Finally, we report genetic and biochemical evidence that the two-ligand mechanism for potentiation of PtdIns 4-OH kinase activity is a broadly conserved feature of plant Sec14-nodulin proteins, and that this strategy appeared only late in plant evolution. Taken together, the data indicate that the PtdIns/PtdCho-exchange mechanism for stimulated PtdIns(4)P synthesis either arose independently during evolution in yeast and in higher plants, or a suitable genetic module was introduced to higher plants from a fungal source and subsequently exploited by them.

Differential response to anodal tDCS and PAS is indicative of impaired focal LTP-like plasticity in schizophrenia

Increasing evidence suggests that neural plasticity impairments, observed in schizophrenia patients, are driven by dysfunctional integration of neural signaling. However, what is less clear is whether this impairment is resultant from a general deficit in plastic induction or whether a specific plastic mechanism is affected. In the current study we aimed to assess whether schizophrenia has a selective impact on focal or non-focal plasticity induction. To pursue this goal we utilized two non-invasive stimulation techniques that differ in the mechanism of long-term potentiation (LTP)-like plasticity induction: focal paired associative stimulation (PAS) and non-focal anodal transcranial direct current stimulation (a-tDCS). 20 schizophrenia patients and 20 matched healthy controls received PAS and a-tDCS in two separate sessions. Cortical excitability and cortical plasticity were assessed by transcranial magnetic stimulation (TMS)-elicited motor evoked potentials (MEP). In both study groups, non-focal a-tDCS resulted in a significant increase of mean MEP magnitude indicating the successful induction of non-focal LTP-like plasticity. In contrast, an increase in mean MEP magnitude following PAS was only observed in the control group, suggesting impaired focal LTP-like plasticity in schizophrenia. Additionally, we observed significantly impaired short-latency intracortical inhibition (SICI) in schizophrenia. This is the first study to comparatively evaluate non-focal and focal plasticity mechanisms in schizophrenia patients. The differential patterns of LTP-like plasticity responses indicate that reduced plasticity in schizophrenia could be ascribed to impairments in spatially and temporally restricted signal integration. This impairment, coupled with an observed reduction of inhibitory circuit efficacy, might further contribute to impairments in coordinating focal signals.

Polyamino-Isoprenic Derivatives Block Intrinsic Resistance of P. aeruginosa to Doxycycline and Chloramphenicol In Vitro.

Multidrug resistant bacteria have been a worldwide concern for decades. Though new molecules that effectively target Gram-positive bacteria are currently appearing on the market, a gap remains in the treatment of infections caused by Gram-negative bacteria. Therefore, new strategies must be developed against these pathogens. The aim of this study was to select an antibiotic for which a bacterium is naturally resistant and to use an escort molecule to restore susceptibility, similarly to the model of β-lactam/ β-lactamase inhibitors. High-content screening was performed on the reference strain PA01, allowing the selection of four polyamino-isoprenic compounds that acted synergistically with doxycycline. They were assayed against clinical isolates and Multi-Drug-Resistant strains. One of these compounds was able to decrease the MIC of doxycycline on the reference strain, efflux pump overproducers and clinical isolates of P. aeruginosa, to the susceptibility level. Similar results were obtained using chloramphenicol as the antibiotic. Membrane permeation assays and real-time efflux experiments were used to characterize the mechanism of doxycycline potentiation.The results showed that the selected compound strongly decreases the efficiency of glucose-triggered efflux associated with a slight destabilization of the outer membrane. According to these data, targeting natural resistance may become an interesting way to combat MDR pathogens and could represent an alternative to already devised strategies.

Fasiglifam (TAK-875) has dual potentiating mechanisms via Gαq-GPR40/FFAR1 signaling branches on glucose-dependent insulin secretion.

Fasiglifam (TAK‐875) is a free fatty acid receptor 1 (FFAR1)/G‐protein–coupled receptor 40 (GPR40) agonist that improves glycemic control in type 2 diabetes with minimum risk of hypoglycemia. Fasiglifam potentiates glucose‐stimulated insulin secretion (GSIS) from pancreatic β‐cells glucose dependently, although the precise mechanism underlying the glucose dependency still remains unknown. Here, we investigated key cross‐talk between the GSIS pathway and FFAR1 signaling, and Ca2+ dynamics using mouse insulinoma MIN6 cells. We demonstrated that the glucose‐dependent insulinotropic effect of fasiglifam required membrane depolarization and that fasiglifam induced a glucose‐dependent increase in intracellular Ca2+ level and amplification of Ca2+ oscillations. This differed from the sulfonylurea glimepiride that induced changes in Ca2+ dynamics glucose independently. Stimulation with cell‐permeable analogs of IP3 or diacylglycerol (DAG), downstream second messengers of Gαq‐FFAR1, augmented GSIS similar to fasiglifam, indicating their individual roles in the potentiation of GSIS pathway. Intriguingly, the IP3 analog triggered similar Ca2+ dynamics to fasiglifam, whereas the DAG analog had no effect. Despite the lack of an effect on Ca2+ dynamics, the DAG analog elicited synergistic effects on insulin secretion with Ca2+ influx evoked by an L‐type voltage‐dependent calcium channel opener that mimics glucose‐dependent Ca2+ dynamics. These results indicate that the Gαq signaling activated by fasiglifam enhances GSIS pathway via dual potentiating mechanisms in which IP3 amplifies glucose‐induced Ca2+ oscillations and DAG/protein kinase C (PKC) augments downstream secretory mechanisms independent of Ca2+ oscillations.

362 Lipopolysaccharide-Mediated Ubiquitin Conjugating Enzyme 9 Phosphorylation as a Novel Mechanism for Potentiation of Alcohol Toxicity

362 Lipopolysaccharide-Mediated Ubiquitin Conjugating Enzyme 9 Phosphorylation as a Novel Mechanism for Potentiation of Alcohol Toxicity

Broad-spectrum antimicrobial photocatalysis mediated by titanium dioxide and UVA is potentiated by addition of bromide ion via formation of hypobromite

Antimicrobial photocatalysis involves the UVA excitation of titanium dioxide (TiO2) nanoparticles (particularly the anatase form) to produce reactive oxygen species (ROS) that kill microbial cells. For the first time we report that the addition of sodium bromide to photoactivated TiO2 (P25) potentiates the killing of Gram-positive, Gram-negative bacteria and fungi by up to three logs. The potentiation increased with increasing bromide concentration in the range of 0–10mM. The mechanism of potentiation is probably due to generation of both short and long-lived oxidized bromine species including hypobromite as shown by the following observations. There is some antimicrobial activity remaining in solution after switching off the light, that lasts for 30min but not 2h, and oxidizes 3,3′,5,5′-tetramethylbenzidine. N-acetyl tyrosine ethyl ester was brominated in a light dose-dependent manner, however no bromine or tribromide ion could be detected by spectrophotometry or LC–MS. The mechanism appears to have elements in common with the antimicrobial system (myeloperoxidase+hydrogen peroxide+bromide).

Exploring the positive allosteric modulation of human α7 nicotinic receptors from a single-channel perspective

Enhancement of α7 nicotinic receptor (nAChR) function by positive allosteric modulators (PAMs) is a promising therapeutic strategy to improve cognitive deficits. PAMs have been classified only on the basis of their macroscopic effects as type I, which only enhance agonist-induced currents, and type II, which also decrease desensitization and reactivate desensitized nAChRs. To decipher the molecular basis underlying these distinct activities, we explored the effects on single-α7 channel currents of representative members of each type and of less characterized compounds. Our results reveal that all PAMs enhance open-channel lifetime and produce episodes of successive openings, thus indicating that both types affect α7 kinetics. Different PAM types show different sensitivity to temperature, suggesting different mechanisms of potentiation. By using a mutant α7 receptor that is insensitive to the prototype type II PAM (PNU-120596), we show that some though not all type I PAMs share the structural determinants of potentiation. Overall, our study provides novel information on α7 potentiation, which is key to the ongoing development of therapeutic compounds.

The effect of chemotherapy on programmed cell death 1/programmed cell death 1 ligand axis: some chemotherapeutical drugs may finally work through immune response.

Most tumors are immunogenic which would trigger some immune response. Chemotherapy also has immune potentiating mechanisms of action. But it is unknown whether the immune response is associated with the efficacy of chemotherapy and the development of chemoresistance. Recently, there is a growing interest in immunotherapy, among which the co-inhibitory molecules, programmed cell death 1/programmed cell death 1 ligand (PD-1/PD-L1) leads to immune evasion. Since some reports showed that conventional chemotherapeutics can induce the expression of PD-L1, we try to summarize the effect of chemotherapy on PD-1/PD-L1 axis and some potential molecules relevant to PD-1/PD-L1 in chemoresistance in this review.

The functional interaction between abaecin and pore-forming peptides indicates a general mechanism of antibacterial potentiation

Long-chain proline-rich antimicrobial peptides such as bumblebee abaecin show minimal activity against Gram-negative bacteria despite binding efficiently to specific intracellular targets. We recently reported that bumblebee abaecin interacts with Escherichia coli DnaK but shows negligible antibacterial activity unless it is combined with sublethal doses of the pore-forming peptide hymenoptaecin. These two bumblebee peptides are co-expressed in vivo in response to a bacterial challenge. Here we investigated whether abaecin interacts similarly with pore-forming peptides from other organisms by replacing hymenoptaecin with sublethal concentrations of cecropin A (0.3μM) or stomoxyn (0.05μM). We found that abaecin increased the membrane permeabilization effects of both peptides, confirming that it can reduce the minimal inhibitory concentrations of pore-forming peptides from other species. We also used atomic force microscopy to show that 20μM abaecin combined with sublethal concentrations of cecropin A or stomoxyn causes profound structural changes to the bacterial cell surface. Our data indicate that the potentiating functional interaction between abaecin and pore-forming peptides is not restricted to specific co-expressed peptides from the same species but is likely to be a general mechanism. Combination therapies based on diverse insect-derived peptides could therefore be used to tackle bacteria that are recalcitrant to current antibiotics.

Regulation of CNGA1 Channel Gating by Interactions with the Membrane

We have previously shown that a histidine (H420) in the first intracellular helix (A’ helix) of cyclic nucleotide-gated (CNGA1) channels is involved in potentiation of CNGA1 currents by transition metal ions. The binding of transition metals stabilizes the open state of the channel, so understanding the details of metal ion coordination will provide insight into the structural changes that occur after ligand binding to promote channel opening. Because structural studies suggest that the A’ helix is proximal to the membrane, we tested the hypothesis that a metal ion is coordinated between H420 and the intracellular leaflet of the bilayer with transition metal FRET (tmFRET) and patch-clamp fluorometry. To label CNGA1 channels, the amber codon suppression strategy was used to incorporate a fluorescent, noncanonical amino acid, L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (L-Anap) near H420. We chose Co2+ (1-30 μM) to potentiate CNGA1 currents and serve as a FRET acceptor for L-Anap in inside-out patches from Xenopus oocytes. We found that Co2+ quenched Anap fluorescence by ∼50%, and the H420Q mutation significantly attenuated quenching, providing further evidence that Co2+ binds to H420. Next, to test whether the membrane provides a second Co2+ ligand, we added high-affinity metal binding sites to the membrane using a novel, metal-chelating fatty acid (C18-NTA). We found that metal potentiation was increased for low (1-3 μM) Co2+ concentrations with C18-NTA-Co2+, and this effect required H420. These results indicate that membrane-bound metal ions interact with the A’ helix, suggesting that the mechanism for metal potentiation is metal ion coordination between H420 and a lipid with an anionic headgroup with lower affinity for Co2+ compared to C18-NTA. This arrangement of the A’ helix relative to the membrane potentiates channel opening and may be an important structural transition that occurs with cyclic nucleotide regulation.

A Mechanism for the Temporal Potentiation of Genipin to the Cytotoxicity of Cisplatin in Colon Cancer Cells.

OBJECTIVES: To investigate the potentiation effect of Genipin to Cisplatin induced cell senescence in HCT-116 colon cancer cells in vitro. METHODS: Cell viability was estimated by Propidium iodide and Hoechst 3342, reactive oxygen species (ROS) with DHE, mitochondrial membrane potential (MMP) with JC-1 MMP assay Kit and electron current production with microbial fuel cells (MFC). RESULTS: Genipin inhibited the UCP2 mediated anti-oxidative proton leak significantly promoted the Cisplatin induced ROS and subsequent cell death, which was similar to that of UCP2-siRNA. Cells treated with Cisplatin alone or combined with Genipin, ROS negatively, while MMP positively correlated with cell viability. Cisplatin induced ROS was significantly decreased by detouring electrons to MFC, or increased by Genipin combined treatment. Compensatory effects of UCP2 up-regulation with time against Genipin treatment were suggested. Shorter the Genipin treatment before Cisplatin better promoted the Cisplatin induced ROS and subsequent cell death. CONCLUSION: The interaction of leaked electron with Cisplatin was important during ROS generation. Inhibition of UCP2-mediated proton leak with Genipin potentiated the cytotoxicity of Cisplatin. Owing to the compensatory effects against Genipin, shorter Genipin treatment before Cisplatin was recommended in order to achieve better potentiation effect.

Mechanisms of Inhibition and Potentiation of α4β2 Nicotinic Acetylcholine Receptors by Members of the Ly6 Protein Family

α4β2 nicotinic acetylcholine receptors (nAChRs) are abundantly expressed throughout the central nervous system and are thought to be the primary target of nicotine, the main addictive substance in cigarette smoking. Understanding the mechanisms by which these receptors are regulated may assist in developing compounds to selectively interfere with nicotine addiction. Here we report previously unrecognized modulatory properties of members of the Ly6 protein family on α4β2 nAChRs. Using a FRET-based Ca(2+) flux assay, we found that the maximum response of α4β2 receptors to agonist was strongly inhibited by Ly6h and Lynx2 but potentiated by Ly6g6e. The mechanisms underlying these opposing effects appear to be fundamentally distinct. Receptor inhibition by Lynx2 was accompanied by suppression of α4β2 expression at the cell surface, even when assays were preceded by chronic exposure of cells to an established chaperone, nicotine. Receptor inhibition by Lynx2 also was resistant to pretreatment with extracellular phospholipase C, which cleaves lipid moieties like those that attach Ly6 proteins to the plasma membrane. In contrast, potentiation of α4β2 activity by Ly6g6e was readily reversible by pretreatment with phospholipase C. Potentiation was also accompanied by slowing of receptor desensitization and an increase in peak currents. Collectively our data support roles for Lynx2 and Ly6g6e in intracellular trafficking and allosteric potentiation of α4β2 nAChRs, respectively.

Identification of the determinants of 2-deoxyglucose sensitivity in cancer cells by shRNA library screening

Combining glycolytic inhibition with other anti-cancer therapies is a potential approach to treating cancer. In this context, we attempted to identify genes that determine sensitivity to 2-deoxyglucose (2DG), a glycolytic inhibitor, in cancer cells using pooled shRNA libraries targeting ∼15,000 genes. The screen revealed that COPB1 and ARCN1, which are essential in retrograde transport, as determinants of sensitivity to 2DG: silencing of COPB1 or ARCN1 expression sensitized cells to 2DG toxicity. To address the mechanism of potentiation of 2DG toxicity by inhibition of COPI-mediated transport, we focused on the role of lipolysis as an alternate source of energy upon inhibition of glycolysis. In the process of lipolysis, COPI-mediated transport is required for localization to lipid droplets of adipose triglyceride lipase (ATGL), a key enzyme that produces fatty acids from triacylglycerol as a substrate for β-oxidation. The ATGL inhibitor atglistatin potentiated 2DG toxicity, consistent with a model in which a defect in COPI-mediated transport of ATGL to lipid droplets inhibits energy supply, thereby sensitizing cells to glycolytic inhibition. Collectively, our data demonstrated that a defect in COPI-mediated transport or pharmacological inhibition of ATGL potentiates 2DG toxicity in cancer cells, possibly due to a reduction in the energy supply.

Chronic Hyponatremia Causes Neurologic and Psychologic Impairments.

Hyponatremia is the most common clinical electrolyte disorder. Once thought to be asymptomatic in response to adaptation by the brain, recent evidence suggests that chronic hyponatremia may be linked to attention deficits, gait disturbances, risk of falls, and cognitive impairments. Such neurologic defects are associated with a reduction in quality of life and may be a significant cause of mortality. However, because underlying diseases such as adrenal insufficiency, heart failure, liver cirrhosis, and cancer may also affect brain function, the contribution of hyponatremia alone to neurologic manifestations and the underlying mechanisms remain unclear. Using a syndrome of inappropriate secretion of antidiuretic hormone rat model, we show here that sustained reduction of serum sodium ion concentration induced gait disturbances; facilitated the extinction of a contextual fear memory; caused cognitive impairment in a novel object recognition test; and impaired long-term potentiation at hippocampal CA3-CA1 synapses. In vivo microdialysis revealed an elevated extracellular glutamate concentration in the hippocampus of chronically hyponatremic rats. A sustained low extracellular sodium ion concentration also decreased glutamate uptake by primary astrocyte cultures, suggesting an underlying mechanism of impaired long-term potentiation. Furthermore, gait and memory performances of corrected hyponatremic rats were equivalent to those of control rats. Thus, these results suggest chronic hyponatremia in humans may cause gait disturbance and cognitive impairment, but these abnormalities are reversible and careful correction of this condition may improve quality of life and reduce mortality.