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KEY POINTS Presynaptic mitochondria not only absorb but also release Ca2+ during high frequency stimulation (HFS) when presynaptic [Ca2+ ] is kept low (

The aim of this study was to introduce the implemented MEDBIZ platform based on the internet of medical things (IoMT) supporting real-time digital health services for precision medicine. In addition, we demonstrated four empirical studies of the digital health ecosystem that could provide real-time healthcare services based on IoMT using real-world data from in-hospital and out-hospital patients. Implemented MEDBIZ platform based on the IoMT devices and big data to provide digital healthcare services to the enterprise and users. The big data platform is consisting of four main components: IoMT, core, analytics, and services. Among the implemented MEDBIZ platform, we performed four clinical trials that designed monitoring services related to chronic obstructive pulmonary disease, metabolic syndrome, arrhythmia, and diabetes mellitus. Of the four empirical studies on monitoring services, two had been completed and the rest were still in progress. In the metabolic syndrome monitoring service, two studies were reported. One was reported that intervention components, especially wearable devices and mobile apps, made systolic blood pressure, diastolic blood pressure, waist circumference, and glycosylated hemoglobin decrease after 6 months. Another one was presented that increasing high-density lipoprotein cholesterol and triglyceride levels were prevented in participants with the pre-metabolic syndrome. Also, self-care using healthcare devices might help prevent and manage metabolic syndrome. In the arrhythmia monitoring service, during the real-time monitoring of vital signs remotely at the monitoring center, 318 (15.9%) general hikers found abnormal signals, and 296 (93.1%) people were recommended for treatment. We demonstrated the implemented MEDBIZ platform based on IoMT supporting digital healthcare services by acquiring real-world data for getting real-world evidence. And then through this platform, we were developing software as a medical device, digital therapeutics, and digital healthcare services, and contributing to the development of the digital health ecosystem.

UNSTRUCTURED The aim of this study is to introduce the implemented big data platform (MEDBIZ) based on the internet of medical things (IoMT) supporting digital healthcare services and to discuss about application cases of this platform. Implemented MEDBIZ platform based on the IoMT devices and big data to provide digital healthcare services to the enterprise and users. The big data platform is consisting of four main components: IoMT, Core, Analytics, and Services. IoMT component is used for lifelog data acquisition and collection from the IoMT devices. The core components are composed of the main functional operations including the metadata, resource brokers and computing elements, virtual file system, security, and system logs. Analytics component is covered data analyzing frameworks such as Hadoop, Spark, R, and TensorFlow. Finally, the service component can support the web-based or mobile app-based digital healthcare services through the Open API to the end-users. As a result, an implemented big data platform can provide various digital healthcare services using various IoMT devices. Among them, we are focusing on detailed empirical studies about chronic obstructive pulmonary disease (COPD), metabolic syndrome, vital sign, arrhythmia, and diabetes monitoring services. We demonstrated the implemented MEDBIZ platform based on IoMT supporting digital healthcare services by acquiring real-world data for getting the real-world evidence. And then through this platform, we are developing Software as a Medical Device (SaMD), digital therapeutics, and digital healthcare services, and contributing to the development of the digital health ecosystem.

Purpose We propose the Lifelog Bigdata Platform as a sustainable digital healthcare system based on individual-centric lifelog datasets and describe the standardization of lifelog and clinical data in its full-cycle management system. Materials and Methods The Lifelog Bigdata Platform was developed by Yonsei Wonju Health System on the cloud to support digital healthcare and precision medicine. It consists of five core components: data acquisition system, de-identification of individual information, lifelog integration, analyzer, and service. We designed a gathering system into a dedicated virtual machine to save lifelog or clinical outcomes and established standard guidelines for maintaining the quality of gathering procedures. We used standard integration keys to integrate the lifelog and clinical data. Metadata were generated from the data warehouse after loading combined or fragmented data on it. We analyzed the de-identified lifelog and clinical data using the lifelog analyzer to prevent and manage acute and chronic diseases through providing results of statistics on analysis. Results The big data centers were built in four hospitals and seven companies for integrating lifelog and clinical data to develop the Lifelog Bigdata Platform. We integrated and loaded lifelog big data and clinical data for 3 years. In the first year, we uploaded 94 types of data on the platform with a total capacity of 221 GB. Conclusion The Lifelog Bigdata Platform is the first to combine lifelog and clinical data. The proposed standardization guidelines can be used for future platforms to achieve a virtuous cycle structure of lifelogging big data and an industrial ecosystem.

A structured XML has been widely used to present services on various Web-services. The XML is also used for digital documents and digital signatures and for the representation of multimedia files in email systems. The XML document should be firstly parsed to access elements in the XML. The parsing is the most compute-instensive task in the use of XML documents. Most of the previous work has focused on hardware based XML parsers in order to improve parsing performance, while a little work has studied parsing techniques. We present the high performance parsing technique which can be used all of XML parsers and design hardware based XML parser using an FPGA. The proposed parsing technique uses element analyzers instead of the state machine and performs multibyte-based element matching. As a result, our parsing technique can reduce the number of clock cycles per byte(CPB) and does not need to require any preprocessing, such as loading XML data into memory. Compared to other parsers, our parser acheives 1.33~1.82 times improvement in the system performance. Therefore, the proposed parsing technique can process XML documents in real time and is suitable for applying to all of XML parsers.

Expression of neuregulin-2 (NRG2) is intense in a few regions of the adult brain where neurogenesis persists; however, little is understood about its role in developments of newborn neurons. To study the role of NRG2 in synaptogenesis at different developmental stages, newborn granule cells in rat hippocampal slice cultures were labeled with retrovirus encoding tetracycline-inducible microRNA targeting NRG2 and treated with doxycycline (Dox) at the fourth or seventh postinfection day (dpi). The developmental increase of GABAergic postsynaptic currents (GPSCs) was suppressed by the early Dox treatment (4 dpi), but not by late treatment (7 dpi). The late Dox treatment was used to study the effect of NRG2 depletion specific to excitatory synaptogenesis. The Dox effect on EPSCs emerged 4 d after the impairment in dendritic outgrowth became evident (10 dpi). Notably, Dox treatment abolished the developmental increases of AMPA-receptor mediated EPSCs and the AMPA/NMDA ratio, indicating impaired maturation of glutamatergic synapses. In contrast to GPSCs, Dox effects on EPSCs and dendritic growth were independent of ErbB4 and rescued by concurrent overexpression of NRG2 intracellular domain. These results suggest that forward signaling of NRG2 mediates GABAergic synaptogenesis and its reverse signaling contributes to dendritic outgrowth and maturation of glutamatergic synapses.SIGNIFICANCE STATEMENTThe hippocampal dentate gyrus is one of special brain regions where neurogenesis persists throughout adulthood. Synaptogenesis is a critical step for newborn neurons to be integrated into preexisting neural network. Because neuregulin-2 (NRG2), a growth factor, is intensely expressed in these regions, we investigated whether it plays a role in synaptogenesis and dendritic growth. We found that NRG2 has dual roles in the development of newborn neurons. For GABAergic synaptogenesis, the extracellular domain of NRG2 acts as a ligand for a receptor on GABAergic neurons. In contrast, its intracellular domain was essential for dendritic outgrowth and glutamatergic synapse maturation. These results imply that NRG2 may play a critical role in network integration of newborn neurons.

Web-services employ XML that is the well-formed structure as a de-facto standard to represent data. SOAP or REST is one of the representative web-services using XML based massage passing systems. The XML parser can be divided into event driven and DOM tree. A streaming parser as an event driven is widely used for high-speed parsing. Since the streaming parser processes XML documents in sequence, they have any limitation to improve system performance. In order to improve speed of streaming XML parser, we present multibyte based streaming XML hardware parser using the element analyzer instead of the state machine. Compared to other parsers, the proposed MStreXHP can achieve about 2.72 times improvement in the number of clock cycles to be consumed in comparison of characters and sustain about 7.8Gbps throughput. Therefore, our MStreXHP is desirable for the streaming XML hardware parser on high-performance systems.

Key points We investigated the cellular mechanisms underlying mossy fibre-induced heterosynaptic long-term potentiation of perforant path (PP) inputs to CA3 pyramidal cells. Here we show that this heterosynaptic potentiation is mediated by downregulation of Kv1.2 channels. The downregulation of Kv1.2 preferentially enhanced PP-evoked EPSPs which occur at distal apical dendrites. Such enhancement of PP-EPSPs required activation of dendritic Na+ channels, and its threshold was lowered by downregulation of Kv1.2. Our results may provide new insights into the long-standing question of how mossy fibre inputs constrain the CA3 network to sparsely represent direct cortical inputs. Abstract A short high frequency stimulation of mossy fibres (MFs) induces long-term potentiation (LTP) of direct cortical or perforant path (PP) synaptic inputs in hippocampal CA3 pyramidal cells (CA3-PCs). However, the cellular mechanism underlying this heterosynaptic modulation remains elusive. Previously, we reported that repetitive somatic firing at 10 Hz downregulates Kv1.2 in the CA3-PCs. Here, we show that MF inputs induce similar somatic firing and downregulation of Kv1.2 in the CA3-PCs. The effect of Kv1.2 downregulation was specific to PP synaptic inputs that arrive at distal apical dendrites. We found that the somatodendritic expression of Kv1.2 is polarized to distal apical dendrites. Compartmental simulations based on this finding suggested that passive normalization of synaptic inputs and polarized distributions of dendritic ionic channels may facilitate the activation of dendritic Na+ channels preferentially at distal apical dendrites. Indeed, partial block of dendritic Na+ channels using 10 nm tetrodotoxin brought back the enhanced PP-evoked excitatory postsynaptic potentials (PP-EPSPs) to the baseline level. These results indicate that activity-dependent downregulation of Kv1.2 in CA3-PCs mediates MF-induced heterosynaptic LTP of PP-EPSPs by facilitating activation of Na+ channels at distal apical dendrites.

Web-services employ an XML to represent data and an XML parser is needed to use data. The DOM(Document Object Model) is widely used to parse an XML, but it is not suitable for any systems with limited resources because it requires a preprocessing to create the DOM and additional memory space. In this paper, we propose the StreXTree(Streaming XML Tree) with failure transitions and without any preprocessing tasks in order to improve the system performance. Compared to other works, our StreXTree parser achieves 2.39x and 3.02x improvement in system performance in Search and RBStreX, respectively. In addition, our StreXTree parser supports Well-Formed checking to verify the syntax and structure of XML.

The intrinsic excitability of neurons plays a critical role in the encoding of memory at Hebbian synapses and in the coupling of synaptic inputs to spike generation. It has not been studied whether somatic firing at a physiologically relevant frequency can induce intrinsic plasticity in hippocampal CA3 pyramidal cells (CA3-PCs). Here, we show that a conditioning train of 20 action potentials (APs) at 10 Hz causes a persistent reduction in the input conductance and an acceleration of the AP onset time in CA3-PCs, but not in CA1-PCs. Induction of such long-term potentiation of intrinsic excitability (LTP-IE) was accompanied by a reduction in the D-type K(+) current, and was abolished by the inhibition of endocytosis or protein tyrosine kinase (PTK). Consistently, the CA3-PCs from Kv1.2 knock-out mice displayed no LTP-IE with the same conditioning. Furthermore, the induction of LTP-IE depended on the back-propagating APs (bAPs) and intact distal apical dendrites. These results indicate that LTP-IE is mediated by the internalization of Kv1.2 channels from the distal regions of apical dendrites, which is triggered by bAP-induced dendritic Ca(2+) signalling and the consequent activation of PTK.

An XML is widely used for web services, such as SOAP(Simple Object Access Protocol) and REST (Representational State Transfer), and also de facto standard for representing data. Since the XML parser using DOM(Document Object Model) requires a preprocessing task creating a DOM-tree, and then storing it into memory, embedded systems with limited resources typically employ a streaming XML parser without preprocessing. In this paper, we propose a new architecture for the streaming XML parser using an APSearch(Adaptive Parallel Search) on FPGA(Field Programmable Gate Array). Compared to other approaches, the proposed APSearch parser dramatically reduces overhead on the software side and achieves about 2.55 and 2.96 times improvement in the time needed for an XML parsing. Therefore, our APSearch parser is suitable for systems to speed up XML parsing.

Leptin is a pivotal regulator of energy and glucose homeostasis, and defects in leptin signaling result in obesity and diabetes. The ATP-sensitive potassium (K ATP ) channels couple glucose metabolism to insulin secretion in pancreatic β-cells. In this study, we provide evidence that leptin modulates pancreatic β-cell functions by promoting K ATP channel translocation to the plasma membrane via AMP-activated protein kinase (AMPK) signaling. K ATP channels were localized mostly to intracellular compartments of pancreatic β-cells in the fed state and translocated to the plasma membrane in the fasted state. This process was defective in leptin-deficient ob /ob mice, but restored by leptin treatment. We discovered that the molecular mechanism of leptin-induced AMPK activation involves canonical transient receptor potential 4 and calcium/calmodulin-dependent protein kinase kinase β. AMPK activation was dependent on both leptin and glucose concentrations, so at optimal concentrations of leptin, AMPK was activated sufficiently to induce K ATP channel trafficking and hyperpolarization of pancreatic β-cells in a physiological range of fasting glucose levels. There was a close correlation between phospho-AMPK levels and β-cell membrane potentials, suggesting that AMPK-dependent K ATP channel trafficking is a key mechanism for regulating β-cell membrane potentials. Our results present a signaling pathway whereby leptin regulates glucose homeostasis by modulating β-cell excitability.

Network Intrusion Detection Systems use regular expression to represent malicious packets and hardware-based pattern matching is required for fast deep packet inspection. Although hardware architectures for implementing constraint repetition operators such as {10} were recently proposed, they have some limitation. In this paper, we propose hardware architecture supporting constraint repetitions of general regular expression sub-patterns with lower logic complexity. The subpatterns supported by the proposed contraint repetition architecture include general regular expression patterns as well as a single character and fixed length patterns. With the proposed building block, we can implement more efficiently regular expression pattern matching hardwares.

Previous studies indicate that boutons from the same axon exhibit distinct Ca2+dynamics depending on the postsynaptic targets. Mossy fibers of hippocampal granule cells innervate synaptic targets via morphologically distinct boutons. We investigated mitochondrial involvement in the generation of post-tetanic residual Ca2+(Cares) at large and smallen passantmossy fiber boutons (MFBs). Mitochondria limited the [Ca2+]ibuild-up during high-frequency stimulation (HFS) at large MFBs, but not at small MFBs. The amount of Cares, quantified as a time integral of residual [Ca2+]i, was significantly larger at large MFBs than at small MFBs, and that at large MFBs was substantially attenuated by inhibitors of mitochondrial Ca2+uniporter and mitochondrial Na+/Ca2+exchanger (mitoNCX). In contrast, blockers of mitoNCX had no effect on the Caresat small MFBs. Post-tetanic Careshas been proposed as a mechanism for post-tetanic potentiation (PTP). We examined mitochondrial involvement in PTP at mossy fiber synapses on hilar mossy cells (MF→MC synapse) and on hilar interneurons (MF→HI synapse), which are presumably innervated via large and small MFBs, respectively. Consistent with the differential contribution of mitochondria to Caresat large and small MFBs, mitoNCX blockers significantly reduced the PTP at the MF→MC synapse, but not at the MF→HI synapse. In contrast, protein kinase C (PKC) inhibitors significantly reduced the PTP at MF→HI synapse, but not at the MF→MC synapse. These results indicate that mitochondria- and PKC-dependent PTP are expressed at distinct hilar mossy fiber synapses depending on postsynaptic targets.

Na(+)/Ca(2+) exchangers are key players for Ca(2+) clearance in pancreatic β-cells, but their molecular determinants and roles in insulin secretion are not fully understood. In the present study, we newly discovered that the Li(+)-permeable Na(+)/Ca(2+) exchangers (NCLX), which were known as mitochondrial Na(+)/Ca(2+) exchangers, contributed to the Na(+)-dependent Ca(2+) movement across the plasma membrane in rat INS-1 insulinoma cells. Na(+)/Ca(2+) exchange activity by NCLX was comparable to that by the Na(+)/Ca(2+) exchanger, NCX. We also confirmed the presence of NCLX proteins on the plasma membrane using immunocytochemistry and cell surface biotinylation experiments. We further investigated the role of NCLX on exocytosis function by measuring the capacitance increase in response to repetitive depolarization. Small interfering (si)RNA-mediated downregulation of NCLX did not affect the initial exocytosis, but significantly suppressed sustained exocytosis and recovery of exocytosis. XIP (NCX inhibitory peptide) or Na(+) replacement for inhibiting Na(+)-dependent Ca(2+) clearance also selectively suppressed sustained exocytosis. Consistent with the idea that sustained exocytosis requires ATP-dependent vesicle recruitment, mitochondrial function, assessed by mitochondrial membrane potential (ΔΨ), was impaired by siNCLX or XIP. However, depolarization-induced exocytosis was hardly affected by changes in intracellular Na(+) concentration, suggesting a negligible contribution of mitochondrial Na(+)/Ca(2+) exchanger. Taken together, our data indicate that Na(+)/Ca(2+) exchanger-mediated Ca(2+) clearance mediated by NCLX and NCX is crucial for optimizing mitochondrial function, which in turn contributes to vesicle recruitment for sustained exocytosis in pancreatic β-cells.

Glutamate, a major neurotransmitter in the brain, activates ionotropic and metabotropic glutamate receptors (iGluRs and mGluRs, respectively). The two types of glutamate receptors interact with each other, as exemplified by the modulation of iGluRs by mGluRs. However, the other way of interaction (i.e., modulation of mGluRs by iGluRs) has not received much attention. In this study, we found that group I mGluR-specific agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) alone is not sufficient to activate phospholipase C (PLC) in rat hippocampus, while glutamate robustly activates PLC. These results suggested that additional mechanisms provided by iGluRs are involved in group I mGluR-mediated PLC activation. A series of experiments demonstrated that glutamate-induced PLC activation is mediated by mGluR5 and is facilitated by local Ca2+signals that are induced by AMPA-mediated depolarization and L-type Ca2+channel activation. Finally, we found that PLC and L-type Ca2+channels are involved in hippocampal mGluR-dependent long-term depression (mGluR-LTD) induced by paired-pulse low-frequency stimulation, but not in DHPG-induced chemical LTD. Together, we propose that AMPA receptors initiate Ca2+influx via the L-type Ca2+channels that facilitate mGluR5-PLC signaling cascades, which underlie mGluR-LTD in rat hippocampus.

1. 1. The authors examined the anticonvulsant effects of MK-801 on the pilocarpine-induced seizure model. Intraperitoneal injection of pilocarpine (400 mg/kg) induced tonic and clonic seizure. Scopolamine (10 mg/kg) and pentobarbital (5 mg/kg) prevented development of pilocarpine-induced behavioral seizure but MK-801 (0.5 mg/kg) did not. 2. 2. An electrical seizure measured with hippocampal EEG appeared in the pilocarpine-treated group. Scopolamine and pentobarbital blocked the pilocarpine-induced electrographic seizure. MK-801 treatment augmented the electrographic seizure induced by pilocarpine. 3. 3. Brain damage was assessed by examining the hippocampus microscopically. Pilocarpine produced neuronal death in the hippocampus, which showed pyknotic changes. Pentobarbital, Scopolamine and MK-801 protected the brain damage by pilocarpine, though in the MK-801-treated group, the pyramidal cells of hippocampus appeared darker than normal. In all treatments, granule cells of the dentate gyrus were not affected. 4. 4. These results indicate that status epilepticus induced by pilocarpine is initiated by cholinergic overstimulation and propagated by glutamatergic transmission, the elevation of which may cause brain damage through an excitatory NMDA receptor-mediated mechanism.

The intrinsic excitability of neurons plays a critical role in the encoding of memory at Hebbian synapses and in the coupling of synaptic inputs to spike generation. It has not been studied whether somatic firing at a physiologically relevant frequency can induce intrinsic plasticity in hippocampal CA3 pyramidal cells (CA3-PCs). Here, we show that a conditioning train of 20 action potentials (APs) at 10 Hz causes a persistent reduction in the input conductance and an acceleration of the AP onset time in CA3-PCs, but not in CA1-PCs. Induction of such long-term potentiation of intrinsic excitability (LTP-IE) was accompanied by a reduction in the D-type K(+) current, and was abolished by the inhibition of endocytosis or protein tyrosine kinase (PTK). Consistently, the CA3-PCs from Kv1.2 knock-out mice displayed no LTP-IE with the same conditioning. Furthermore, the induction of LTP-IE depended on the back-propagating APs (bAPs) and intact distal apical dendrites. These results indicate that LTP-IE is mediated by the internalization of Kv1.2 channels from the distal regions of apical dendrites, which is triggered by bAP-induced dendritic Ca(2+) signalling and the consequent activation of PTK.

We have previously reported that the surface expression of K+-dependent Na+/Ca2+ exchanger 2 (NCKX2) in the somatodendritic compartment is kept low by constitutive endocytosis, which results in the polarization of surface NCKX2 to the axon. Clathrin-mediated endocytosis is initiated by interaction of the mu subunit of adaptor protein complex 2 (AP-2) with the canonical tyrosine motif (Yxx Phi) of a target molecule. We examined whether endocytosis of NCKX2 involves two putative tyrosine motifs ((365)YGKL and (YDTM)-Y-371) in the cytoplasmic loop of NCKX2. Coimmunoprecipitation assay revealed that the (365)YGKL motif is essential for the interaction with the mu subunit of AP2 (AP2M1). Consistently, either overexpression of NCKX2-Y365A mutant or knockdown of AP2M1 in cultured hippocampal neurons significantly reduced the internalization of NCKX2 from the somatodendritic surface and thus abolished the axonal polarization of surface NCKX2. Next, we tested whether the interaction between the tyrosine motif and AP2M1 is regulated by phosphorylation of the 365th tyrosine residue (Tyr-365). Tyrosine phosphorylation of heterologously expressed NCKX2-WT, but not NCKX2-Y365A, was increased by carbachol (CCh) in PC-12 cells. The effect of CCh was inhibited by PP2, a Src family kinase (SFK) inhibitor. Moreover, PP2 facilitated the endocytosis of NCKX2 in both the somatodendritic and axonal compartments, suggesting that tyrosine phosphorylation of NCKX2 by SFK negatively regulates its endocytosis. Supporting this idea, activation of SFK enhanced the NCKX activity in the proximal dendrites of dentate granule cells (GCs). These results suggest that endocytosis of somatodendritic NCKX2 is regulated by SFK-dependent phosphorylation of Tyr-365.

We have previously shown that K+-dependent Na+/Ca2+exchanger (NCKX) is a major calcium clearance mechanism at the large axon terminals of central neurons, whereas their somata display little NCKX activity. We investigated mechanisms underlying the axonal polarization of NCKX2 in rat hippocampal neurons. We identified NCKX2 as the first neuron-specific cargo molecule of kinesin family member 21A (KIF21A). The intracellular loop of NCKX2 specifically interacted with the WD-40 repeats, a putative cargo-binding domain, of KIF21A. Dominant-negative mutant or depletion of KIF21A inhibited the transport of NCKX2-GFP to axon fibers. Knockdown of KIF21A caused calcium dysregulation at axonal boutons but not at somatodendritic regions. Despite the axonal polarization of the NCKX activity, both somatodendritic and axonal regions were immunoreactive to NCKX2. The surface expression of NCKX2 revealed by live-cell immunocytochemistry, however, displayed highly polarized distribution to the axon. Inhibition of endocytosis increased the somatodendritic surface NCKX2 and thus abolished the axonal polarization of surface NCKX2. These results indicate that KIF21A-mediated axonal transport and selective somatodendritic endocytosis underlie the axonal polarized surface expression of NCKX2.

In this paper we present regular expression pattern matching architecture supporting constrained repetitions. The proposed architecture can implement the constrained repetitions of a regular expression while previous works support only the constrained repetitions of a single character or a multi-cycle, fixed length pattern. The blocks for the constrained repetitions are simply implemented using a counter. The beginning constrained repetition blocks are implemented differently from the non-beginning blocks to support overlapped matching. The proposed architecture can implement all constrained repetitions used in Snort rule-sets with smaller resources.

We have isolated a new prenylated chalcone from the roots of Sophora flavescens (Leguminosae). We determined that structure of this compound is 7,9,2',4'-tetrahydroxy-8-isopentenyl-5-methoxychalcone (1) on the basis of spectroscopic analysis (1D and 2D NMR data). Compound 1 exhibited potent cytotoxicity against human acute promyelocytic (HL60), mouse lymphocytic (L1210) and human histiocytic (U937) leukemia cells.

Different members of the Na+/Ca2++K+ exchanger (NCKX) family are present in distinct brain regions, suggesting that they may have cell-specific functions. Many neuronal channels and transporters are regulated via phosphorylation. Regulation of the rat brain NCKXs by protein kinases, however, has not been described. Here, we report an increase in NCKX2 activity in response to protein kinase C (PKC) activation. Outward current of NCKX2 heterologously expressed in HEK293 cells was enhanced by beta-phorbol dibutyrate (PDBu), whereas PDBu had little effect on activity of NCKX3 or NCKX4. The PDBu-induced enhancement (PIE) of NCKX2 activity was abolished by PKC inhibitors and significantly reduced when the dominant negative mutant of PKCepsilon (K437R) was overexpressed. Moreover, PDBu accelerated the decay rate of the Ca2+ transient at the calyx of Held, where NCKX is the major Ca2+-clearance mechanism. Intracellular perfusion with alkaline phosphatase completely inhibited PIE. Consistently, beta-phorbol myristate acetate (PMA), but not 4alpha-PMA, induced a 3-fold stimulation of 32P incorporation into NCKX2 expressed in HEK293 cells. To investigate the sites involved, PIE of wild-type NCKX2 was compared with mutant NCKX2 in which the three putative PKC consensus sites were replaced with alanine, either individually or in combination. Double-site mutation involving Thr-476 (T166A/T476A and T476A/S504A) disrupted PIE, whereas single mutation of Thr-166, Thr-476, or Ser-504 or the double mutant T166A/S504A failed to completely prevent PIE. These findings suggest that PKC-mediated activation of NCKX2 is sensitive to mutation of multiple PKC consensus sites via a mechanism that may involve several phosphorylation events.

To elucidate the cellular functions of phospholipase A(2) in plants, an Arabidopsis cDNA encoding a secretory low molecular weight phospholipase A(2) (AtsPLA(2)beta) was isolated. Phenotype analyses of transgenic plants showed that overexpression of AtsPLA(2)beta promotes cell elongation, resulting in prolonged leaf petioles and inflorescence stems, whereas RNA interference-mediated silencing of AtsPLA(2)beta expression retards cell elongation, resulting in shortened leaf petioles and stems. AtsPLA(2)beta is expressed in the cortical, vascular, and endodermal cells of the actively growing tissues of inflorescence stems and hypocotyls. AtsPLA(2)beta then is secreted into the extracellular spaces, where signaling for cell wall acidification is thought to occur. AtsPLA(2)beta-overexpressing or -silenced transgenic plants showed altered gravitropism in inflorescence stems and hypocotyls. AtsPLA(2)beta expression is induced rapidly by auxin treatment and in the curving regions of inflorescence stems undergoing the gravitropic response. These results suggest that AtsPLA(2)beta regulates the process of cell elongation and plays important roles in shoot gravitropism by mediating auxin-induced cell elongation.