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Intracellular calcium (Ca.sup.2+) signaling regulates many cellular functions, including cell proliferation and migration, in both normal cells and cancer cells. Store-operated Ca.sup.2+ entry (SOCE) is a major mechanism by which Ca.sup.2+ is imported from the extracellular space to the intracellular space, especially in nonexcitable cells. Store-operated Ca.sup.2+ entry (SOCE) is also a receptor-regulated Ca.sup.2+ entry pathway that maintains Ca.sup.2+ homeostasis by sensing reduced Ca.sup.2+ levels in the endoplasmic reticulum (ER). In general, the activation of G protein-coupled receptors (GPCRs) or immunoreceptors, such as T-cell, B-cell and Fc receptors, results in the production of inositol 1,4,5-trisphosphate (IP.sub.3). IP.sub.3 binds to IP.sub.3 receptors located in the ER membrane. The, IP.sub.3 receptors in the ER membrane trigger a rapid and transient release of Ca.sup.2+ from the ER store. The resulting depletion of ER Ca.sup.2+ concentrations is sensed by the EF-hand motif of stromal interaction molecule (STIM), i.e., calcium sensor, which then translocates to the plasma membrane (PM). STIM interacts with Orai Ca.sup.2+ channel subunits (also known as CRACM1) on the PM, leading to Ca.sup.2+ influx from the extracellular space to increase intracellular Ca.sup.2+ concentrations. The physiological functions of Orai and STIM have been studied mainly with respect to their roles in the immune system. Based on numerous previous studies, Orai channels (Orai1, Orai2 and Orai3 channels) control Ca.sup.2+ release-activated Ca.sup.2+ (CRAC) currents and contribute to SOCE currents in other types of cells, including various cancer cells. There are many reports that Orai1 is involved in cell proliferation, migration, metastasis, apoptosis and epithelial-mesenchymal transition (EMT) in various cancers. We previously found that Orai1 plays important roles in cell apoptosis and migration in melanoma. Recently, we reported novel evidence of Orai1 in human oral squamous cell carcinoma (OSCC) cells and human cardiac fibroblasts (HCFs). In this review, we present multiple physiological functions of Orai1 in various cancer cells and cardiac fibroblasts, including our findings. Keywords: Calcium, Orai1, EP4, Store-operated-calcium entry (SOCE), Cancer, Cardiac fibroblast, Author(s): Masanari Umemura[sup.1], Rina Nakakaji[sup.1] and Yoshihiro Ishikawa[sup.1] Introduction Calcium (Ca[sup.2+]) plays important roles as a second messenger in almost all cell types. Ca[sup.2+] release-activated Ca[sup.2+] (CRAC) channels mediate a [...]

How altered metabolism contributes to chemotherapy resistance in cancer cells remains unclear. Through a metabolism-related kinome RNAi screen, we identified inositol-trisphosphate 3-kinase B (ITPKB) as a critical enzyme that contributes to cisplatin-resistant tumor growth. We demonstrated that inositol 1,3,4,5- tetrakisphosphate (IP4), the product of ITPKB, plays a critical role in redox homeostasis upon cisplatin exposure by reducing cisplatin-induced ROS through inhibition of a ROS-generating enzyme, NADPH oxidase 4 (NOX4), which promotes cisplatin- resistant tumor growth. Mechanistically, we identified that IP4 competes with the NOX4 cofactor NADPH for binding and consequently inhibits NOX4. Targeting ITPKB with shRNA or its small-molecule inhibitor resulted in attenuation of NOX4 activity, imbalanced redox status, and sensitized cancer cells to cisplatin treatment in patient-derived xenografts. Our findings provide insight into the crosstalk between kinase-mediated metabolic regulation and platinum-based chemotherapy resistance in human cancers. Our study also suggests a distinctive signaling function of IP4 that regulates NOX4. Furthermore, pharmaceutical inhibition of ITPKB displayed synergistic attenuation of tumor growth with cisplatin, suggesting ITPKB as a promising synthetic lethal target for cancer therapeutic intervention to overcome cisplatin resistance., Introduction Cisplatin kills rapidly dividing cells by damaging their DNA and plays a crucial role in the chemotherapy of human cancers (1-3). Nevertheless, cisplatin is often accompanied by side effects, [...]

Inositol-1,4,5-trisphosphate receptors (Ins[P.sub.3]Rs) and ryanodine receptors (RyRs) are tetrameric intracellular [Ca.sup.2+] channels (1). In each of these receptor families, the pore, which is formed by carboxy-terminal transmembrane domains, is regulated by signals that are detected by large cytosolic structures. Ins[P.sub.3]R gating is initiated by Ins[P.sub.3] binding to the Ins[P.sub.3]-binding core (IBC, residues 224-604 of Ins[P.sub.3]R1) (2) and it requires the suppressor domain (SD, residues 1-223 of Ins[P.sub.3]R1) (2-8). Here we present structures of the amino-terminal region (NT, residues 1-604) of rat Ins[P.sub.3]R1 with (3.6 Å) and without (3.0 [angstrom])Ins[P.sub.3] bound. The arrangement of the three NT domains, SD, IBC-β and IBC-α, identifies two discrete interfaces (a and b) between the IBC and SD. Similar interfaces occur between equivalent domains (A, B and C) in RyR1 (ref. 9). The orientations of the three domains when docked into a tetrameric structure of Ins[P.sub.3]R (10) and of the ABC domains docked into RyR (9) are remarkably similar. The importance of the α-interface for activation of Ins[P.sub.3]R and RyR is confirmed by mutagenesis and, for RyR, by disease-causing mutations (9,11,12). Binding of Ins[P.sub.3] causes partial closure of the clam-like IBC, disrupting the β-interface and pulling the SD towards the IBC. This reorients an exposed SD loop ('hotspot' (HS) loop) that is essential for Ins[P.sub.3]R activation (7). The loop is conserved in RyR and includes mutations that are associated with malignant hyperthermia and central core disease (9,11,12). The HS loop interacts with an adjacent NT, suggesting that activation re-arranges inter-subunit interactions. The A domain of RyR functionally replaced the SD in full-length Ins[P.sub.3]R, and an Ins[P.sub.3]R in which its C-terminal transmembrane region was replaced by that from RyR1 was gated by Ins[P.sub.3] and blocked by ryanodine. Activation mechanisms are conserved between Ins[P.sub.3]R and RyR. Allosteric modulation of two similar domain interfaces within an N-terminal subunit reorients the first domain (SD or A domain), allowing it, through interactions of the second domain of an adjacent subunit (IBC-β or B domain), to gate the pore., The essential role of the SD in linking Ins[P.sub.3] binding to Ins[P.sub.3]R- gating highlights the need to define the structural consequences of Ins[P.sub.3] binding to the N-terminal region (NT, residues [...]

Glycosylphosphatidyl inositol (GPI)-anchored proteins in Candida albicans are responsible for a vast range of functions, and deletions in certain GPI-anchored proteins severely reduce adhesion and virulence of this organism. In addition, completely modified GPIs are necessary for virulence. GPI anchor biosynthesis is essential for viability and starts with the transfer of N-acetylglucosamine to phosphatidylinositol. This step is catalysed by a multi-subunit complex, GPI--N-acetylglucosarninyltransferase (GPI-GnT). In this, the first report to our knowledge on a subunit of the Candida GPI--GnT complex, we show that CaGpil 9p is the functional equivalent of the Saccharomyces cerevisiae Gpi19p. An N-terminal truncation mutant of CaGpi19p functionally complements a conditionally lethal S. cerevisiae gpi19 mutant. Further, we constructed a conditional null mutant of CaGPI19 by disrupting one allele and placing the remaining copy under the control of the MET3 promoter. Repression leads to growth defects, cell wall biogenesis aberrations, azole sensitivity and hyperfilamention. In addition, there is a noticeable gene dosage effect, with the heterozygote also displaying intermediate degrees of most phenotypes. The mutants also displayed a reduced susceptibility to the antifungal agent amphotericin B. Collectively, the results suggest that CaGPI19 is required for normal morphology and cell wall architecture. DOI 10.1099/mic.0.039628-0

scyllo-lnositol (SI) is a stereoisomer of inositol whose catabolism has not been characterized in bacteria. We found that Bacillus subtilis 168 was able to grow using SI as its sole carbon source and that this growth was dependent on a functional iol operon for catabolism of myo-inositol (MI; another inositol isomer, which is abundant in nature). Previous studies elucidated the MI catabolic pathway in B. subtilis as comprising multiple stepwise reactions catalysed by a series of lol enzymes. The first step of the pathway converts MI to scyllo-inosose (SIS) and involves the MI dehydrogenase IolG. Since IolG does not act on SI, we suspected that there could be another enzyme converting SI into SIS, namely an SI dehydrogenase. Within the whole genome, seven genes paralogous to iolG have been identified and two of these, iolX and iolW (formerly known as yisS and yvaA, respectively), were selected as candidate genes for the putative SI dehydrogenase since they were both prominently expressed when B. subtilis was grown on medium containing SI. iolX and iolW were cloned in Escherichia coil and both were shown to encode a functional enzyme, revealing the two distinct SI dehydrogenases in B. subtilis. Since inactivation of ioIX impaired growth with SI as the carbon source, IoIX was identified as a catabolic enzyme required for SI catabolism and it was shown to be [NAD.sup.+] dependent. The physiological role of IoIW remains unclear, but it may be capable of producing SI from SIS with NADPH oxidation. DOI 10.1099/mic.0.037499-0

Recently, we identified, for the first time, two-pore channels (TPCs, TPCN for gene name) as a novel family of nicotinic acid adenine dinucleotide phosphate (NAADP)-gated, endolysosome-targeted calcium release channels. Significantly, three subtypes of TPCs have been characterized, TPCI-3, with each being targeted to discrete acidic calcium stores, namely lysosomes (TPC2) and endosomes (TPC1 and TPC3). That TPCs act as NAADP-gated calcium release channels is clear, given that NAADP binds to high--and low-affinity sites associated with TPC2 and thereby induces calcium release and homologous desensitization, as observed in the case of endogenous NAADP receptors. Moreover, NAADP-evoked calcium signals via TPC2 are ablated by short hairpin RNA knockdown of TPC2 and by depletion of acidic calcium stores with bafilomycin. Importantly, however, NAADP-evoked calcium signals were biphasic in nature, with an initial phase of calcium release from lysosomes via TPC2, being subsequently amplified by calcium-induced calcium release (CICR) from the endoplasmic reticulum (ER). In marked contrast, calcium release via endosome-targeted TPC1 induced only spatially restricted calcium signals that were not amplified by CICR from the ER. These findings provide new insights into the mechanisms that cells may utilize to 'filter' calcium signals via junctional complexes to determine whether a given signal remains local or is converted into a propagating global signal. Essentially, endosomes and lysosomes represent vesicular calcium stores, quite unlike the ER network, and TPCs do not themselves support CICR or, therefore, propagating regenerative calcium waves. Thus 'quantal' vesicular calcium release via TPCs must subsequently recruit inositol 1,4,5-trisphoshpate receptors and/or ryanodine receptors on the ER by CICR to evoke a propagating calcium wave. This may call for a revision of current views on the mechanisms of intracellular calcium signaling. The purpose of this review is, therefore, to provide an appropriate framework for future studies in this area. two-pore segment channel; nicotinic acid adenine dinucleotide phosphate; sarco (endo)plasmic reticulum; lysosome doi:10.1152/ajpcell.00475.2009

The Caenorhabditis elegans intestinal epithelium generates rhythmic inositol 1,4,5-trisphosphate ([IP.sub.3])-dependent [Ca.sup.2+] oscillations that control muscle contractions required for defecation. Two highly [Ca.sup.2+]-selective transient receptor potential (TRP) melastatin (TRPM) channels, GON-2 and GTL-1, function with PLC[gamma] in a common signaling pathway that regulates [IP.sub.3]-dependent intracellular [Ca.sup.2+] release. A second PLC, PLC[beta], is also required for [IP.sub.3]-dependent [Ca.sup.2+] oscillations, but functions in an independent signaling mechanism. PLC[gamma] generates [IP.sub.3] that regulates [IP.sub.3] receptor activity. We demonstrate here that PLC[gamma], via hydrolysis of phosphatidylinositol 4,5-bisphosphate ([PIP.sub.2]) also regulates GON-2/GTL-1 function. Knockdown of PLC[gamma] but not PLC[beta] activity by RNA interference (RNAi) inhibits channel activity ~80%. Inhibition is fully reversed by agents that deplete [PIP.sub.2] levels. [PIP.sub.2] added to the patch pipette has no effect on channel activity in PLC[gamma] RNAi cells. However, in control cells, 10 [micro]M [PIP.sub.2] inhibits whole cell current ~80%. Channel inhibition by phospholipids is selective for [PIP.sub.2] with an [IC.sub.50] value of 2.6 [micro]M. Elevated [PIP.sub.2] levels have no effect on channel voltage and [Ca.sup.2+] sensitivity and likely inhibit by reducing channel open probability, single-channel conductance, and/or trafficking. We conclude that hydrolysis of [PIP.sub.2] by PLC[gamma] functions in the activation of both the [IP.sub.3] receptor and GON-2/GTL-1 channels. GON-2/GTL-1 functions as the major intestinal cell [Ca.sup.2+] influx pathway. Calcium influx through the channel feedback regulates its activity and likely functions to modulate [IP.sub.3] receptor function. [PIP.sub.2]-dependent regulation of GON-2/ GTL-1 may provide a mechanism to coordinate plasma membrane [Ca.sup.2+] influx with PLC[gamma] and [IP.sub.3] receptor activity as well as intracellular [Ca.sup.2+] store depletion. Caenorhabditis elegans; inositol 1,4,5-trisphosphate; transient receptor potential channels; patch-clamp electrophysiology; phospholipase C; phosphatidylinositol 4,5-bisphosphate doi: 10.1152/ajpcell.00394.2009

Inositol is essential in eukaryotes, and must be imported or synthesized. Inositol biosynthesis in Saccharomyces cerevisiae is controlled by three non-essential genes that make up the inositol regulon: SclNO2 and SclNO4, which together encode a heterodimeric transcriptional activator, and ScOPI1, which encodes a transcriptional repressor. ScOpi1p inhibits the Sclno2-Sclno4p activator in response to extracellular inositol levels. An important gene controlled by the inositol regulon is SclNO1, which encodes inositol-3-phosphate synthase, a key enzyme in inositol biosynthesis. In the pathogenic yeast Candida albicans, homologues of the S. cerevisiae inositol regulon genes are 'transcriptionally rewired'. Instead of regulating the CAINO1 gene, CAINO2 and CAINO4 regulate ribosomal genes. Another Candida species that is a prevalent cause of infections is Candida glabrata; however, C. glabrata is phylogenetically more closely related to S. cerevisiae than C. albicans. Experiments were designed to determine if C. glabrata homologues of the inositol regulon genes function similarly to S. cerevisiae or are transcriptionally rewired. CglNO2, CglNO4 and CgOPI1 regulate CglNO1 in a manner similar to that observed in S. cerevisiae. However, unlike in S. cerevisiae, CgOPI1 is essential. Genetic data indicate that CgOPI1 is a repressor that affects viability by regulating activation of a target of the inositol regulon. DOI 10.1099/mic.0.030072-0

In Salmonella enterica serovar Typhimurium, the genomic island GEI4417/4436 has recently been identified to be responsible for myo-inositol (MI) utilization. Here, two of the four island-encoded permeases are identified as the MI transporters of this pathogen. In-frame deletion of iolT1 (STM4418) led to a severe growth defect, and deletion of iolT1 (STM4419) to a slight growth defect in the presence of MI. These phenotypes could be complemented by providing the putative transporter genes in trans. Bioluminescence-based reporter assays demonstrated a strong induction of their promoters [P.sub.iolT1] and [P.sub.iolT2] in the presence of MI but not of glucose. Deletion of iolR, which encodes the negative regulator of most genes involved in MI degradation, resulted in upregulation of PioiT1 and PioIT2, indicating that the expression of IolT1 and IolT2 is repressed by IolR. This finding was supported by bandshift assays using purified IolR. Both transporters are located in the membrane when expressed in Escherichia coli. Heterologously expressed IolT1 had its optimal activity at pH 5.5. Together with the strongly reduced MI uptake in the presence of protonophores, this indicates that IolT1 operates as a proton symporter. Using myo-[1,2-[[sup.3]H](N)]inositol, a saturable uptake activity of IolT1 with a [K.sub.m] value between 0.49 and 0.79 mM was determined in DH5[alpha] expressing IolT1, in S. enterica serovar Typhimurium strain 14028, and in mutant 14028 [DELTA]iolT2. Phylogenetic analysis of IolT1 identified putative MI transporters in Gram-negative bacteria also able to utilize MI. DOI 10.1099/mic.0.032250-0

Evidence for a modulatory effect of cyclosporin A (CsA) on calcium signaling and cell survival in dystrophin-deficient cells is presented. Our previous works strongly supported the hypothesis of an overactivation of [Ca.sup.2+] release via inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) in dystrophin-deficient cells, both during membrane depolarization and at rest, through spontaneous [Ca.sup.2+] release events. Forced expression of mini-dystrophin in these cells contributed, during stimulation and in resting condition, to the recovery of a controlled calcium homeostasis. In the present work, we demonstrate that CsA exposure displayed a dual-modulator effect on calcium signaling in dystrophin-deficient cells. Short-time incubation induced a decrease of IP3-dependent calcium release, leading to patterns of release similar to those observed in myotubes expressing mini-dystrophin, whereas long-time incubation reduced the expression of the type I of IP3 receptors (IP3R-1) RNA levels. Moreover, both IP3R-1 knockdown and blockade through 2-aminoethoxydiphenyle borate or CsA induced improved survival of dystrophin-deficient myotubes, demonstrating the cell death dependence on the IP3-dependent calcium signaling as well as the protective effect of CsA. Inhibition of the IP3 pathway could be a very interesting approach for reducing the natural cell death of dystrophin-deficient cells in development. calcium regulation; cyclosporin A; muscular dystrophy doi: 10.1152/ajpcell.00048.2009.

Nonglucose carbohydrates such as mannose and inositol are important in early growth and development, although little is known about their metabolism. Our aim in this study was to determine the plasma appearance rates (Ra) for mannose and inositol in newborns as an index of utilization and as an improved guide to supplementation practices. We studied late- preterm (n = 9) and term (n = 5) infants (median 34 wk gestation, range 33-41 wk) using a multiple isotope infusion start time protocol to determine Ra for each carbohydrate. The plasma mannose concentration [median (range)] was 69.83 (48.60-111.75) [micro]mol/L and the Ra was 0.59 (0.42-0.98) [micro]mol x [kg.sup.-1] x [min.sup.-1] (854 [micro]mol x [kg.sup.-1] x [d.sup.-1] ).The plasma inositol concentration was 175.74 (59.71-300.60) [micro]mol/L and Ra was 1.06 (0.33-1.75) [micro]mol x [kg.sup.-1] x [min.sup.-1] (1521 [micro]mol x [kg.sup.-1] x [d.sup.-1]). The Ra for mannose and inositol are >10-fold higher than the amounts a breast-fed infant typically ingests, which are ~6 [micro]mol x [kg.sup.-1] x [d.sup.-1] mannose and 150 [micro]mol x [kg.sup.-1] x [d.sup.-1] inositol. Thus, for both mannose and inositol, the newborn infant must produce these compounds from glucose at rates sufficient to meet nutritional requirements.

myo-Inositol (inositol) is an essential nutrient that is used for building phosphatidylinositol and its derivatives in eukaryotes and even in some eubacteria such as the mycobacteria. As a consequence, fungal, protozoan and mycobacterial pathogens must be able to acquire inositol in order to proliferate and cause infection in their hosts. There are two primary mechanisms for acquiring inositol. One is to synthesize inositol from glucose 6-phosphate using two sequentially acting enzymes: inositol-3-phosphate synthase (Ino1p) converts glucose 6-phosphate to inositol 3-phosphate, and then inositol monophosphatase (IMPase) dephosphorylates inositol 3-phosphate to generate inositol. The other mechanism is to import inositol from the environment via inositol transporters. Inositol is readily abundant in the bloodstream of mammalian hosts, providing a source from which many pathogens could potentially import inositol. However, despite this abundance of inositol in the host, some pathogens such as the bacterium Mycobacterium tuberculosis and the protist parasite Trypanosoma brucei must be able to make inositol de novo in order to cause disease (M. tuberculosis) or even grow (T. brucei). Other pathogens such as the fungus Candida albicans are equally adept at causing disease by importing inositol or by making it de novo. The role of inositol acquisition in the biology and pathogenesis of the parasite Leishmania and the fungus Cryptococcus are being explored as well. The specific strategies used by these pathogens to acquire inositol while in the host are discussed in relation to each pathogen's unique metabolic requirements.

MicroRNA-155 (miR-155) has emerged asa critical regulator of immune cell development, function, and disease. However, the mechanistic basis for its impact on the hematopoietic system remains largely unresolved. Because miRNAs function by repressing specific mRNAs through direct 3'UTR interactions, we have searched for targets of miR-155 implicated in the regulation of hematopoiesis. In the present study, we identify Src homology-2 domain-containing inositol 5-phosphatase 1 (SHIP1) asa direct target of miR-155, and, using gain and loss of function approaches, show that miR-155 represses SHIP1 through direct 3'UTR interactions that have been highly conserved throughout evolution. Repression of endogenous SHIP1 by miR-155 occurred following sustained over-expression of miR-155 in hematopoietic cells both in vitro and in vivo, and resulted in increased activation of the kinase Akt during the cellular response to LPS. Furthermore, SHIP1 was also repressed by physiologically regulated miR-155, which was observed in LPS-treated WT versus [miR-155.sup.-/-] primary macrophages. In mice, specific knockdown of SHIP1 in the hematopoietic system following retroviral delivery of a miR-155-formatted siRNA against SHIP1 resulted in a myeloproliferative disorder, with striking similarities to that observed in miR-155-expressing mice. Our study unveils a molecular link between miR-155 and SHIP1 and provides evidence that repression of SHIP1 is an important component of miR-155 biology. cancer | hematopoiesis | inflammation | miRNA | myeloid

Knockout mutation of STM4432 resulted in a growth-deficient phenotype of Salmonella enterica serovar Typhimurium in the presence of myo-inositol (MI) as the sole carbon source. STM4432 is part of a 22.6-kb genomic island which spans STM4417 to STM4436 (genomic island 4417/4436) and is responsible for MI degradation. Genome comparison revealed the presence of this island in only six Salmonella strains and a high variability of the iol gene organization in gram-negative bacteria. Upon nonpolar deletion of 11 island loci, the genes involved in six enzymatic steps of the MI pathway were identified. The generation time of S. enterica serovar Typhimurium in minimal medium with MI decreases with higher concentrations of this polyol. Reverse transcriptase PCR showed five separate transcriptional units encompassing the genes iolA-iolB, iolE-iolG1, iolC1-iolC2, iolD1-iolD2-iolG2, and iolI2-iolH. Luciferase reporter assays revealed a strong induction of their promoters in the presence of MI but not glucose. The main regulator, Io1R, was identified due to a reduced lag phase of a strain mutated in STM4417 (iolR). Deletion of iolR resulted in stimulation of the iol operons, indicating its negative effect on the iol genes of S. enterica serovar Typhimurium in rich medium at a transcriptional level. Bandshift assays demonstrated the binding of this putative repressor to promoter sequences of iolA, iolC1, and iolD1. Binding of IolR to its own promoter and induced iolR expression in an IolR-negative background demonstrate that its transcription is autoregulated. This is the first characterization of MI degradation in a gram-negative bacterium, revealing a complex transcriptional organization and regulation of the S. enterica serovar Typhimurium iol genes.

Calcium ([Ca.sup.2+]) release through inositol 1,4,5-trisphosphate receptors ([IP.sub.3]Rs) regulates the function of virtually every mammalian cell. Unlike ryanodine receptors, which generate local [Ca.sup.2+] events ('sparks') that transmit signals to the juxtaposed cell membrane, a similar functional architecture has not been reported for [IP.sub.3]Rs. Here, we have identified spatially fixed, local [Ca.sup.2+] release events ('pulsars') in vascular endothelial membrane domains that project through the internal elastic lamina to adjacent smooth muscle membranes. [Ca.sup.2+] pulsars are mediated by [IP.sub.3]Rs in the endothelial endoplasmic reticulum of these membrane projections. Elevation of [IP.sub.3] by the endothelium-dependent vasodilator, acetylcholine, increased the frequency of [Ca.sup.2+] pulsars, whereas blunting [IP.sub.3] production, blocking [IP.sub.3]Rs, or depleting endoplasmic reticulum [Ca.sup.2+] inhibited these events. The elementary properties of [Ca.sup.2+] pulsars were distinct from ryanodine-receptor-mediated [Ca.sup.2+] sparks in smooth muscle and from [IP.sub.3]-mediated [Ca.sup.2+] puffs in Xenopus oocytes. The intermediate conductance, [Ca.sup.2+]-sensitive potassium ([K.sub.Ca]3.1) channel also colocalized to the endothelial projections, and blockage of this channel caused an 8-mV depolarization. Inhibition of [Ca.sup.2+] pulsars also depolarized to a similar extent, and blocking [K.sub.Ca] 3.1 channels was without effect in the absence of pulsars. Our results support a mechanism of [IP.sub.3] signaling in which [Ca.sup.2+] release is spatially restricted to transmit intercellular signals. calcium | endothelium | calcium biosensor | intermediate conductance [Ca.sup.2+]-sensitive potassium channel | calcium pulsar

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2007.10.029 Byline: David Weinkove (b)(c), Michael Bastiani (a), Tamara A.M. Chessa (b), Deepa Joshi (a), Linda Hauth (a), Frank T. Cooke (d), Nullin Divecha (b), Kim Schuske (a) Keywords: PI(4,5)P.sub.2; PIP5K; Axon outgrowth; Caenorhabditis elegans; Neuron maintenance; Neuron degeneration Abstract: Growth cones are dynamic membrane structures that migrate to target tissue by rearranging their cytoskeleton in response to environmental cues. The lipid phosphatidylinositol (4,5) bisphosphate (PIP.sub.2) resides on the plasma membrane of all eukaryotic cells and is thought to be required for actin cytoskeleton rearrangements. Thus PIP.sub.2 is likely to play a role during neuron development, but this has never been tested in vivo. In this study, we have characterized the PIP.sub.2 synthesizing enzyme Type I PIP kinase (ppk-1) in Caenorhabditis elegans. PPK-1 is strongly expressed in the nervous system, and can localize to the plasma membrane. We show that PPK-1 purified from C. elegans can generate PIP.sub.2 in vitro and that overexpression of the kinase causes an increase in PIP.sub.2 levels in vivo. In developing neurons, PPK-1 overexpression leads to growth cones that become stalled, produce ectopic membrane projections, and branched axons. Once neurons are established, PPK-1 overexpression results in progressive membrane overgrowth and degeneration during adulthood. These data suggest that overexpression of the Type I PIP kinase inhibits growth cone collapse, and that regulation of PIP.sub.2 levels in established neurons may be important to maintain structural integrity and prevent neuronal degeneration. Author Affiliation: (a) Department of Biology, University of Utah, Salt Lake City, UT 84112-0840, USA (b) Division of Cellular Biochemistry, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands (c) Department of Biology, University College London, Gower Street, London WC1E 6BT, UK (d) Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK Article History: Received 11 September 2007; Revised 17 October 2007; Accepted 23 October 2007

Cilia project from cells as membranous extensions, with microtubule structural cores assembling from basal bodies by intraflagellar transport (IFT). Here, we report a ciliary role for the inositol 1,3,4,5,6-pentakisphosphate 2-kinase (Ipk1) that generates inositol hexakisphosphate. In zebrafish embryos, reducing Ipk1 levels inhibited ciliary beating in Kupffer's vesicle and decreased ciliary length in the spinal canal, pronephric ducts, and Kupffer's vesicle. Electron microscopy showed that ciliary axonemal structures were not grossly altered. However, coincident knockdown of Ipk1 and IFT88 or IFT57 had synergistic perturbations. With GFP-Ipk1 enriched in centrosomes and basal bodies, we propose that Ipk1 plays a previously uncharacterized role in ciliary function. cilia | inositol hexakisphosphate | kidney development | left-right asymmetry | zebrafish

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tibs.2005.11.003 Byline: Shamshad Cockcroft Abstract: Members of the phosphoinositide-specific phospholipase C (PLC) family have key roles in cell signalling. In response to many extracellular stimuli, such as hormones, neurotransmitters, antigens and growth factors, PLCs catalyse the hydrolysis of phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P.sub.2], thereby generating two well-established second messengers, inositol (1,4,5)-trisphosphate and diacylglycerol. Eleven PLC isozymes encoded by different genes have been identified in mammals and, on the basis of their structure and sequence relationships, have been classified into five families designated PLC[beta] (1-4), PLC[gamma] (1 and 2), PLC[delta] (1, 3 and 4), PLCI[micro] (1) and PLCI[paragraph] (1). All PLCs contain the catalytic X and Y domain, in addition to other regulatory domains including the C2 domain and the EF-hand domain. In 2005, four groups independently identified an entirely new family of PLCs - I*1 and I*2 - using data mining of mammalian genomes. The properties of the PLCI* enzyme suggest that it might act as a Ca.sup.2+ sensor, in particular, functioning during formation and maintenance of the neuronal network in the postnatal brain. Author Affiliation: Department of Physiology, University College London, London WC1E 6JJ, UK

Inositol is considered a growth factor in yeast cells and it plays an important role in Candida as an essential precursor for phospholipomannan, a glycophosphatidylinositol (GPI)-anchored glycolipid on the cell surface of Candida which is involved in the pathogenicity of this opportunistic fungus and which binds to and stimulates human macrophages. In addition, inositol plays an essential role in the phosphatidylinositol signal transduction pathway, which controls many cell cycle events. Here, high-affinity myo-inositol uptake in Candida albicans has been characterized, with an apparent [K.sub.m] value of 240 [+ or -] 15 [mu]M, which appears to be active and energy-dependent as revealed by inhibition with azide and protonophores (FCCP, dinitrophenol). Candida myo-inositol transport was sodium-independent but proton-coupled with an apparent [K.sub.m] value of 11.04 [+ or -] 1.1 nM for [H.sup.+], equal pH 7.96 [+ or -] 0.05, suggesting that the C. albicans myo-inositoI-[H.sup.+] transporter is fully activated at physiological pH. C. albicans inositol transport was not affected by cytochalasin B, phloretin or phlorizin, an inhibitor of mammalian sodium-dependent inositol transport. Furthermore, myo-inositol transport showed high substrate specificity for inositol and was not significantly affected by hexose or pentose sugars as competitors, despite their structural similarity. Transport kinetics in the presence of eight different inositol isomers as competitors revealed that proton bonds between the C-2, C-3 and C-4 hydroxyl groups of myo-inositol and the transporter protein play a critical role for substrate recognition and binding. It is concluded that C. albicans myo-inositoI-[H.sup.+] transport differs kinetically and pharmacologically from the human sodium-dependent myo-inositol transport system and constitutes an attractive target for delivery of cytotoxic inositol analogues in this pathogenic fungus.

The experiments reported here were designed to answer the question of whether inositol 1,4,5-trisphosphate (I[P.sub.3])-induced calcium release is necessary for generating the entire light response of Limulus ventral photoreceptors. For this purpose the membrane-permeable I[P.sub.3] receptor antagonist 2-aminoethoxydiphenyl borate (2APB) (Maruyama, T., T. Kanaji, S. Nakade, T. Kanno, and K. Mikoshiba. 1997. J. Biochem. (Tokyo). 122:498-505) was used. Previously, 2APB was found to inhibit the light activated current of Limulus ventral photoreceptors and reversibly inhibit both light and I[P.sub.3] induced calcium release as well as the current activated by pressure injection of calcium into the light sensitive lobe of the photoreceptor (Wang, Y., M. Deshpande, and R. Payne. 2002. Cell Calcium. 32:209). In this study 2APB was found to inhibit the response to a flash of light at all light intensities and to inhibit the entire light response to a step of light, that is, both the initial transient and the steady-state components of the response to a step of light were inhibited. The light response in cells injected with the calcium buffer 1,2-bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) was reversibly inhibited by 2APB, indicating that these light responses result from I[P.sub.3]-mediated calcium release giving rise to an increase in [Ca.sub.i]. The light response obtained from cells after treatment with 100 [micro]M cyclopiazonic acid (CPA), which acts to empty intracellular calcium stores, was reversibly inhibited by 2APB, indicating that the light response alter CPA treatment results from I[P.sub.3]-mediated calcium release and a consequent rise in [Ca.sub.i]. Together these findings imply that I[P.sub.3]-induced calcium release is necessary for generating the entire light response of Limulus ventral photoreceptors. KEYWORDS: phototransduction * microvillar photoreceptors * inositol 1,4,5-trisphosphate receptor * 2-aminoethoxydiphenyl borate * calcium buffers

The Ins[P.sub.3]R proteins have three recognized domains, the Ins[P.sub.3]-binding, regulatory/coupling, and channel domains (Mignery, G.A., and TC. Sudhof. 1990. EMBOJ. 9:3893-3898). The Ins[P.sub.3] binding domain and the channel-forming domain are at opposite ends of the protein. Ligand regulation of the channel must involve communication between these different regions of the protein. This communication likely involves the interceding sequence (i.e., the regulatory/coupling domain). The single channel functional attributes of the full-length recombinant type-1, -2, and -3 Ins[P.sup.3]R channels have been defined. Here, two type-I/type-2 Ins[P.sup.3]R regulatory/coupling domain chimeras were created and their single channel function defined. One chimera (1-2-1) contained the type-2 regulatory/coupling domain in a type-1 backbone. The other chimera (2-1-2) contained the type-1 regulatory/coupling domain in a type-2 backbone. These chimeric proteins were expressed in COS cells, isolated, and then reconstituted in proteoliposomes. The proteoliposomes were incorporated into artificial planar lipid bilayers and the single-channel function of the chimeras defined. The chimeras had permeation properties like that of wild-type channels. The ligand regulatory properties of the chimeras were altered. The Ins[P.sup.3] and [Ca.sup.2+] regulation had some unique features but also had features in common with wild-type channels. These results suggest that different independent structural determinants govern Ins[P.sup.3]R permeation and ligand regulation. It also suggests that ligand regulation is a multideterminant process that involves several different regions of the protein. This study also demonstrates that a chimera approach can be applied to define Ins[P.sup.3]R structure-function. KEY WORDS: inositol trisphosphate receptor * [Ca.sup.2+] release channel * intracellular [Ca.sup.2+] signaling

Maturation of mouse oocytes is accompanied by an increase in sensitivity to inositol 1,4,5-trisphosphate (I[P.sub.3])-mediated release of intracellular calcium. To test the hypothesis that the maturation-associated 1.5- to 2.0-fold increase in the mass of the type 1 I[P.sub.3] receptor (I[P.sub.3]R-1) confers this increase in I[P.sub.3] sensitivity, we employed RNA interference to prevent this change in I[P.sub.3]R-1 protein level. Microinjection into germinal vesicle (GV)-intact oocytes of dsRNA corresponding to the I[P.sub.3]R-1 sequence resulted in a >90% reduction in the amount of maternal I[P.sub.3]R-1 mRNA and prevented the maturation-associated increase in the mass of the I[P.sub.3]R-1 protein. These injected oocytes matured to metaphase II, and there was no effect on the maturation-associated increases in p[34.sup.cdc2]/cyclin B kinase and MAP kinase activities or the global pattern of protein synthesis. I[P.sub.3]-induced cortical granule exocytosis was significantly decreased in these eggs when compared with controls previously injected with enhanced green fluorescent protein (EGFP) dsRNA. Following insemination, the I[P.sub.3]R-1 dsRNA-injected eggs displayed significantly fewer [Ca.sup.2+] transients than controls, and the duration of the first [Ca.sup.2+] transient was about half that of controls. These results support the hypothesis that the maturation-associated increase in the mass of I[P.sub.3]R-1 confers the increase in I[P.sub.3]-sensitivity that is observed following oocyte maturation and is necessary for the proper [Ca.sup.2+] oscillatory pattern following insemination. Keywords: I[P.sub.3] receptor; Egg activation; Cortical granule exocytosis; RNAi

The coupling mechanism between depletion of [Ca.sup.2+] stores in the endoplasmic reticulum and plasma membrane store-operated ion channels is fundamental to [Ca.sup.2+] signaling in many cell types and has yet to be completely elucidated. Using [Ca.sup.2+] release-activated [Ca.sup.2+] (CRAC) channels in RBL2H3 cells as a model system, we have shown that CRAC channels are maintained in the closed state by an inhibitory factor rather than being opened by the inositol 1,4,5-trisphosphate receptor. This inhibitory role can be fulfilled by the Drosophila protein INAD (inactivation-no after potential D). The action of INAD requires [Ca.sup.2+] and can be reversed by a diffusible [Ca.sup.2+] influx factor. Thus the coupling between the depletion of [Ca.sup.2+] stores and the activation of CRAC channels may involve a mammalian homologue of INAD and a low-molecular-weight, diffusible store-depletion signal. store-operated ion channels; inositol 1,4,5-trisphosphate receptor

Research has been conducted on Plasmodium glycosylphosphatidylinositol biosynthetic enzymes. The study of the enzymes' substrate specificities has been carried out and the results indicate the existence of similarity between Plasmodium and mammalian enzymes.

Research has been conducted on the optically active regioisomers. The complete synthesis of all possible IP (sub)3 and IP (sub)4 optically active regioisomers has been carried out via the use of CRL-catalyzed enzymatic resolution and benzoyl migration strategy and the details are discussed.