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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

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

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

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

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.

Research has been conducted on the alpha-D-Galp-(1(approaches)3)-D-Man. The synthesis of this compound which is present in type-2 glycoinositolphospholipids and in the lipophosphoglycan core of Leishmania has been carried out and is described.

PI 3-kinases and PTEN are discussed in this minireview, which considers how opposites attract by chemical means. Phosphatidylinositol lipids are central as mediators in diverse intracellular signaling pathways. Recent research into how the metabolism of these lipids by phosphatidylinositol 3-kinases and the PTEN 3-phosphoinositide phosphatase may coordinate G protein coupled signaling pathways during eukaryotic chemotaxis.

Research has been conducted on the inositol phosphate derivatives. The synthesis of the inositol stereoisomers' derivatives and all conduritol diastereomers has been carried out via the enzymatic resolution of conduritol B and C derivatives and the details are reported.

Research has been conducted on the spicamycin congeners. The synthesis of these congeners has been carried out starting from the cyclitol myo-inositol and the results demonstrate the N-glycoside linkage construction in spicamycin.

The dynamic properties of the inositol (1,4,5)-trisphosphate (I[P.sub.3]) receptor are crucial for the control of intracellular [Ca.sup.2+], including the generation of [Ca.sup.2+] oscillations and waves. However, many models of this receptor do not agree with recent experimental data on the dynamic responses of the receptor. We construct a model of the I[P.sub.3] receptor and fit the model to dynamic and steady-state experimental data from type-2 I[P.sub.3] receptors. Our results indicate that, (i) [Ca.sup.2+] binds to the receptor using saturating, not mass-action, kinetics; (i) [Ca.sup.2+] decreases the rate of I[P.sub.3] binding while simultaneously increasing the steady-state sensitivity of the receptor to I[P.sub.3]; (iii) the rate of [Ca.sup.2+]-induced receptor activation increases with [Ca.sup.2+] and is faster than [Ca.sup.2+]-induced receptor inactivation; and (iv) I[P.sub.3] receptors are sequentially activated and inactivated by [Ca.sup.2+] even when I[P.sub.3] is bound. Our results emphasize that measurement of steady-state properties alone is insufficient to characterize the functional properties of the receptor.

Results show that iolT and iolF genes encode inositol transporter proteins with the former mediating inositol uptake and utilization. Data indicate that iolT is regulated by the IolR repressor.

Research has been conducted on the alkali metal ions Li (super)+, Na (super)+ and K (super)+ in the constrained inositol orthoformate derivatives. The structural features conductive to the complexation of these metal ions have been investigated in solution, in the solid state and in the gas phase via the electrospray ionization mass spectroscopy and the details are discussed.

Research has been conducted on the fully phosphorylated glycosylphosphatidyl inositol anchor pseudohexasaccharide. The retrosynthesis of this compound has led to the building blocks and the results are discussed.

Research has been conducted on the Bacillus subtilis inositol operon involved in the myo-inositol catabolism. The results indicate that the catabolite repression is mediated by CcpA and that CcpA binds to the catabolite-responsive elements.

Research has been conducted on the inositol polyphosphates. The synthesis of the inositol diastereoisomers as enantiomerically pure forms carried out starting with 6-O-acetyl-5-enopyranosides is described.

Results demonstrate that a functional myo-inositol dehydrogenase gene is essential for efficient nitrogen fixation and nodulation of soybean by Sinorhizobium fredii USDA191 as shown by the inability of mutants to establish and nodulate even when population of 10-fold higher is used.

The endoplasmic reticulum (ER) of the mature mouse egg consists of a fine tubular network and pronounced accumulations in the cortex. The ER was visualized both in intact eggs and with in vitro preparations of the cortex using the fluorescent lipophilic dye, DiI. Immunofiuorescent labeling of the ER in isolated cortical preparations demonstrated that the ER clusters contain inositol 1,4,5-trisphosphate (IP(sub 3)) receptors, indicating an important involvement in sperm-induced Ca(super 2+). transients, which are triggered by IP(sub 3). We imaged the ER during fertilization and the subsequent Ca(super 2+) transients and found that the clusters remained intact throughout this period. Recovery of fluorescence after photobleaching established that the ER clusters are continuous with the reticular ER network and that these structures remain stable and continuous throughout the time of fertilization-induced Ca(super 2+) transients; continuity also remained during IP(sub 3) injection. These results indicate that, in contrast to echinoderm eggs, the ER of mouse eggs does not become disrupted when it releases Ca(super 2+) at fertilization. The localization and apparent stability of the cortical ER clusters may be important in generating Ca(super 2+) oscillations, which are characteristic of fertilized mammalian eggs. Imaging of intracellular Ca(super 2+) revealed that Ca(super 2+) transients originate in the hemisphere of the egg that contains abundant ER clusters, thus the mouse contains a stable cortical pacemaker responsible for generating Ca(super 2+) waves. Key Words: endoplasmic reticulum; mouse egg; calcium waves; inositol trisphosphate; calcium stores; fertilization.

Disruption of sblA in Streptomyces lividans is discussed. This disruption makes expression of a heterologous alpha-amylase gene in the presence of glucose possible. In a transposition mutant of S. lividans TK24, the usually glucose-repressible expression of a heterologous alpha amylase gene was rendered resistant to glucose repression.

A study was conducted to determine the single-channel properties of inositol (1,4,5)-triphosphate receptor (InsP3R) which is heterologously expressed in HEK-293 cells. Findings showed that recombinant InsP3R retained specific InsP3 binding properties and were recognized by anti-InsP3R rabbit polyclonal antibody. Gating and conductance properties of recombinant neuronal rat InsP3R-I were similar to properties of native rate cerebellar InsP3R. These suggested that rat neuronal InsP3R-I cDNA encodes a protein sufficient to produce InsP3-gated channel.

Byline: Carmen J. Williams (a), Lisa M. Mehlmann (a ), Laurinda A. Jaffe (a ), Gregory S. Kopf (a), Richard M. Schultz (a)(a ) Abstract: Embryonic development is initiated after the fertilizing sperm contacts the egg and triggers a process termed 'egg activation,' resulting in calcium release, cortical granule exocytosis, recruitment of maternal mRNAs, and cell cycle resumption. Heterotrimeric guanine nucleotide-binding proteins (G proteins) may be involved in mouse egg activation since inhibition of G protein [beta][gamma] subunits partially inhibits sperm-induced cell cycle resumption. In addition, specific events of egg activation can be initiated in the absence of sperm by acetylcholine stimulation of mouse eggs overexpressing the human m1 muscarinic receptor, a G protein-coupled receptor. In somatic cells, G protein in the G.sub.q family couple ligand stimulation of the m1 muscarinic receptor to activation of phospholipase C, resulting in the production of inositol 1,4,5-trisphosphate (IP.sub.3) and IP.sub.3-mediated release of intracellular calcium. Since IP.sub.3-mediated calcium release is involved in egg activation at fertilization, we have examined the role of G.sub.q family G proteins in both sperm-independent (muscarinic receptor-mediated) and sperm-induced egg activation using a function-blocking antibody raised against the common C-terminal region of G.sub.q and G.sub.11 proteins. We show that this antibody effectively inhibits G.sub.q family G proteins in mouse eggs by demonstrating that the antibody inhibits egg activation in response to stimulation of the m1 muscarinic receptor. This same antibody, however, does not inhibit sperm-induced egg activation events. These results indicate that although activation of G.sub.q family G proteins can result in egg activation in the mouse, it is unlikely that these proteins are used by the sperm to initiate egg activation at fertilization. Author Affiliation: (a) Center for Research on Reproduction and Women's Health and Department of Ob/Gyn, University of Pennsylvania Health System, University of Pennsylvania, Philadelphia, Pennsylvania 19104 (a ) Department of Physiology, University of Connecticut Health Center, Farmington, Connecticut 06032 (a ) Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Article History: Received 15 January 1998; Accepted 5 March 1998

Scs2p is an integral membrane protein in the endoplasmic reticulum of the yeast, Saccharomyces cerevisiae. The protein is necessary in the metabolism of inositol as it activates the expression of the INO1. This protein is coded by the gene SCS2 and weighs 35 kilodalton. Although Scs2p is a component of the endoplasmic reticulum, there is no evidence that it functions in protein transport and secretion.

The intracellular calcium (Ca) release channel inositol 1,4,5-trisphosphate (InsPsub3) receptor, is found in most cell types. An increase in cytoplasmic Ca is observed on activation of the InsPsub3-gated channel. The Ca dependence of InsPsub3-gated channel activity was measured as a function of InsPsub3 concentration. InsPsub3 binding and channel activity was also compared. Interactions between Ca and InsPsub3 with the InsPsub3-gated channel were found to be complex.

Valproic acid and lithium are effective anti-bipolar drugs. We recently showed that lithium stimulated the release of glutamate in monkey and mouse cerebral cortex slices, which, through activation of the N-methyl-D-aspartate receptor, increased accumulation of inositol 1,4,5-trisphosphate [Ins(1,4,5)[P.sub.3]]. We show here that valproate behaves similarly to lithium in that at therapeutic concentrations it stimulates glutamate release and Ins(1,4,5)[P.sub.3] accumulation in mouse cerebral cortex slices. The fact that these two effects are a common denominator for two structurally unrelated anti-bipolar drugs suggests that these effects are important in their antibipolar action. The effects of maximal concentrations of lithium and valproate on glutamate release are additive, suggesting different mechanisms for release, which are discussed. The additivity of the two drugs on glutamate release is consistent with the clinical benefit of combining the two drugs in the treatment of subsets of bipolar patients, e.g., in rapid cycling manic-depression. Unlike lithium, valproate does not increase accumulation of inositol monophosphates, inositol bisphosphates, or inositoi 1,3,4-trisphosphate. This is additional evidence against the 'inositol depletion' hypothesis, which states that, by trapping inositol in the form of inositol monophosphates and certain inositol polyphosphates, lithium exerts its antimanic action by inhibiting resynthesis of phosphoinositides with resultant blunting of Ins(1,4,5)[P.sub.3] signaling.

The modulation of the expression of the genes that prepare phospholipid in the presence of inositol in yeasts probably involves the release of the cognate activator gene, INO2, and the inhibition of the negative-acting gene, OPI1. The biosynthesis of phospholipids is affected by the regulation of transcription of INO2 and OPI1. However, transcription of the INO2 gene which is under the control of the GALI promoter gene, is unaffected by inositol but is influenced by galactose.

Enzyme assays suggest the involvement of 2-keto-myo-inositol and D-2,3-diketo-4-deoxy epi-inositol in the myo-inositol catabolism of Rhizobium leguminosarum bv. viciae. Growth of R. leguminosarum on myo-inositol increases the enzyme's myo-inositol dehydrogenase and 2-keto-myo-inositol dehydratase. Examination of the growth in glucose as carbon source and resuspension studies in myo-inositol show that O2 consumption is an inducible route. Mutation studies reveal the necessity of intra-cellular dicarboxylation for the repression of these enzymes. Isolation and studies of three transposon mutants that do not induce both enzymes suggest that myo-inositol is not vital as an energy source in bacteriods.

The role of inositol lipids in mediating some of the effects of extracellular matrix on cellular form and functions was investigated. The study protocol involved the measurement of the effects of adhesion to fibronectin (FN) on the lipid phosphatidylinositol bisphosphate (PIP2) and its hydrolysis products diacylglycerol and inositol triphosphate. PIP2 and its hydrolysis products have functions in cytoskeletal regulation and as second messengers, respectively. The reults showed that adhesion to FN stimulates inositol lipid synthesis and increases PDGF-induced inositol lipid breakdown.