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Efficacious and accessible sources of natural killer (NK) cells would widen their use as immunotherapeutics, particularly for solid cancers. Here, we show that human somatic cells can be directly reprogrammed into NK cells with a CD56brightCD16bright phenotype using pluripotency transcription factors and an optimized reprogramming medium. The directly reprogrammed NK cells have strong innate-adaptive immunomodulatory activity and are highly potent against a wide range of cancer cells, including difficult-to-treat solid cancers and cancer stem cells. Both directly reprogrammed NK cells bearing a cancer-specific chimeric antigen receptor and reprogrammed NK cells in combination with antibodies competent for antibody-dependent cell-mediated cytotoxicity led to selective anticancer effects with augmented potency. The direct reprogramming of human somatic cells into NK cells is amenable to the production of autologous and allogeneic NK cells, and will facilitate the design and testing of cancer immunotherapies and combination therapies.

Microglial activation is known to be an important event during innate immunity, but microglial inflammation is also thought to play a role in the etiology of neurodegenerative diseases. Recently, it was reported that autophagy could influence inflammation and activation of microglia. However, little is known about the regulation of autophagy during microglial activation. In this study, we demonstrated that mitochondrial fission-induced ROS can promote autophagy in microglia. Following LPS-induced autophagy, GFP-LC3 puncta were increased, and this was suppressed by inhibiting mitochondrial fission and mitochondrial ROS. Interestingly, inhibition of mitochondrial fission and mitochondrial ROS also resulted in decreased p62 expression, but Beclin1 and LC3B were unaffected. Taken together, these results indicate that ROS induction due to increased LPS-stimulated mitochondrial fission triggers p62 mediated autophagy in microglial cells. Our findings provide the first important clues towards understanding the correlation between mitochondrial ROS and autophagy. Abbreviations: Drp1; Dynamin related protein 1, LPS; Lipopolysaccharide, ROS; Reactive Oxygen Species, GFP; Green Fluorescent Protein, CNS; Central Nervous System, AD; Alzheimer’s Disease, PD; Parkinson’s Disease, ALIS; Aggresome-like induced structures, iNOS; inducible nitric oxide synthase, Cox-2; Cyclooxygenase-2, MAPK; Mitogen-activated protein kinase; SODs; Superoxide dismutase, GPXs; Glutathione Peroxidase, Prxs; Peroxiredoxins

BackgroundSchwann cells (SCs) are primarily responsible for regeneration and repair of the peripheral nervous system (PNS). Renewable and lineage-restricted SC precursors (SCPs) are considered highly desirable and promising cell sources for the production of SCs and for studies of SC lineage development, but SCPs are extremely limited. Here, we present a novel direct conversion strategy for the generation of human SCPs, capable of differentiating into functional SCs.MethodsEasily accessible human skin fibroblast cells were directly induced into integration-free SCPs using episomal vectors (Oct3/4, Klf4, Sox2, L-Myc, Lin28 and p53 shRNA) under SCP lineage-specific chemically defined medium conditions. Induced SCPs (iSCPs) were further examined for their ability to differentiate into SCs. The identification and functionality of iSCPs and iSCP-differentiated SCs (iSCs) were confirmed according to morphology, lineage-specific markers, neurotropic factor secretion, and/or standard functional assays.ResultsHighly pure, Sox 10-positive of iSCPs (more than 95% purity) were generated from human skin fibroblasts within 3 weeks. Established iSCPs could be propagated in vitro while maintaining their SCP identity. Within 1 week, iSCPs could efficiently differentiate into SCs (more than 95% purity). The iSCs were capable of secreting various neurotrophic factors such as GDNF, NGF, BDNF, and NT-3. The in vitro myelinogenic potential of iSCs was assessed by myelinating cocultures using mouse dorsal root ganglion (DRG) neurons or human induced pluripotent stem cell (iPSC)-derived sensory neurons (HSNs). Furthermore, iSC transplantation promoted sciatic nerve repair and improved behavioral recovery in a mouse model of sciatic nerve crush injury in vivo.ConclusionsWe report a robust method for the generation of human iSCPs/iSCs that might serve as a promising cellular source for various regenerative biomedical research and applications, such as cell therapy and drug discovery, especially for the treatment of PNS injury and disorders.

Testis-specific protein, Y-encoded like (TSPYL) family proteins (TSPYL1-6), which are members of the nucleosome assembly protein superfamily, have been determined to be involved in the regulation of various cellular functions. However, the potential role of TSPYL family proteins in endothelial cells (ECs) has not been determined. Here, we demonstrated that the expression of TSPYL5 is highly enriched in human ECs such as human umbilical vein endothelial cells (HUVECs) and human pluripotent stem cell-differentiated ECs (hPSC-ECs). Importantly, TSPYL5 overexpression was shown to promote EC proliferation and functions, such as migration and tube formation, by downregulating p53 expression. Adriamycin-induced senescence was markedly blocked by TSPYL5 overexpression. In addition, the TSPYL5 depletion-mediated loss of EC functions was blocked by p53 inhibition. Significantly, TSPYL5 overexpression promoted angiogenesis in Matrigel plug and wound repair in a mouse skin wound healing model in vivo. Our results suggest that TSPYL5, a novel angiogenic regulator, plays a key role in maintaining endothelial integrity and function. These findings extend the understanding of TSPYL5-dependent mechanisms underlying the regulation of p53-related functions in ECs.

In various neuronal diseases, the activation of microglia contributes to the production of excessive neurotoxic factors, such as pro-inflammatory mediators. In particular, the overproduction of pro-inflammatory cytokines and nitric oxide (NO) has critical effects on the development of neurodegenerative diseases and gliomas in the brain. Recent studies have suggested that isocitrate dehydrogenase 2 (IDH2) plays a key role in inducing gliomas and neurodegeneration. IDH2 dysfunction has been linked to various cancers and neurodegenerative diseases associated with uncontrolled inflammatory responses, such as the excessive generation of pro-inflammatory cytokines. In this study, we demonstrate that IDH2 contributes to the regulation of pro-inflammatory mediators in microglia. The downregulation of IDH2 decreased the lipopolysaccharide (LPS)-induced pro-inflammatory response in BV-2 and primary microglial cells. Furthermore, IDH2 deficiency downregulated pro-inflammatory mediators via modulation of the ERK and NF-κB pathways. These results indicate that IDH2 is a potential target for the regulation of pro-inflammatory responses in LPS-activated microglial cells. Our findings also provide a basis for the development of new therapies for pro-inflammatory responses in dysfunction-associated neuronal diseases.

Summary Schwann cells play a crucial role in successful nerve repair and regeneration by supporting both axonal growth and myelination. However, the sources of human Schwann cells are limited both for studies of Schwann cell development and biology and for the development of treatments for Schwann cell-associated diseases. Here, we provide a rapid and scalable method to produce self-renewing Schwann cell precursors (SCPs) from human pluripotent stem cells (hPSCs), using combined sequential treatment with inhibitors of the TGF-β and GSK-3 signaling pathways, and with neuregulin-1 for 18 days under chemically defined conditions. Within 1 week, hPSC-derived SCPs could be differentiated into immature Schwann cells that were functionally confirmed by their secretion of neurotrophic factors and their myelination capacity in vitro and in vivo. We propose that hPSC-derived SCPs are a promising, unlimited source of functional Schwann cells for treating demyelination disorders and injuries to the peripheral nervous system., Highlights • hPSC-SCPs are highly expandable under chemically defined medium condition • hPSC-SCPs can rapidly and efficiently differentiate into functional Schwann cells • SCP-SCs myelinate axon and secrete various neurotrophic factors • SCP-SCs promote axonal regeneration in sciatic nerve-damaged mice, In this article, Cho and colleagues show that an efficient strategy for producing directly an unlimited supply of functional human Schwann cells (SCs) via successful derivation of expandable Schwann cell precursors (SCPs) from human pluripotent stem cells (hPSC-SCPs). Functional and molecular characteristic of SCs from hPSC-SCPs (SCP-SCs) were shown both in vitro and in vivo.

In this paper, we present OLED display technologies for large-size and premium TVs. Since high-quality TV needs total accuracy to reproduce input images, OLED panels must achieve stable luminance, uniformity at low luminance, and color uniformity. Using various OLED and TFT compensation technologies, we have successfully developed an OLED display with the “Real Black” image quality and high color uniformity. Additionally we have been developing OLED displays for broadcast monitors, which require much higher image quality than commercial TVs. We are now focusing on cost competitiveness of premium OLED displays.

Additional file 2: Table S1. Primers for qRT-PCR. Table S2. List of antibodies.

Although comparative genome-wide transcriptomic analysis has provided insight into the biology of human induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs), the distinct alternative splicing (AS) signatures of iMSCs remain elusive. Here, we performed Illumina RNA sequencing analysis to characterize AS events in iMSCs compared with tissue-derived MSCs. A total of 4586 differentially expressed genes (|FC| &gt, 2) were identified between iMSCs and umbilical cord blood-derived MSCs (UCB-MSCs), including 2169 upregulated and 2417 downregulated genes. Of these, 164 differentially spliced events (BF &gt, 20) in 112 genes were identified between iMSCs and UCB-MSCs. The predominant type of AS found in iMSCs was skipped exons (43.3%), followed by retained introns (19.5%), alternative 3′ (15.2%) and 5′ (12.8%) splice sites, and mutually exclusive exons (9.1%). Functional enrichment analysis showed that the differentially spliced genes (|FC| &gt, 2 and BF &gt, 20) were mainly enriched in functions associated with focal adhesion, extracellular exosomes, extracellular matrix organization, cell adhesion, and actin binding. Splice isoforms of selected genes including TRPT1, CNN2, and AP1G2, identified in sashimi plots, were further validated by RT-PCR analysis. This study provides valuable insight into the biology of iMSCs and the translation of mechanistic understanding of iMSCs into therapeutic applications.

In this paper, we present novel organic light-emitting diode (OLED) display panel compensation technologies for large-sized ultra-high-definition OLED TVs considering variations of threshold voltage, mobility, channel size, OLED efficiency, and OLED uniformity. Using these technologies, we have successfully launched 55-, 65- and 77-in. ultra-high-definition OLED TVs.

In this paper, we present advanced OLED display technologies for large-size semi-flexible TVs, including a coplanar TFT backplane, a high-performance RGBW structure, compensation technologies and a high dynamic range technology for enhancing image qualities. By using them we have developed various displays and TVs such as curved, transparent, dual plate, and semi-flexible display applications. Currently we concentrate on technologies for premium displays and cost competitiveness for UHD OLED TVs.

Iron is an essential element for neuronal as well as cellular functions. However, Iron overload has been known to cause neuronal toxicity through mitochondrial fission, dysregulation of Ca2+, endoplasmic reticulum (ER) stress, and reactive oxygen species (ROS) production. Nevertheless, the precise mechanisms of iron-induced oxidative stress and mitochondria- and ER-related iron toxicity in neuronal cells are not fully understood. In this study, we demonstrated that iron overload induces ROS production earlier in the ER than in the mitochondria, and peroxiredoxin 5 (Prx5), which is a kind of antioxidant induced by iron overload, prevents iron overload-induced mitochondrial fragmentation mediated by contact with ER and translocation of Drp1, by inhibiting ROS production and calcium/calcineurin pathway in HT-22 mouse hippocampal neuronal cells. Moreover, Prx5 also prevented iron overload-induced ER-stress and cleavage of caspase-3, which consequently attenuated neuronal cell death. Therefore, we suggested that iron overload induces oxidative stress in the ER earlier than in the mitochondria, thereby increasing ER stress and calcium levels, and consequently causing mitochondrial fragmentation and neuronal cell death. So we thought that this study is essential for understanding iron toxicity in neurons, and Prx5 may serve as a new therapeutic target to prevent iron overload-induced diseases and neurodegenerative disorders.

In this paper, we present novel OLED display technologies for large-size UHD OLED TVs, including an IGZO TFT backplane, an RGBW pixel structure with white OLED, driving schemes and compensation methods applied to the panel. We also discuss technical issues and challenges in panel design and driving methods to compensate non-uniformities of oxide TFTs, OLED devices, color, and luminance. Using these technologies, we have successfully launched 55-, 65- and 77-inch UHD OLED TVs. Currently we focus on technologies to achieve higher image qualities, a wider color range and a higher cost competitiveness in the TV market.

The importance of alternative splicing (AS) events in pluripotency regulation has been highlighted by the determination of different roles and contributions of different splice isoforms of pluripotency-related genes and by the identification of distinct pluripotency-related splicing factors. In particular, epithelial splicing regulatory protein 1 (ESRP1) has been characterized as an essential splicing factor required for the regulation of human pluripotency and differentiation. Nevertheless, a detailed molecular characterization of ESRP1 (mRNA splice variants 1–6) in human pluripotency is lacking. In this study, we determined that ESRP1 splice variants are differentially expressed in undifferentiated and differentiated human pluripotent stem cells (PSCs). Undifferentiated human PSCs predominantly expressed the ESRP1 v1, v4, and v5, and their expression was downregulated upon differentiation. Ectopic expression of ESRP1 v1, v4, or v5 enhanced the pluripotent reprogramming of human fibroblasts and restored the ESRP1 knockdown-mediated reduction of reprogramming efficiency. Notably, undifferentiated human PSCs expressed the cell surface protein CD44 variant 3 (CD44 v3), and isoform switching from CD44 v3 to CD44 variant 6 (CD44 v6) occurred upon differentiation. Importantly, the human PSC-specific ESRP1 variants influenced CD44 v3 expression. CD44 knockdown or inhibition of binding of CD44 with its major ligand, hyaluronan, significantly induced the loss of human PSC pluripotency and the reduction of reprogramming efficiency. Our results demonstrate that the effect of ESRP1 and CD44 on human PSC pluripotency is isoform-dependent and that ESRP1-induced CD44 v3 is functionally associated with human PSC pluripotency control.

The development of various flexible and stretchable materials has attracted interest for promising applications in biomedical engineering and electronics industries. This interest in wearable electronics, stretchable circuits, and flexible displays has created a demand for stable, easily manufactured, and cheap materials. However, the construction of flexible and elastic electronics, on which commercial electronic components can be mounted through simple and cost-effective processing, remains challenging. We have developed a nanocomposite of carbon nanotubes (CNTs) and polydimethylsiloxane (PDMS) elastomer. To achieve uniform distributions of CNTs within the polymer, an optimized dispersion process was developed using isopropyl alcohol (IPA) and methyl-terminated PDMS in combination with ultrasonication. After vaporizing the IPA, various shapes and sizes can be easily created with the nanocomposite, depending on the mold. The material provides high flexibility, elasticity, and electrical conductivity without requiring a sandwich structure. It is also biocompatible and mechanically stable, as demonstrated by cytotoxicity assays and cyclic strain tests (over 10,000 times). We demonstrate the potential for the healthcare field through strain sensor, flexible electric circuits, and biopotential measurements such as EEG, ECG, and EMG. This simple and cost-effective fabrication method for CNT/PDMS composites provides a promising process and material for various applications of wearable electronics.

Ultra-high definition (UHD) curved organic light-emitting diode (OLED) TV requires advanced technologies to realize mass production. White, red, green, and blue (WRGB) OLED TV based on stripe WRGB sub-pixel structure has distinct advantages in luminance of white, faster response time, wider viewing angle, and potential higher resolution. In this paper, we will introduce technological progress for commercializing large-sized and UHD curved OLED TV. Those technologies including oxide thin film transistors, white OLEDs, compensation circuit, and solid phase encapsulation enable panel size scalability as well as mass production with lifetime reliability.

The world's first 77-inch UHD OLED TV with an excellent image quality has been developed, using an IGZO TFT backplane and white OLEDs with an RGBW pixel structure and a novel compensation method applied to the panel. In this paper, we discuss technical issues and challenges in panel design and driving methods to compensate the non-uniformity of oxide TFTs, OLED devices, colors, and luminance, including our recent technologies that have realized a panel size scalability and a product reliability for commercializing large-size OLED TVs.

Small molecules enable OCT4-mediated direct reprogramming into expandable human neural stem cells

Individual pharmacokinetic differences for herb–drug interaction have been associated with genetic variations of the multidrug resistance (MDR) gene. A high level expression of MDR protein increases cellular efflux and might decrease drug sensitivity. This study investigated the drug efflux activity difference of human MDR1 triallelic variant 2677G/T/A (rs2032582), as a nonsynonymous 893Ala/Ser/Thr, using Xenopus laevis oocytes and MDR1 overexpressing LLC-PK1 cells. Two MDR1 variants (2667T/893Ser and 2667A/893Thr) were generated using human MDR1 cDNA (2677G/893Ala). No significant difference in the expression of MDR1 893Ala/Ser/Thr was found in X. laevis oocytes. However, the MDR1 2667A/893Thr variant interestingly showed a significant decrease of efflux activity for both digoxin and daunorubicin compared with those of 893Ala and 893Ser variants. In further investigation assessing the inhibitory effects of three herbal extracts on MDR1, 893Ala and 893Ser showed significant decreases of efflux activities in treatments with P. cocos (p = 0.005 for 893Ser) and D. dasycarpus (p = 0.0009 for 893Ala; p = 0.002 for 893Ser) in X. laevis oocytes. The results in this study suggest that herbal medicines could interact with other drugs and change the therapeutic effects depending on the genetic polymorphisms of individuals. Copyright © 2011 John Wiley & Sons, Ltd.

This study was designed to determine whether early gabapentin treatment has a protective analgesic effect on neuropathic pain and compared its effect to the late treatment in a rat neuropathic model, and as the potential mechanism of protective action, the alpha(2)delta(1)-subunit of the voltage-dependent calcium channel (alpha(2)delta(1)-subunit) was evaluated in both sides of the L5 dorsal root ganglia (DRG). Neuropathic pain was induced in male Sprague-Dawley rats by a surgical ligation of left L5 nerve. For the early treatment group, rats were injected with gabapentin (100 mg/kg) intraperitoneally 15 min prior to surgery and then every 24 hr during postoperative day (POD) 1-4. For the late treatment group, the same dose of gabapentin was injected every 24 hr during POD 8-12. For the control group, L5 nerve was ligated but no gabapentin was administered. In the early treatment group, the development of allodynia was delayed up to POD 10, whereas allodynia was developed on POD 2 in the control and the late treatment group (p0.05). The alpha(2)delta(1)-subunit was up-regulated in all groups, however, there was no difference in the level of the alpha(2)delta(1)-subunit among the three groups. These results suggest that early treatment with gabapentin offers some protection against neuropathic pain but it is unlikely that this action is mediated through modulation of the alpha(2)delta(1)-subunit in DRG.

This study shows the effects of L-carnitine treatment on cell proliferation with hepa1c1c7 mouse cancer cells and NCTC 1469 normal cells. In an MTT assay, L-carnitine increased the number of dead hepa1c1c7 cells, while there was no difference in the number of NCTC 1469 cells. mRNA and protein levels of TNF-alpha, Fas, and caspase-8, which are closely related to cell apoptosis by a death ligand/receptor-dependent apoptosis pathway, were increased by L-carnitine treatment. In addition, L-carnitine treatment regulated mitochondria-dependent apoptosis pathways by inducing the up-regulation of caspase-9 and caspase-3 and the down-regulation of Bcl-2 in hepa1c1c 7 cells. Taken together, the findings of this study have demonstrated that L-carnitine could induce apoptosis in hepa1c1c7 cells by regulating Fas ligands and inhibiting the expression of Bcl-2. These results suggest that L: -carnitine treatment could be related to both a mitochondrion-dependent and a death ligand/receptor-dependent apoptosis pathway in hepa1c1c7 cells. Our results could give information for understanding the L-carnitine-induced apoptosis mechanism in some cancer cells.

The newly synthesized N-substituted derivative of 3-arylpyrrolidine LB50053, 2--butyl}-1,2-benzisothiazol-3(2H)-one-1,1-dioxide, was studied in receptor-binding assays and in electrophysiological measurements. Competitive binding experiments with various radioligands to the rat forebrain revealed that the (S)-enantiomer of LB50053 had a high affinity (Ki 4.2 nmol/l) and a high selectivity for 5-HT1A receptors as compared with 5-HT2A, D1 dopamine, D2 dopamine, or α2-adrenergic receptor. In Xenopus oocytes, where coupling of the 5-HT1A receptor to the G protein activated inwardly rectifying K+ channel 1 (GIRK1) was established, (S)-LB50053 evoked an inward K+ current through GIRK1 in a manner consistent with a partial agonism. The Kd value deduced from the dose-response relationships of the 5-HT1A receptor full agonist 8-OH-2-(di-n-propylamino)-1,2,3,4-tetrahydronaphthalene and (S)-LB50053 according to Waud analysis was 64.60 nmol/l. These results demonstrate that LB50053 is a 5-HT1A receptor partial agonist and thus can be used as a therapeutic or pharmacological research tool for 5-HT1A receptor mediated events in the future.

Xenopus oocyte is one of the widely used heterologous expression systems of ion channels for electrophysiological studies. Here we describe a new method in which cRNA produced by polymerase chain reaction (PCR) and in vitro transcription is injected to express ion channels in oocytes. This method enables us (1) to eliminate all or a part of the untranslated region of the cDNA and to replace it with a known sequence which helps increase the expression level in oocytes, and (2) to use the PCR product for in vitro transcription without subcloning. Using this method, the expression level of one of the neuronal nicotinic acetyfcholine receptors (nAChRs) α7 subtype in oocytes was systematically increased by more than 100‐fold, which was confirmed both by the α‐Bungarotoxin (αBgt) binding assay and the current measurement

The G protein‐activated inwardly rectifying K+ channel (GIRK1) was coex‐pressed in Xenopus oocytes along with the 5‐HTia receptor, a 7‐helix receptor known to be coupled to K+ channels in many neural tissues. Thus, the activation of the 5‐HT1A receptor by its agonist leads to the opening of GIRK1. The GIRK1 current was measured using the two electrode voltage clamp technique with bath application of 5‐HT in the presence of various external potassium concentrations [K+]o. GIRK1 showed a strong inward rectification since only hyperpolarizing voltages evoked inward currents. K+ was the major ion carrier as evidenced by about 44 mV voltage shift corresponding to a 10‐fold external [K+] change. 5‐HT induced a concentration‐dependent inward K+ current (EC 50=10.7nM) which was blocked by Ba2+ Pertussis toxin (PTX) pre‐treatment reduced the K+ current by as much as about 70%, suggesting that PTX‐sensitive G protein (Gi or Go type) are involved in the 5‐HT1A receptor‐GIRK1 coupling in Xenopus oocytes

The direct lineage reprogramming of somatic cells to other lineages by defined factors has led to innovative cell-fate-change approaches for providing patient-specific cells. Recent reports have demonstrated that four pluripotency factors (Oct4, Sox2, Klf4, and c-Myc) are sufficient to directly reprogram fibroblasts to other specific cells, including induced neural stem cells (iNSCs). Here, we show that mouse fibroblasts can be directly reprogrammed into midbrain dopaminergic neuronal progenitors (DPs) by temporal expression of the pluripotency factors and environment containing sonic hedgehog and fibroblast growth factor 8. Within thirteen days, self-renewing and functional induced DPs (iDPs) were generated. Interestingly, the inhibition of both Jak and Gsk3β notably enhanced the iDP reprogramming efficiency. We confirmed the functionality of the iDPs by showing that the dopaminergic neurons generated from iDPs express midbrain markers, release dopamine, and show typical electrophysiological profiles. Our results demonstrate that the pluripotency factors-mediated direct reprogramming is an invaluable strategy for supplying functional and proliferating iDPs and may be useful for other neural progenitors required for disease modeling and cell therapies for neurodegenerative disorders.

Previously, we reported the phosphorylation of moesin induced by electroconvulsive shock in rat brain and by glutamate in immortalized rat hippocampal cells. However, the function of phosphorylated moesin in differentiated neurons is not well understood. In this study, we observed that glutamate induces phosphorylation of ezrin/radixin/moesin proteins (ERM) in cultured hippocampal cells and that phosphorylated ERM localizes at the newly formed filopodia of neurites. The glutamate-induced phosphorylation of ERM is calcium-dependent, and inhibition of protein kinase C abolishes ERM phosphorylation as well as RhoA activation. The inhibitions of RhoA and RhoA kinase also diminishes the glutamate-induced ERM phosphorylation in cultured hippocampal cells. The knock-down of moesin or the inhibition of ERM phosphorylation results in the reduction of glutamate-induced filopodia protrusion and diminishes the increase in active synaptic boutons induced by glutamate treatment. These results indicate that glutamate-induced phosphorylation of ERM proteins in primary cultured differentiated hippocampal neurons is mediated by calcium-dependent protein kinase C, RhoA and RhoA kinase, and the phosphorylated ERM protein is necessary for the formation of filopodial protrusion and may be involved in pre-synaptic trafficking.

Previously, we reported the phosphorylation of moesin induced by electroconvulsive shock in rat brain and by glutamate in immortalized rat hippocampal cells. However, the function of phosphorylated moesin in differentiated neurons is not well understood. In this study, we observed that glutamate induces phosphorylation of ezrin/radixin/moesin proteins (ERM) in cultured hippocampal cells and that phosphorylated ERM localizes at the newly formed filopodia of neurites. The glutamate-induced phosphorylation of ERM is calcium-dependent, and inhibition of protein kinase C abolishes ERM phosphorylation as well as RhoA activation. The inhibitions of RhoA and RhoA kinase also diminishes the glutamate-induced ERM phosphorylation in cultured hippocampal cells. The knock-down of moesin or the inhibition of ERM phosphorylation results in the reduction of glutamate-induced filopodia protrusion and diminishes the increase in active synaptic boutons induced by glutamate treatment. These results indicate that glutamate-induced phosphorylation of ERM proteins in primary cultured differentiated hippocampal neurons is mediated by calcium-dependent protein kinase C, RhoA and RhoA kinase, and the phosphorylated ERM protein is necessary for the formation of filopodial protrusion and may be involved in pre-synaptic trafficking.

Magnesium-inhibited, non-selective cation current (I(MIC)) is activated by depletion of intracellular Mg(2+) and ATP. I(MIC) transports various divalent cations including Mg(2+) and Ca(2+), and is involved in cell viability. We investigated the effect of actin dynamics on I(MIC). Formation of a stable cortical actin network by calyculin A inhibited the activation of I(MIC), while the actin depolymerizing reagent, cytochalasin D, reversed the inhibition. Induction of a dense cortical actin layer by transfecting the constitutively active form of RhoA also inhibited the activation of I(MIC). These results suggest that the activation of I(MIC) may be dynamically regulated by actin cytoskeleton rearrangement.

The newly synthesized N-substituted derivative of 3-aryl-pyrrolidine LB50053, 2-[4-[3-(4-fluoro)-phenylpyrrolidine-1-yl] - butyl]-1,2- benzisothiazol -3(2H)-one-1,1-dioxide, was studied in receptor-binding assays and in electrophysiological measurements. Competitive binding experiments with various radioligands to the rat fore-brain revealed that the (S)-enantiomer of LB50053 had a high affinity (Ki 4.2 nmol/l) and a high selectivity for 5-HT(1A) receptors as compared with 5-HT(2A), D(1) dopamine, D(2) dopamine, or (alpha(2)-adrenergic receptor. In Xenopus oocytes, where coupling of the 5-HT(1A) receptor to the G protein activated inwardly rectifying K(+) channel 1(GIRK1) was established, (S)-LB50053 evoked an inward K(+) current through GIRK1 in a manner consistent with a partial agonism. The K(d) value deduced from the dose-response relationships of the 5-HT(1A) receptor full agonist 8-OH-2-(di-n-propylamino)-1,2,3,4-tetrahydronaphthalene and (S)-LB50053 according to Waud analysis was 64.60 nmol/l. These results demonstrate that LB50053 is a 5-HT(1A) receptor partial agonist and thus can be used asa therapeutic or pharmacological research tool for 5-HT(1A) receptor mediated events in the future.

Phosphoinositide-specific phospholipase Cgamma (PLCgamma) is a key regulatory enzyme that binds to the phosphoryl-tyrosine residues in the cytoplasmic domain of certain activated receptors and catalyses the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] forming IP3 and diacylglycerol (DAG) in response to several mitogenic factors. Previously, we determined that microinjected PLCgamma induces DNA synthesis in G0-arrested NIH 3T3 cells, suggesting the possibility that PLCgamma may have an oncogenic potential. In this report, we demonstrate that overexpression of PLCgamma in NIH 3T3 cells results in altered growth properties and cellular transformation. The PLCgamma/3T3 transfectants do not require serum growth factors to proliferate, display anchorage-independent growth in soft agar and induce tumors when transplanted into nude mice. These findings suggest that overexpression of PLCgamma facilitates the transformation of NIH 3T3 cells. Furthermore, PLCgamma expression and activity have been shown to be elevated in many human tumors. Thus, PLCgamma signaling may contribute to the promotion and/or progression of human cancers.