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Sample records for del inositol fosfoglicano

  1. Inositol transport proteins.

    PubMed

    Schneider, Sabine

    2015-04-28

    The cyclic polyol myo-inositol is a key molecule in many different metabolic pathways among all organisms; in addition, it is fundamental for osmotic balance in the mammalian brain. This review sums up inositol transporters from eukaryotic organisms, elucidating their vital role in regulating the intracellular distribution and uptake of inositol. They can be divided into two groups according to their transport mechanisms: (1) sodium ion coupled inositol transporters that belong to the Solute Carrier Families 5 and 6-like Superfamily and, (2) proton coupled inositol symporters that are members of the Major Facilitator Superfamily. Intriguingly members of both families offer promising targets for medical treatment of a variety of diseases.

  2. Inositol pyrophosphate pyrotechnics.

    PubMed

    Bhandari, Rashna; Chakraborty, Anutosh; Snyder, Solomon H

    2007-05-01

    Physiologic roles of highly phosphorylated inositol phosphates, including those containing pyrophosphate groups, have been the focus of much recent interest. In the April 6, 2007 issue of Science, two papers (Lee et al., 2007; Mulugu et al., 2007) demonstrate the occurrence of a novel inositol pyrophosphate molecule in yeast and elucidate its role in phosphate homeostasis.

  3. Inositol phosphates in the environment.

    PubMed Central

    Turner, Benjamin L; Papházy, Michael J; Haygarth, Philip M; McKelvie, Ian D

    2002-01-01

    The inositol phosphates are a group of organic phosphorus compounds found widely in the natural environment, but that represent the greatest gap in our understanding of the global phosphorus cycle. They exist as inositols in various states of phosphorylation (bound to between one and six phosphate groups) and isomeric forms (e.g. myo, D-chiro, scyllo, neo), although myo-inositol hexakisphosphate is by far the most prevalent form in nature. In terrestrial environments, inositol phosphates are principally derived from plants and accumulate in soils to become the dominant class of organic phosphorus compounds. Inositol phosphates are also present in large amounts in aquatic environments, where they may contribute to eutrophication. Despite the prevalence of inositol phosphates in the environment, their cycling, mobility and bioavailability are poorly understood. This is largely related to analytical difficulties associated with the extraction, separation and detection of inositol phosphates in environmental samples. This review summarizes the current knowledge of inositol phosphates in the environment and the analytical techniques currently available for their detection in environmental samples. Recent advances in technology, such as the development of suitable chromatographic and capillary electrophoresis separation techniques, should help to elucidate some of the more pertinent questions regarding inositol phosphates in the natural environment. PMID:12028785

  4. Two inositol hexakisphosphate kinases drive inositol pyrophosphate synthesis in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inositol pyrophosphates are novel cellular signaling molecules with newly discovered roles in energy sensing and metabolic control. Studies in eukaryotes have revealed that these compounds turn over rapidly, and thus only small amounts accumulate. Inositol pyrophosphates have not been the subject of...

  5. 21 CFR 184.1370 - Inositol.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Inositol. 184.1370 Section 184.1370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Substances Affirmed as GRAS § 184.1370 Inositol. (a) Inositol, or myo-inositol (C6H12O6, CAS Reg. No....

  6. Preparation of quality inositol pyrophosphates.

    PubMed

    Loss, Omar; Azevedo, Cristina; Szijgyarto, Zsolt; Bosch, Daniel; Saiardi, Adolfo

    2011-01-01

    Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells are inositol pentakisphosphate (IP(5;)) and inositol hexakisphosphate (phytic acid or IP(6;)). IP(5;) and IP(6;) are the precursors of inositol pyrophosphate molecules that contain one or more pyrophosphate bonds(1). Phosphorylation of IP(6;) generates diphoshoinositolpentakisphosphate (IP(7;) or PP-IP(5;)) and bisdiphoshoinositoltetrakisphosphate (IP(8;) or (PP)(2;)-IP(4;)). Inositol pyrophosphates have been isolated from all eukaryotic organisms so far studied. In addition, the two distinct classes of enzymes responsible for inositol pyrophosphate synthesis are highly conserved throughout evolution(2-4). The IP(6;) kinases (IP(6;)Ks) posses an enormous catalytic flexibility, converting IP(5;) and IP(6;) to PP-IP(4;) and IP(7;) respectively and subsequently, by using these products as substrates, promote the generation of more complex molecules(5,6). Recently, a second class of pyrophosphate generating enzymes was identified in the form of the yeast protein VIP(1;) (also referred as PP-IP(5;)K), which is able to convert IP(6;) to IP(7;) and IP(8;)(7,8). Inositol pyrophosphates regulate many disparate cellular processes such as insulin secretion(9), telomere length(10,11), chemotaxis(12), vesicular trafficking(13), phosphate homeostasis(14) and HIV-1 gag release(15). Two mechanisms of actions have been proposed for this class of molecules. They can affect cellular function by allosterically interacting with specific proteins like AKT(16). Alternatively, the pyrophosphate group can donate a phosphate to pre-phosphorylated proteins(17). The enormous potential of this research field is hampered by the absence of a commercial source of inositol pyrophosphates, which is preventing many scientists from studying these molecules and this new post-translational modification. The methods currently available to isolate

  7. Broad Spectrum Anticancer Activity of Myo-Inositol and Inositol Hexakisphosphate

    PubMed Central

    Dinicola, Simona

    2016-01-01

    Inositols (myo-inositol and inositol hexakisphosphate) exert a wide range of critical activities in both physiological and pathological settings. Deregulated inositol metabolism has been recorded in a number of diseases, including cancer, where inositol modulates different critical pathways. Inositols inhibit pRB phosphorylation, fostering the pRB/E2F complexes formation and blocking progression along the cell cycle. Inositols reduce PI3K levels, thus counteracting the activation of the PKC/RAS/ERK pathway downstream of PI3K activation. Upstream of that pathway, inositols disrupt the ligand interaction between FGF and its receptor as well as with the EGF-transduction processes involving IGF-II receptor and AP-1 complexes. Additionally, Akt activation is severely impaired upon inositol addition. Downregulation of both Akt and ERK leads consequently to NF-kB inhibition and reduced expression of inflammatory markers (COX-2 and PGE2). Remarkably, inositol-induced downregulation of presenilin-1 interferes with the epithelial-mesenchymal transition and reduces Wnt-activation, β-catenin translocation, Notch-1, N-cadherin, and SNAI1 release. Inositols interfere also with the cytoskeleton by upregulating Focal Adhesion Kinase and E-cadherin and decreasing Fascin and Cofilin, two main components of pseudopodia, leading hence to invasiveness impairment. This effect is reinforced by the inositol-induced inhibition on metalloproteinases and ROCK1/2 release. Overall, these effects enable inositols to remodel the cytoskeleton architecture. PMID:27795708

  8. The inositols and polycystic ovary syndrome

    PubMed Central

    Kalra, Bharti; Kalra, Sanjay; Sharma, J. B.

    2016-01-01

    This review describes the rationale, biochemical, and clinical data related to the use of inositols in polycystic ovary syndrome (PCOS). It covers studies related to the mechanism of action of myo-inositol and D-chiro-inositol (MDI), with randomized controlled trials conducted in women with PCOS, and utilizes these data to suggest pragmatic indications and methods for using MDI combination in PCOS. Rationally crafted inositol combinations have a potential role to play in maintaining metabolic, endocrine, and reproductive health in women with PCOS. PMID:27730087

  9. Novel inositol catabolic pathway in Thermotoga maritima.

    PubMed

    Rodionova, Irina A; Leyn, Semen A; Burkart, Michael D; Boucher, Nathalie; Noll, Kenneth M; Osterman, Andrei L; Rodionov, Dmitry A

    2013-08-01

    myo-inositol (MI) is a key sugar alcohol component of various metabolites, e.g. phosphatidylinositol-based phospholipids that are abundant in animal and plant cells. The seven-step pathway of MI degradation was previously characterized in various soil bacteria including Bacillus subtilis. Through a combination of bioinformatics and experimental techniques we identified a novel variant of the MI catabolic pathway in the marine hyperthermophilic bacterium Thermotoga maritima. By using in vitro biochemical assays with purified recombinant proteins we characterized four inositol catabolic enzymes encoded in the TM0412-TM0416 chromosomal gene cluster. The novel catabolic pathway in T. maritima starts as the conventional route using the myo-inositol dehydrogenase IolG followed by three novel reactions. The first 2-keto-myo-inositol intermediate is oxidized by another, previously unknown NAD-dependent dehydrogenase TM0412 (named IolM), and a yet unidentified product of this reaction is further hydrolysed by TM0413 (IolN) to form 5-keto-l-gluconate. The fourth step involves epimerization of 5-keto-l-gluconate to d-tagaturonate by TM0416 (IolO). T. maritima is unable to grow on myo-inositol as a single carbon source. The determined in vitro specificity of the InoEFGK (TM0418-TM0421) transporter to myo-inositol-phosphate suggests that the novel pathway in Thermotoga utilizes a phosphorylated derivative of inositol.

  10. Inositol pyrophosphates: between signalling and metabolism.

    PubMed

    Wilson, Miranda S C; Livermore, Thomas M; Saiardi, Adolfo

    2013-06-15

    The present review will explore the insights gained into inositol pyrophosphates in the 20 years since their discovery in 1993. These molecules are defined by the presence of the characteristic 'high energy' pyrophosphate moiety and can be found ubiquitously in eukaryotic cells. The enzymes that synthesize them are similarly well distributed and can be found encoded in any eukaryote genome. Rapid progress has been made in characterizing inositol pyrophosphate metabolism and they have been linked to a surprisingly diverse range of cellular functions. Two decades of work is now beginning to present a view of inositol pyrophosphates as fundamental, conserved and highly important agents in the regulation of cellular homoeostasis. In particular it is emerging that energy metabolism, and thus ATP production, is closely regulated by these molecules. Much of the early work on these molecules was performed in the yeast Saccharomyces cerevisiae and the social amoeba Dictyostelium discoideum, but the development of mouse knockouts for IP6K1 and IP6K2 [IP6K is IP6 (inositol hexakisphosphate) kinase] in the last 5 years has provided very welcome tools to better understand the physiological roles of inositol pyrophosphates. Another recent innovation has been the use of gel electrophoresis to detect and purify inositol pyrophosphates. Despite the advances that have been made, many aspects of inositol pyrophosphate biology remain far from clear. By evaluating the literature, the present review hopes to promote further research in this absorbing area of biology.

  11. How inositol pyrophosphates control cellular phosphate homeostasis?

    PubMed

    Saiardi, Adolfo

    2012-05-01

    Phosphorus in his phosphate PO(4)(3-) configuration is an essential constituent of all life forms. Phosphate diesters are at the core of nucleic acid structure, while phosphate monoester transmits information under the control of protein kinases and phosphatases. Due to these fundamental roles in biology it is not a surprise that phosphate cellular homeostasis is under tight control. Inositol pyrophosphates are organic molecules with the highest proportion of phosphate groups, and they are capable of regulating many biological processes, possibly by controlling energetic metabolism and adenosine triphosphate (ATP) production. Furthermore, inositol pyrophosphates influence inorganic polyphosphates (polyP) synthesis. The polymer polyP is solely constituted by phosphate groups and beside other known functions, it also plays a role in buffering cellular free phosphate [Pi] levels, an event that is ultimately necessary to generate ATP and inositol pyrophosphate. Although it is not yet clear how inositol pyrophosphates regulate cellular metabolism, understanding how inositol pyrophosphates influence phosphates homeostasis will help to clarify this important link. In this review I will describe the recent literature on this topic, with in the hope of inspiring further research in this fascinating area of biology.

  12. Myo-inositol content of common foods: development of a high-myo-inositol diet.

    PubMed

    Clements, R S; Darnell, B

    1980-09-01

    Since virtually no information is available concerning the myo-inositol content of dietary constituents, we have measured the amount of this material present in 487 foods by gas-liquid chromatography. We observed that the greatest amounts of myo-inositol were present in fruits, beans, grains, and nuts. Fresh vegetables and fruits were found to contain more myo-inositol than did frozen, canned, or salt-free products. The data provided in this report were used to develop diets that contained varying, but known amounts of myo-inositol. The myo-inositol intake that could be provided by such diets ranged from 225 to 1500 mg/day per 1800 kcal and within this range the agreement between the calculated and measured amounts of this material was excellent (r = 0.98). Since abnormalities in the metabolism of myo-inositol have been speculated to play a role in the pathogenesis of the polyneuropathies associated with diabetes mellitus and chronic renal failure, it is possible that the natural history of these neuropahties can be altered by modifying the amount of dietary myo-inositol that is ingested by patients with these diseases.

  13. Both myo-inositol to chiro-inositol epimerase activities and chiro-inositol to myo-inositol ratios are decreased in tissues of GK type 2 diabetic rats compared to Wistar controls.

    PubMed

    Sun, Tie-hua; Heimark, Douglas B; Nguygen, Thang; Nadler, Jerry L; Larner, Joseph

    2002-05-10

    Previous data from our and other labs demonstrated a decreased chiro-inositol content in urine and tissues of human subjects and animals with type 2 diabetes. In urine this decrease in chiro-inositol was accompanied by an increase in myo-inositol content. Decreased urine levels of chiro-inositol in monkeys were next correlated with the severity of underlying insulin resistance determined by five separate assays. To investigate the decreased chiro-inositol and the accompanying increased myo-inositol excretions in urine in humans and monkeys, we postulated a defect in the epimerization of myo-inositol to chiro-inositol. [(3)H]Myo-inositol was then shown to be converted to [(3)H]chiro-inositol in rats in vivo and in fibroblasts in vitro in a process stimulated by insulin. We next demonstrated that the conversion of [(3)H]myo-inositol to [(3)H]chiro-inositol in vivo was markedly decreased in GK type 2 diabetic rats compared to Wistar controls in liver, muscle, and fat, insulin sensitive tissues. Decreases of 20-25% conversion to baseline levels of under 5% conversion were observed. In the present work, we initially compared the total contents of myo-inositol and chiro-inositol in GK type 2 diabetic rat kidney, liver, and muscle compared to Wistar controls. We demonstrated a consistent decreased total chiro-inositol to myo-inositol ratio in kidney, liver, and muscle compared to controls. We next established the presence of a myo-inositol to chiro-inositol epimerase activity in rat liver cytosol. Enzyme activity was shown to be time and enzyme concentration dependent with a broad pH optimum. It required NADH and NADPH for full activity, which is compatible with its action via an oxido-reductive mechanism. Lastly, we demonstrated that the epimerase enzyme bioactivity was significantly decreased in muscle, liver, and fat cytosolic extracts of GK type 2 diabetic rats versus Wistar controls. Decreased myo-inositol to chiro-inositol epimerase activity may therefore play a

  14. The Effectiveness of Myo-Inositol and D-Chiro Inositol Treatment in Type 2 Diabetes

    PubMed Central

    Di Vieste, Giacoma; Bonomo, Matteo

    2016-01-01

    Inositol has been used as a supplement in treating several pathologies such as PCOS, metabolic syndrome, and gestational diabetes. Both myo-inositol and its isomer d-chiro-inositol showed insulin mimetic effects in conditions of insulin resistance. Type 2 diabetes (T2DM) is a condition typically caused by insulin resistance. There is a lack of evidence of inositol use in T2DM. We evaluated the effectiveness and safety of myo-inositol and d-chiro-inositol treatment in T2DM. This was a pilot study involving a consecutive sample of patients with T2DM with suboptimal glycemic control (HbA1c 7.0–10.0%) already treated with glucose-lowering agents. Patients (23.1% males, mean age of 60.8 ± 11.7 years) took for three months a combination of myo-inositol (550 mg) and d-chiro-inositol (13.8 mg) orally twice a day as add-on supplement to their glucose-lowering drugs. Possible occurrence of side effects was investigated. After three months of treatment fasting blood glucose (192.6 ± 60.2 versus 160.9 ± 36.4; p = 0.02) and HbA1c levels (8.6 ± 0.9 versus 7.7 ± 0.9; p = 0.02) significantly decreased compared to baseline. There was no significant difference in blood pressure, lipid profile, and BMI levels. None of the participants reported side effects. In conclusion, a supplementation with a combination of myo- and d-chiro-inositol is an effective and safe strategy for improving glycemic control in T2DM. PMID:27807448

  15. Inositol metabolism and cell growth in a Chinese hamster ovary cell myo-inositol auxotroph.

    PubMed

    Jackowski, S; Voelker, D R; Rock, C O

    1988-11-15

    The intracellular concentrations of polyphosphoinositides and inositol phosphates were determined, and their role in growth factor-initiated cell division was investigated in a Chinese hamster ovary cell inositol auxotroph (CHO-K1-Ins). Metabolic labeling experiments during inositol starvation of CHO-K1-Ins cells showed that 1) the lipid-linked inositol component was maintained at the expense of the soluble inositol pool, 2) the decreasing cellular content of phosphatidylinositol was replaced by phosphatidylglycerol, and 3) the concentrations of inositol polyphosphates and polyphosphoinositides were conserved at the expense of inositol and phosphatidylinositol. These data show that homeostatic mechanisms exist for the maintenance of the polyphosphoinositide and inositol phosphate pools at the expense of inositol and phosphatidylinositol. The addition of alpha-thrombin to growth-arrested (serum-starved) CHO-K1-Ins cells stimulated the incorporation of [3H]thymidine into DNA to the same extent as that observed following serum readdition. gamma-Thrombin was also an effective mitogen, but active site-inhibited alpha-thrombin was not. Both alpha- and gamma-thrombin, but not catalytic site-inhibited alpha-thrombin, initiated phosphatidylinositol turnover in vivo and increased phosphatidylinositol 4,5-bisphosphate phospholipase C activity in vitro. Serum and insulin were potent CHO-K1-Ins cell mitogens, but neither triggered phosphatidylinositol turnover in vivo nor activated phospholipase C in vitro. The activation of phospholipase C plays a determinant role in thrombin-initiated cell cycle progression in Chinese hamster ovary cells, although other growth factor-signaling pathways exist that are independent of polyphosphoinositide catabolism.

  16. 21 CFR 582.5370 - Inositol.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Inositol. 582.5370 Section 582.5370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  17. 21 CFR 582.5370 - Inositol.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Inositol. 582.5370 Section 582.5370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  18. 21 CFR 582.5370 - Inositol.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Inositol. 582.5370 Section 582.5370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  19. 21 CFR 582.5370 - Inositol.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Inositol. 582.5370 Section 582.5370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  20. 21 CFR 582.5370 - Inositol.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Inositol. 582.5370 Section 582.5370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  1. 21 CFR 184.1370 - Inositol.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... this ingredient as generally recognized as safe (GRAS) as a direct human food ingredient is based upon... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Inositol. 184.1370 Section 184.1370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DIRECT...

  2. Myo-Inositol content determined by myo-inositol biosynthesis and oxidation in blueberry fruit.

    PubMed

    Song, Fangyuan; Su, Hongyan; Yang, Nan; Zhu, Luying; Cheng, Jieshan; Wang, Lei; Cheng, Xianhao

    2016-11-01

    Myo-inositol metabolism in plant edible organs has become the focus of many recent studies because of its benefits to human health and unique functions in plant development. In this study, myo-inositol contents were analyzed during the development of two blueberry cultivars, cv 'Berkeley' and cv 'Bluecrop'. Furthermore, two VcMIPS 1/2 (Vaccinium corymbosum MIPS) genes, one VcIMP (Vaccinium corymbosum IMP) gene and one VcMIOX (Vaccinium corymbosum MIOX) gene were isolated for the first time from blueberry. The expression patterns of VcMIPS2, VcIMP and VcMIOX genes showed a relationship with the change profiles of myo-inositol content during fruit ripening. The results were further confirmed by the analyses of the enzyme activity. Results indicated that both myo-inositol biosynthesis and oxidation played important roles in determining of myo-inositol levels during the development of blueberry. To our knowledge, this report is the first to discuss myo-inositol levels in fruits in terms of biosynthesis and catabolism. PMID:27211661

  3. The inositol phosphate/diacylglycerol signalling pathway in Trypanosoma cruzi.

    PubMed Central

    Docampo, R; Pignataro, O P

    1991-01-01

    Using [32P]Pi and [3H]inositol as precursors, we have detected the presence of phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, and their derivatives inositol phosphate, inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate respectively, in Trypanosoma cruzi epimastigotes. Using digitonin-permeabilized cells it was possible to detect a stimulation in the formation of inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate as well as an increased generation of diacylglycerol in the presence of 1 mM-CaCl2. These results are consistent with the operation of a functional inositol phosphate/diacylglycerol pathway in T. cruzi, and constitute the first demonstration of the presence and activation of this pathway in a parasitic protozoan. These results also indicate that this pathway is conserved during evolution from lower to higher eukaryotic organisms. Images Fig. 1. PMID:2025225

  4. Inositol pyrophosphates inhibit synaptotagmin-dependent exocytosis.

    PubMed

    Lee, Tae-Sun; Lee, Joo-Young; Kyung, Jae Won; Yang, Yoosoo; Park, Seung Ju; Lee, Seulgi; Pavlovic, Igor; Kong, Byoungjae; Jho, Yong Seok; Jessen, Henning J; Kweon, Dae-Hyuk; Shin, Yeon-Kyun; Kim, Sung Hyun; Yoon, Tae-Young; Kim, Seyun

    2016-07-19

    Inositol pyrophosphates such as 5-diphosphoinositol pentakisphosphate (5-IP7) are highly energetic inositol metabolites containing phosphoanhydride bonds. Although inositol pyrophosphates are known to regulate various biological events, including growth, survival, and metabolism, the molecular sites of 5-IP7 action in vesicle trafficking have remained largely elusive. We report here that elevated 5-IP7 levels, caused by overexpression of inositol hexakisphosphate (IP6) kinase 1 (IP6K1), suppressed depolarization-induced neurotransmitter release from PC12 cells. Conversely, IP6K1 depletion decreased intracellular 5-IP7 concentrations, leading to increased neurotransmitter release. Consistently, knockdown of IP6K1 in cultured hippocampal neurons augmented action potential-driven synaptic vesicle exocytosis at synapses. Using a FRET-based in vitro vesicle fusion assay, we found that 5-IP7, but not 1-IP7, exhibited significantly higher inhibitory activity toward synaptic vesicle exocytosis than IP6 Synaptotagmin 1 (Syt1), a Ca(2+) sensor essential for synaptic membrane fusion, was identified as a molecular target of 5-IP7 Notably, 5-IP7 showed a 45-fold higher binding affinity for Syt1 compared with IP6 In addition, 5-IP7-dependent inhibition of synaptic vesicle fusion was abolished by increasing Ca(2+) levels. Thus, 5-IP7 appears to act through Syt1 binding to interfere with the fusogenic activity of Ca(2+) These findings reveal a role of 5-IP7 as a potent inhibitor of Syt1 in controlling the synaptic exocytotic pathway and expand our understanding of the signaling mechanisms of inositol pyrophosphates. PMID:27364007

  5. Analysis of Dictyostelium discoideum inositol pyrophosphate metabolism by gel electrophoresis.

    PubMed

    Pisani, Francesca; Livermore, Thomas; Rose, Giuseppina; Chubb, Jonathan Robert; Gaspari, Marco; Saiardi, Adolfo

    2014-01-01

    The social amoeba Dictyostelium discoideum was instrumental in the discovery and early characterization of inositol pyrophosphates, a class of molecules possessing highly-energetic pyrophosphate bonds. Inositol pyrophosphates regulate diverse biological processes and are attracting attention due to their ability to control energy metabolism and insulin signalling. However, inositol pyrophosphate research has been hampered by the lack of simple experimental procedures to study them. The recent development of polyacrylamide gel electrophoresis (PAGE) and simple staining to resolve and detect inositol pyrophosphate species has opened new investigative possibilities. This technology is now commonly applied to study in vitro enzymatic reactions. Here we employ PAGE technology to characterize the D. discoideum inositol pyrophosphate metabolism. Surprisingly, only three major bands are detectable after resolving acidic extract on PAGE. We have demonstrated that these three bands correspond to inositol hexakisphosphate (IP₆ or Phytic acid) and its derivative inositol pyrophosphates, IP₇ and IP₈. Biochemical analyses and genetic evidence were used to establish the genuine inositol phosphate nature of these bands. We also identified IP₉ in D. discoideum cells, a molecule so far detected only from in vitro biochemical reactions. Furthermore, we discovered that this amoeba possesses three different inositol pentakisphosphates (IP₅) isomers, which are largely metabolised to inositol pyrophosphates. Comparison of PAGE with traditional Sax-HPLC revealed an underestimation of the cellular abundance of inositol pyrophosphates by traditional methods. In fact our study revealed much higher levels of inositol pyrophosphates in D. discoideum in the vegetative state than previously detected. A three-fold increase in IP₈ was observed during development of D. discoideum a value lower that previously reported. Analysis of inositol pyrophosphate metabolism using ip6k null amoeba

  6. Inositol: history of an effective therapy for Polycystic Ovary Syndrome.

    PubMed

    Bizzarri, M; Carlomagno, G

    2014-07-01

    Inositol is a physiological compound belonging to the sugar family. The two inositol stereoisomers, myo-inositol and D-chiroinositol are the two main stereisomers present in our body. Myo-inositol is the precursor of inositol triphosphate, a second messenger regulating many hormones such as TSH, FSH and insulin. D-chiroinositol is synthetized by an insulin dependent epimerase that converts myo-inositol into D-chiro-inositol. Polycistic Ovary Syndrome (PCOS) is a metabolic and hormonal disorder and a common cause of infertility. Insulin resistance and the consequent hyperinsulinaemia contribute to hyperandrogenism development, typical marker of PCOS. In these patients myo and/or D-chiro-inositol administration improves insulin sensivity while only myo-inositol is a quality marker for oocytes evaluation. Myo-inositol produces second messengers for FSH and glucose uptake, while D-chiroinositol provides second messengers promoting glucose uptake and glycogen synthesis. The physiological ratio of these two isomers is 40:1 (MI/DCI) and seems to be an optimal approach for the treatment of PCOS disorders.

  7. A new inositol triester from Taraxacum mongolicum.

    PubMed

    Liu, Jifeng; Zhang, Nenling; Liu, Mengqi

    2014-01-01

    One new inositol triester, 4,5,6-tri-O-p-hydroxyphenylacetyl-chiro-inositol (1), was isolated from the ethanolic extract of Taraxacum mongolicum, along with two known compounds, 11β,13-dihydrotaraxinic acid (2) and taraxinic acid β-d-glucopyranosyl ester (3). The isolates were tested for their anti-hepatitis B virus (HBV) activities; 11β,13-dihydrotaraxinic acid (2) exhibited an IC50 value of 0.91 mM inhibiting the secretion of the HBV surface antigen and an IC50 value of 0.34 mM inhibiting the secretion of the HBV e antigen using HBV transfected Hep G2.2.15 cell line.

  8. Partial purification and characterization of indol-3-ylacetylglucose:myo-inositol indol-3-ylacetyltransferase (indoleacetic acid-inositol synthase)

    NASA Technical Reports Server (NTRS)

    Kesy, J. M.; Bandurski, R. S.

    1990-01-01

    A procedure is described for the purification of the enzyme indol-3-ylacetylglucose:myo-inositol indol-3-ylacetyltransferase (IAA-myo-inositol synthase). This enzyme catalyzes the transfer of indol-3-ylacetate from 1-0-indol-3-ylacetyl-beta-d-glucose to myo-inositol to form indol-3-ylacetyl-myo-inositol and glucose. A hexokinase or glucose oxidase based assay system is described. The enzyme has been purified approximately 16,000-fold, has an isoelectric point of pH 6.1 and yields three catalytically inactive bands upon acrylamide gel electrophoresis of the native protein. The enzyme shows maximum transferase activity with myo-inositol but shows some transferase activity with scyllo-inositol and myo-inosose-2. No transfer of IAA occurs with myo-inositol-d-galactopyranose, cyclohexanol, mannitol, or glycerol as acyl acceptor. The affinity of the enzyme for 1-0-indol-3-ylacetyl-beta-d-glucose is, Km = 30 micromolar, and for myo-inositol is, Km = 4 millimolar. The enzyme does not catalyze the exchange incorporation of glucose into IAA-glucose indicating the reaction mechanism involves binding of IAA glucose to the enzyme with subsequent hydrolytic cleavage of the acyl moiety by the hydroxyl of myo-inositol to form IAA myo-inositol ester.

  9. Inositols in the Treatment of Insulin-Mediated Diseases

    PubMed Central

    Muscogiuri, Giovanna; Palomba, Stefano

    2016-01-01

    A growing body of research is currently focused on the role of inositol isomers and in particular myo-inositol (MYO-INS) and D-chiroinositol (DCI) in the treatment of insulin resistance states. Both isomers have been shown to exert insulin-mimetic action and to lower postprandial glucose. Further, insulin resistance-related diseases were associated to derangements in inositol metabolism. Thus, the aim of this review is to provide current evidence on the potential benefits of inositol isomers (MYO-INS and DCI) in the treatment of disease associated to insulin resistance such as polycystic ovary syndrome (PCOS), gestational diabetes, and metabolic syndrome. Finally, molecular insights into inositol insulin-sensitizing effects will be covered focusing on the possible role of inositol glycans as insulin second messengers. PMID:27688754

  10. Inositol hexakisphosphate kinase products contain diphosphate and triphosphate groups.

    PubMed

    Draskovic, Petra; Saiardi, Adolfo; Bhandari, Rashna; Burton, Adam; Ilc, Gregor; Kovacevic, Miroslav; Snyder, Solomon H; Podobnik, Marjetka

    2008-03-01

    Eukaryotic cells produce a family of diverse inositol polyphosphates (IPs) containing pyrophosphate bonds. Inositol pyrophosphates have been linked to a wide range of cellular functions, and there is growing evidence that they act as second messengers. Inositol hexakisphosphate kinase (IP6K) is able to convert the natural substrates inositol pentakisphosphate (IP 5) and inositol hexakisphosphate (IP 6) to several products with an increasing number of phospho-anhydride bonds. In this study, we structurally analyzed IPs synthesized by three mammalian isoforms of IP6K from IP 5 and IP 6. The NMR and mass analyses showed a number of products with diverse, yet specific, stereochemistry, defined by the architecture of IP6K's active site. We now report that IP6K synthesizes both pyrophosphate (diphospho) as well as triphospho groups on the inositol ring. All three IP6K isoforms share the same activities both in vitro and in vivo.

  11. Extraction and analysis of soluble inositol polyphosphates from yeast.

    PubMed

    Azevedo, Cristina; Saiardi, Adolfo

    2006-01-01

    Soluble inositol polyphosphates are implicated in the regulation of many important cellular functions. This protocol to extract and separate inositol polyphosphates from Saccharomyces cerevisiae is divided into three steps: labeling of yeast, extraction of soluble inositol polyphosphates and chromatographic separation. Yeast cells are incubated with tritiated inositol, which is taken up and metabolized into different phosphorylated forms. Soluble inositol polyphosphates are then acid-extracted and fractionated by high-performance liquid chromatography. The radioactivity of each fraction is determined by scintillation counting. This highly sensitive and reproducible method allows the accurate detection of subtle changes in the inositol polyphosphate profile and takes less than 48 h. It can easily be applied to other systems and we have included two adaptations of the protocol, one optimized for mammalian cells and the other for Arabidopsis thaliana. PMID:17406485

  12. Inositols in the Treatment of Insulin-Mediated Diseases

    PubMed Central

    Muscogiuri, Giovanna; Palomba, Stefano

    2016-01-01

    A growing body of research is currently focused on the role of inositol isomers and in particular myo-inositol (MYO-INS) and D-chiroinositol (DCI) in the treatment of insulin resistance states. Both isomers have been shown to exert insulin-mimetic action and to lower postprandial glucose. Further, insulin resistance-related diseases were associated to derangements in inositol metabolism. Thus, the aim of this review is to provide current evidence on the potential benefits of inositol isomers (MYO-INS and DCI) in the treatment of disease associated to insulin resistance such as polycystic ovary syndrome (PCOS), gestational diabetes, and metabolic syndrome. Finally, molecular insights into inositol insulin-sensitizing effects will be covered focusing on the possible role of inositol glycans as insulin second messengers.

  13. Inositols in the Treatment of Insulin-Mediated Diseases.

    PubMed

    Muscogiuri, Giovanna; Palomba, Stefano; Laganà, Antonio Simone; Orio, Francesco

    2016-01-01

    A growing body of research is currently focused on the role of inositol isomers and in particular myo-inositol (MYO-INS) and D-chiroinositol (DCI) in the treatment of insulin resistance states. Both isomers have been shown to exert insulin-mimetic action and to lower postprandial glucose. Further, insulin resistance-related diseases were associated to derangements in inositol metabolism. Thus, the aim of this review is to provide current evidence on the potential benefits of inositol isomers (MYO-INS and DCI) in the treatment of disease associated to insulin resistance such as polycystic ovary syndrome (PCOS), gestational diabetes, and metabolic syndrome. Finally, molecular insights into inositol insulin-sensitizing effects will be covered focusing on the possible role of inositol glycans as insulin second messengers. PMID:27688754

  14. Characterization of the inositol monophosphatase gene family in Arabidopsis

    PubMed Central

    Nourbakhsh, Aida; Collakova, Eva; Gillaspy, Glenda E.

    2015-01-01

    Synthesis of myo-inositol is crucial in multicellular eukaryotes for production of phosphatidylinositol and inositol phosphate signaling molecules. The myo-inositol monophosphatase (IMP) enzyme is required for the synthesis of myo-inositol, breakdown of inositol (1,4,5)-trisphosphate, a second messenger involved in Ca2+ signaling, and synthesis of L-galactose, a precursor of ascorbic acid. Two myo-inositol monophosphatase -like (IMPL) genes in Arabidopsis encode chloroplast proteins with homology to the prokaryotic IMPs and one of these, IMPL2, can complement a bacterial histidinol 1-phosphate phosphatase mutant defective in histidine synthesis, indicating an important role for IMPL2 in amino acid synthesis. To delineate how this small gene family functions in inositol synthesis and metabolism, we sought to compare recombinant enzyme activities, expression patterns, and impact of genetic loss-of-function mutations for each. Our data show that purified IMPL2 protein is an active histidinol-phosphate phosphatase enzyme in contrast to the IMPL1 enzyme, which has the ability to hydrolyze D-galactose 1-phosphate, and D-myo-inositol 1-phosphate, a breakdown product of D-inositol (1,4,5) trisphosphate. Expression studies indicated that all three genes are expressed in multiple tissues, however, IMPL1 expression is restricted to above-ground tissues only. Identification and characterization of impl1 and impl2 mutants revealed no viable mutants for IMPL1, while two different impl2 mutants were identified and shown to be severely compromised in growth, which can be rescued by histidine. Analyses of metabolite levels in impl2 and complemented mutants reveals impl2 mutant growth is impacted by alterations in the histidine biosynthesis pathway, but does not impact myo-inositol synthesis. Together, these data indicate that IMPL2 functions in the histidine biosynthetic pathway, while IMP and IMPL1 catalyze the hydrolysis of inositol- and galactose-phosphates in the plant cell

  15. Arginine transcriptional response does not require inositol phosphate synthesis.

    PubMed

    Bosch, Daniel; Saiardi, Adolfo

    2012-11-01

    Inositol phosphates are key signaling molecules affecting a large variety of cellular processes. Inositol-polyphosphate multikinase (IPMK) is a central component of the inositol phosphate biosynthetic routes, playing essential roles during development. IPMK phosphorylates inositol 1,4,5-trisphosphate to inositol tetrakisphosphate and subsequently to inositol pentakisphosphate and has also been described to function as a lipid kinase. Recently, a catalytically inactive mammalian IPMK was reported to be involved in nutrient signaling by way of mammalian target of rapamycin and AMP-activated protein kinase. In yeast, the IPMK homologue, Arg82, is the sole inositol-trisphosphate kinase. Arg82 has been extensively studied as part of the transcriptional complex regulating nitrogen sensing, in particular arginine metabolism. Whether this role requires Arg82 catalytic activity has long been a matter of contention. In this study, we developed a novel method for the real time study of promoter strength in vivo and used it to demonstrate that catalytically inactive Arg82 fully restored the arginine-dependent transcriptional response. We also showed that expression in yeast of catalytically active, but structurally very different, mammalian or plant IPMK homologue failed to restore arginine regulation. Our work indicates that inositol phosphates do not regulate arginine-dependent gene expression. PMID:22992733

  16. Characterization of inositol phosphates in carrot (Daucus carota L. ) cells

    SciTech Connect

    Rincon, M.; Chen, Q.; Boss, W.F. )

    1989-01-01

    We have shown previously that inositol-1,4,5-trisphosphate (IP{sub 3}) stimulates an efflux of {sup 45}Ca{sup 2+} from fusogenic carrot protoplasts. In light of these results, we suggested that IP{sub 3} might serve as a second messenger for the mobilization of intracellular Ca{sup 2+} in higher plant cells. To determine whether or not IP{sub 3} and other inositol phosphates were present in the carrot cells, the cells were labeled with myo-(2-{sup 3}H)inositol for 18 hours and extracted with ice-cold 10% trichloroacetic acid. The inositol metabolites were separated by anion exchange chromatography and by paper electrophoresis. We found that ({sup 3}H)inositol metabolites coeluted with inositol bisphosphate (IP{sub 2}) and IP{sub 3} when separated by anion exchange chromatography. However, we could not detect IP{sub 2} or IP{sub 3} when the inositol metabolites were analyzed by paper electrophoresis even though the polyphosphoinositides, which are the source of IP{sub 2} and IP{sub 3}, were present in these cells. Thus, ({sup 3}H)inositol metabolites other than IP{sub 2} and IP{sub 3} had coeluted on the anion exchange columns. The data indicate that either IP{sub 3} is rapidly metabolized or that it is not present at a detectable level in the carrot cells.

  17. Structural basis for an inositol pyrophosphate kinase surmounting phosphate crowding

    SciTech Connect

    Wang, Huanchen; Falck, J.R.; Hall, Traci M. Tanaka; Shears, Stephen B.

    2012-01-11

    Inositol pyrophosphates (such as IP7 and IP8) are multifunctional signaling molecules that regulate diverse cellular activities. Inositol pyrophosphates have 'high-energy' phosphoanhydride bonds, so their enzymatic synthesis requires that a substantial energy barrier to the transition state be overcome. Additionally, inositol pyrophosphate kinases can show stringent ligand specificity, despite the need to accommodate the steric bulk and intense electronegativity of nature's most concentrated three-dimensional array of phosphate groups. Here we examine how these catalytic challenges are met by describing the structure and reaction cycle of an inositol pyrophosphate kinase at the atomic level. We obtained crystal structures of the kinase domain of human PPIP5K2 complexed with nucleotide cofactors and either substrates, product or a MgF{sub 3}{sup -} transition-state mimic. We describe the enzyme's conformational dynamics, its unprecedented topological presentation of nucleotide and inositol phosphate, and the charge balance that facilitates partly associative in-line phosphoryl transfer.

  18. Regioselective deprotection of orthobenzoates for the synthesis of inositol phosphates.

    PubMed

    Swarbrick, Joanna M; Cooper, Samuel; Bultynck, Geert; Gaffney, Piers R J

    2009-04-21

    Synthetic myo-inositol 1,4,5-triphosphate, Ins(1,4,5)P(3), and myo-inositol 1,3,4,5-tetraphosphate, Ins(1,3,4,5)P(4), continue to be valuable in biological studies. Inositol orthoesters have proved an important class of intermediate to access these compounds. We investigated the ability of steric bulk from a 4-O protecting group to direct DIBAL-H reduction of inositol orthobenzoates to generate the natural Ins(1,4,5)P(3) precursor 2,3,6-O-tribenzyl myo-inositol. Introduction of an equatorial 4-C-methyl group imparts totally selective reduction and we report the synthesis of novel 4-C-methyl-Ins(1,4,5)P(3) and 4-C-methyl-Ins(1,3,4,5)P(4).

  19. Influence of inositol pyrophosphates on cellular energy dynamics.

    PubMed

    Szijgyarto, Zsolt; Garedew, Assegid; Azevedo, Cristina; Saiardi, Adolfo

    2011-11-11

    With its high-energy phosphate bonds, adenosine triphosphate (ATP) is the main intracellular energy carrier. It also functions in most signaling pathways, as a phosphate donor or a precursor for cyclic adenosine monophosphate. We show here that inositol pyrophosphates participate in the control of intracellular ATP concentration. Yeasts devoid of inositol pyrophosphates have dysfunctional mitochondria but, paradoxically, contain four times as much ATP because of increased glycolysis. We demonstrate that inositol pyrophosphates control the activity of the major glycolytic transcription factor GCR1. Thus, inositol pyrophosphates regulate ATP concentration by altering the glycolytic/mitochondrial metabolic ratio. Metabolic reprogramming through inositol pyrophosphates is an evolutionary conserved mechanism that is also preserved in mammalian systems.

  20. Myo-Inositol-Dependent Sodium Uptake in Ice Plant1

    PubMed Central

    Nelson, Donald E.; Koukoumanos, Michelle; Bohnert, Hans J.

    1999-01-01

    In salt-stressed ice plants (Mesembryanthemum crystallinum), sodium accumulates to high concentrations in vacuoles, and polyols (myo-inositol, d-ononitol, and d-pinitol) accumulate in the cytosol. Polyol synthesis is regulated by NaCl and involves induction and repression of gene expression (D.E. Nelson, B. Shen, and H.J. Bohnert [1998] Plant Cell 10: 753–764). In the study reported here we found increased phloem transport of myo-inositol and reciprocal increased transport of sodium and inositol to leaves under stress. To determine the relationship between increased translocation and sodium uptake, we analyzed the effects of exogenous application of myo-inositol: The NaCl-inducible ice plant myo-inositol 1-phosphate synthase is repressed in roots, and sodium uptake from root to shoot increases without stimulating growth. Sodium uptake and transport through the xylem was coupled to a 10-fold increase of myo-inositol and ononitol in the xylem. Seedlings of the ice plant are not salt-tolerant, and yet the addition of exogenous myo-inositol conferred upon them patterns of gene expression and polyol accumulation observed in mature, salt-tolerant plants. Sodium uptake and transport through the xylem was enhanced in the presence of myo-inositol. The results indicate an interdependence of sodium uptake and alterations in the distribution of myo-inositol. We hypothesize that myo-inositol could serve not only as a substrate for the production of compatible solutes but also as a leaf-to-root signal that promotes sodium uptake. PMID:9880357

  1. Content of methylated inositols in familiar edible plants.

    PubMed

    Negishi, Osamu; Mun'im, Abdul; Negishi, Yukiko

    2015-03-18

    Familiar plants contain large amounts of inositols; soybean, white clover, red clover, bush clover, locust tree, wisteria, and kudzu of the legume family contain pinitol (3-O-methyl-chiro-inositol) at approximately 200-600 mg/100 g fresh weight (FW). The contents of pinitol in other plants were 260 mg/100 g FW for sticky mouse-ear, 275 mg/100 g FW for chickweed, and 332 mg/100 g FW for ginkgo. chiro-Inositol of 191 and 156 mg/100 g FW was also found in dandelion and Japanese mallotus, respectively. Ononitol (4-O-methyl-myo-inositol) of 166 mg/100 g FW was found in sticky mouse-ear. Furthermore, young leaves of ginkgo contained sequoyitol (5-O-methyl-myo-inositol) of 287 mg/100 g FW. Hydroxyl radical scavenging activities of the methylated inositols were higher than those of the original inositols. Effective uses of these familiar edible plants are expected to promote good health. PMID:25734537

  2. scyllo-Inositol promotes robust mutant Huntingtin protein degradation.

    PubMed

    Lai, Aaron Y; Lan, Cynthia P; Hasan, Salwa; Brown, Mary E; McLaurin, Joanne

    2014-02-01

    Huntington disease is characterized by neuronal aggregates and inclusions containing polyglutamine-expanded huntingtin protein and peptide fragments (polyQ-Htt). We have used an established cell-based assay employing a PC12 cell line overexpressing truncated exon 1 of Htt with a 103-residue polyQ expansion that yields polyQ-Htt aggregates to investigate the fate of polyQ-Htt-drug complexes. scyllo-Inositol is an endogenous inositol stereoisomer known to inhibit accumulation and toxicity of the amyloid-β peptide and α-synuclein. In light of these properties, we investigated the effect of scyllo-inositol on polyQ-Htt accumulation. We show that scyllo-inositol lowered the number of visible polyQ-Htt aggregates and robustly decreased polyQ-Htt protein abundance without concomitant cellular toxicity. We found that scyllo-inositol-induced polyQ-Htt reduction was by rescue of degradation pathways mediated by the lysosome and by the proteasome but not autophagosomes. The rescue of degradation pathways was not a direct result of scyllo-inositol on the lysosome or proteasome but due to scyllo-inositol-induced reduction in mutant polyQ-Htt protein levels.

  3. Biosynthesis and possible functions of inositol pyrophosphates in plants.

    PubMed

    Williams, Sarah P; Gillaspy, Glenda E; Perera, Imara Y

    2015-01-01

    Inositol phosphates (InsPs) are intricately tied to lipid signaling, as at least one portion of the inositol phosphate signaling pool is derived from hydrolysis of the lipid precursor, phosphatidyl inositol (4,5) bisphosphate. The focus of this review is on the inositol pyrophosphates, which are a novel group of InsP signaling molecules containing diphosphate or triphosphate chains (i.e., PPx) attached to the inositol ring. These PPx-InsPs are emerging as critical players in the integration of cellular metabolism and stress signaling in non-plant eukaryotes. Most eukaryotes synthesize the precursor molecule, myo-inositol (1,2,3,4,5,6)-hexakisphosphate (InsP6), which can serve as a signaling molecule or as storage compound of inositol, phosphorus, and minerals (referred to as phytic acid). Even though plants produce huge amounts of precursor InsP6 in seeds, almost no attention has been paid to whether PPx-InsPs exist in plants, and if so, what roles these molecules play. Recent work has delineated that Arabidopsis has two genes capable of PP-InsP5 synthesis, and PPx-InsPs have been detected across the plant kingdom. This review will detail the known roles of PPx-InsPs in yeast and animal systems, and provide a description of recent data on the synthesis and accumulation of these novel molecules in plants, and potential roles in signaling.

  4. Biosynthesis and possible functions of inositol pyrophosphates in plants

    PubMed Central

    Williams, Sarah P.; Gillaspy, Glenda E.; Perera, Imara Y.

    2015-01-01

    Inositol phosphates (InsPs) are intricately tied to lipid signaling, as at least one portion of the inositol phosphate signaling pool is derived from hydrolysis of the lipid precursor, phosphatidyl inositol (4,5) bisphosphate. The focus of this review is on the inositol pyrophosphates, which are a novel group of InsP signaling molecules containing diphosphate or triphosphate chains (i.e., PPx) attached to the inositol ring. These PPx-InsPs are emerging as critical players in the integration of cellular metabolism and stress signaling in non-plant eukaryotes. Most eukaryotes synthesize the precursor molecule, myo-inositol (1,2,3,4,5,6)-hexakisphosphate (InsP6), which can serve as a signaling molecule or as storage compound of inositol, phosphorus, and minerals (referred to as phytic acid). Even though plants produce huge amounts of precursor InsP6 in seeds, almost no attention has been paid to whether PPx-InsPs exist in plants, and if so, what roles these molecules play. Recent work has delineated that Arabidopsis has two genes capable of PP-InsP5 synthesis, and PPx-InsPs have been detected across the plant kingdom. This review will detail the known roles of PPx-InsPs in yeast and animal systems, and provide a description of recent data on the synthesis and accumulation of these novel molecules in plants, and potential roles in signaling. PMID:25729385

  5. Phosphorylation regulates myo-inositol-3-phosphate synthase: a novel regulatory mechanism of inositol biosynthesis.

    PubMed

    Deranieh, Rania M; He, Quan; Caruso, Joseph A; Greenberg, Miriam L

    2013-09-13

    myo-Inositol-3-phosphate synthase (MIPS) plays a crucial role in inositol homeostasis. Transcription of the coding gene INO1 is highly regulated. However, regulation of the enzyme is not well defined. We previously showed that MIPS is indirectly inhibited by valproate, suggesting that the enzyme is post-translationally regulated. Using (32)Pi labeling and phosphoamino acid analysis, we show that yeast MIPS is a phosphoprotein. Mass spectrometry analysis identified five phosphosites, three of which are conserved in the human MIPS. Analysis of phosphorylation-deficient and phosphomimetic site mutants indicated that the three conserved sites in yeast (Ser-184, Ser-296, and Ser-374) and humans (Ser-177, Ser-279, and Ser-357) affect MIPS activity. Both S296A and S296D yeast mutants and S177A and S177D human mutants exhibited decreased enzymatic activity, suggesting that a serine residue is critical at that location. The phosphomimetic mutations S184D (human S279D) and S374D (human S357D) but not the phosphodeficient mutations decreased activity, suggesting that phosphorylation of these two sites is inhibitory. The double mutation S184A/S374A caused an increase in MIPS activity, conferred a growth advantage, and partially rescued sensitivity to valproate. Our findings identify a novel mechanism of regulation of inositol synthesis by phosphorylation of MIPS.

  6. Inositol trisphosphate, a novel second messenger in cellular signal transduction

    NASA Astrophysics Data System (ADS)

    Berridge, Michael J.; Irvine, Robin F.

    1984-11-01

    There has recently been rapid progress in understanding receptors that generate intracellular signals from inositol lipids. One of these lipids, phosphatidylinositol 4,5-bisphosphate, is hydrolysed to diacylglycerol and inositol trisphosphate as part of a signal transduction mechanism for controlling a variety of cellular processes including secretion, metabolism, phototransduction and cell proliferation. Diacylglycerol operates within the plane of the membrane to activate protein kinase C, whereas inositol trisphosphate is released into the cytoplasm to function as a second messenger for mobilizing intracellular calcium.

  7. Inositol-deficient food augments a behavioral effect of long-term lithium treatment mediated by inositol monophosphatase inhibition: an animal model with relevance for bipolar disorder.

    PubMed

    Shtein, Liza; Agam, Galila; Belmaker, R H; Bersudsky, Yuly

    2015-04-01

    Lithium treatment in rodents markedly enhances cholinergic agonists such as pilocarpine. This effect can be reversed in a stereospecific manner by administration of inositol, suggesting that the effect of lithium is caused by inositol monophosphatase inhibition and consequent inositol depletion. If so, inositol-deficient food would be expected to enhance lithium effects. Inositol-deficient food was prepared from inositol-free ingredients. Mice with a homozygote knockout of the inositol monophosphatase 1 gene unable to synthesize inositol endogenously and mimicking lithium-treated animals were fed this diet or a control diet. Lithium-treated wild-type animals were also treated with the inositol-deficient diet or control diet. Pilocarpine was administered after 1 week of treatment, and behavior including seizures was assessed using rating scale. Inositol-deficient food-treated animals, both lithium treated and with inositol monophosphatase 1 knockout, had significantly elevated cholinergic behavior rating and significantly increased or earlier seizures compared with the controls. The effect of inositol-deficient food supports the role of inositol depletion in the effects of lithium on pilocarpine-induced behavior. However, the relevance of this behavior to other more mood-related effects of lithium is not clear.

  8. Inositol synthesis regulates the activation of GSK-3α in neuronal cells.

    PubMed

    Ye, Cunqi; Greenberg, Miriam L

    2015-04-01

    The synthesis of inositol provides precursors of inositol lipids and inositol phosphates that are pivotal for cell signaling. Mood stabilizers lithium and valproic acid, used for treating bipolar disorder, cause cellular inositol depletion, which has been proposed as a therapeutic mechanism of action of both drugs. Despite the importance of inositol, the requirement for inositol synthesis in neuronal cells is not well understood. Here, we examined inositol effects on proliferation of SK-N-SH neuroblastoma cells. The essential role of inositol synthesis in proliferation is underscored by the findings that exogenous inositol was dispensable for proliferation, and inhibition of inositol synthesis decreased proliferation. Interestingly, the inhibition of inositol synthesis by knocking down INO1, which encodes inositol-3-phosphate synthase, the rate-limiting enzyme of inositol synthesis, led to the inactivation of GSK-3α by increasing the inhibitory phosphorylation of this kinase. Similarly, the mood stabilizer valproic acid effected transient decreases in intracellular inositol, leading to inactivation of GSK-3α. As GSK-3 inhibition has been proposed as a likely therapeutic mechanism of action, the finding that inhibition of inositol synthesis results in the inactivation of GSK-3α suggests a unifying hypothesis for mechanism of mood-stabilizing drugs. Inositol is an essential metabolite that serves as a precursor for inositol lipids and inositol phosphates. We report that inhibition of the rate-limiting enzyme of inositol synthesis leads to the inactivation of glycogen synthase kinase (GSK) 3α by increasing inhibitory phosphorylation of this kinase. These findings have implications for the therapeutic mechanisms of mood stabilizers and suggest that inositol synthesis and GSK 3α activity are intrinsically related.

  9. Beryllium competitively inhibits brain myo-inositol monophosphatase, but unlike lithium does not enhance agonist-induced inositol phosphate accumulation.

    PubMed Central

    Faraci, W S; Zorn, S H; Bakker, A V; Jackson, E; Pratt, K

    1993-01-01

    Despite limiting side-effects, lithium is the drug of choice for the treatment of bipolar depression. Its action may be due, in part, to its ability to dampen phosphatidylinositol turnover by inhibiting myo-inositol monophosphatase. Beryllium has been identified as a potent inhibitor of partially purified myo-inositol monophosphatase isolated from rat brain (Ki = 150 nM), bovine brain (Ki = 35 nM), and from the human neuroblastoma cell line SK-N-SH (Ki = 85 nM). It is over three orders of magnitude more potent than LiCl (Ki = 0.5-1.2 mM). Kinetic analysis reveals that beryllium is a competitive inhibitor of myo-inositol monophosphatase, in contrast with lithium which is an uncompetitive inhibitor. Inhibition of exogenous [3H]inositol phosphate hydrolysis by beryllium (IC50 = 250-300 nM) was observed to the same maximal extent as that seen with lithium in permeabilized SK-N-SH cells, reflecting inhibition of cellular myo-inositol monophosphatase. However, in contrast with that observed with lithium, agonist-induced accumulation of inositol phosphate was not observed with beryllium in permeabilized and non-permeabilized SK-N-SH cells and in rat brain slices. Similar results were obtained in permeabilized SK-N-SH cells when GTP-gamma-S was used as an alternative stimulator of inositol phosphate accumulation. The disparity in the actions of beryllium and lithium suggest that either (1) selective inhibition of myo-inositol monophosphatase does not completely explain the action of lithium on the phosphatidylinositol cycle, or (2) that uncompetitive inhibition of myo-inositol monophosphatase is a necessary requirement to observe functional lithium mimetic activity. PMID:8387266

  10. Redistribution of tritium during germination of grain harvested from myo-(2-/sup 3/H)inositol- and scyllo-(R-/sup 3/H)inositol-labeled wheat

    SciTech Connect

    Sasaki, K.; Loewus, F.A.

    1982-01-01

    Wheat kernels from myo-(2-/sup 3/H)inositol- or scyllo-(R-/sup 3/H)inositol-labeled plants (Sasaki and Loewus 1980 Plant Physiol 66: 740-745) were used to study redistribution of /sup 3/H into growing regions during germination. Most of the labeled 1-..cap alpha..-galactinol (or the analogous scyllo-inositol galatoside) was hydrolyzed within 1 day. Water-soluble phytate was dephosphorylated within 3 days. A large reserve of bound phytate continued to release myo-inositol over several days. Translocation of free myo-inositol to growing regions provided substrate for the myo-inositol oxidation pathway and incorporation of /sup 3/H into new cell wall polysaccharides. Cell wall polysaccharides in the kernel were degraded during germination. The labeled residues were translocated to growing regions and reutilized for new cell wall formation. Pentosyl residues accounted for most of this label. Free scyllo-inositol followed a path of translocation from kernal to seeding similar to that of myo-inositol. Unlike myo-inositol, it did not furnish substrate for the myo-inositol oxidation pathway but accumulated as free scyllo-inositol in the seeding. The fate of phytate-derived myo-inositol during germination of wheat is discussed in relation to a recent scheme of phytate metabolism proposed by De and Biswas (1979 J Biol Chem 254 :8717-8719) for germinating mung bean seedlings.

  11. Dictyostelium uses ether-linked inositol phospholipids for intracellular signalling

    PubMed Central

    Clark, Jonathan; Kay, Robert R; Kielkowska, Anna; Niewczas, Izabella; Fets, Louise; Oxley, David; Stephens, Len R; Hawkins, Phillip T

    2014-01-01

    Inositol phospholipids are critical regulators of membrane biology throughout eukaryotes. The general principle by which they perform these roles is conserved across species and involves binding of differentially phosphorylated inositol head groups to specific protein domains. This interaction serves to both recruit and regulate the activity of several different classes of protein which act on membrane surfaces. In mammalian cells, these phosphorylated inositol head groups are predominantly borne by a C38:4 diacylglycerol backbone. We show here that the inositol phospholipids of Dictyostelium are different, being highly enriched in an unusual C34:1e lipid backbone, 1-hexadecyl-2-(11Z-octadecenoyl)-sn-glycero-3-phospho-(1'-myo-inositol), in which the sn-1 position contains an ether-linked C16:0 chain; they are thus plasmanylinositols. These plasmanylinositols respond acutely to stimulation of cells with chemoattractants, and their levels are regulated by PIPKs, PI3Ks and PTEN. In mammals and now in Dictyostelium, the hydrocarbon chains of inositol phospholipids are a highly selected subset of those available to other phospholipids, suggesting that different molecular selectors are at play in these organisms but serve a common, evolutionarily conserved purpose. PMID:25180230

  12. Structures, stability and hydrogen bonding in inositol conformers.

    PubMed

    Siddiqui, Nazia; Singh, Vijay; Deshmukh, Milind M; Gurunath, Ramanathan

    2015-07-28

    Various ab initio calculations using the density-functional (DFT), the second order Möller-Plesset perturbation (MP2) and self-consistent reaction field (SCRF) theories were performed on thirteen theoretically possible inositol stereoisomers. Gas phase calculations reveal that the myo- and neo-isomers of inositol (bearing one and two axial hydroxyl groups, respectively) are marginally more stable (by 0.5 kcal mol(-1)) than the all equatorially substituted scyllo-inositol. The calculations when done in different polar solvents show that the scyllo-inositol becomes the most stable inositol isomer, a fact attributed to weaker intramolecular hydrogen bonds. The individual hydrogen bond energy in all the isomers of inositol was also estimated using the molecular tailoring approach (MTA). The calculated hydrogen bond energies in these isomers are in excellent agreement with reported O-H···O hydrogen bond distances and ν(O-H) stretching frequencies. The estimated H-bond energy values suggest that the order of the intramolecular hydrogen bond strength follows: axial-axial > equatorial-axial > axial-equatorial > equatorial-equatorial hydrogen bonds. The intramolecular hydrogen bonds in the scyllo isomer are much weaker than those in other conformers, thus making this isomer more stable in polar solvents.

  13. Inositol depletion restores vesicle transport in yeast phospholipid flippase mutants.

    PubMed

    Yamagami, Kanako; Yamamoto, Takaharu; Sakai, Shota; Mioka, Tetsuo; Sano, Takamitsu; Igarashi, Yasuyuki; Tanaka, Kazuma

    2015-01-01

    In eukaryotic cells, type 4 P-type ATPases function as phospholipid flippases, which translocate phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of the lipid bilayer. Flippases function in the formation of transport vesicles, but the mechanism remains unknown. Here, we isolate an arrestin-related trafficking adaptor, ART5, as a multicopy suppressor of the growth and endocytic recycling defects of flippase mutants in budding yeast. Consistent with a previous report that Art5p downregulates the inositol transporter Itr1p by endocytosis, we found that flippase mutations were also suppressed by the disruption of ITR1, as well as by depletion of inositol from the culture medium. Interestingly, inositol depletion suppressed the defects in all five flippase mutants. Inositol depletion also partially restored the formation of secretory vesicles in a flippase mutant. Inositol depletion caused changes in lipid composition, including a decrease in phosphatidylinositol and an increase in phosphatidylserine. A reduction in phosphatidylinositol levels caused by partially depleting the phosphatidylinositol synthase Pis1p also suppressed a flippase mutation. These results suggest that inositol depletion changes the lipid composition of the endosomal/TGN membranes, which results in vesicle formation from these membranes in the absence of flippases.

  14. The Inositol-3-Phosphate Synthase Biosynthetic Enzyme Has Distinct Catalytic and Metabolic Roles

    PubMed Central

    Frej, Anna D.; Clark, Jonathan; Le Roy, Caroline I.; Lilla, Sergio; Thomason, Peter A.; Otto, Grant P.; Churchill, Grant; Insall, Robert H.; Claus, Sandrine P.; Hawkins, Phillip; Stephens, Len

    2016-01-01

    Inositol levels, maintained by the biosynthetic enzyme inositol-3-phosphate synthase (Ino1), are altered in a range of disorders, including bipolar disorder and Alzheimer's disease. To date, most inositol studies have focused on the molecular and cellular effects of inositol depletion without considering Ino1 levels. Here we employ a simple eukaryote, Dictyostelium discoideum, to demonstrate distinct effects of loss of Ino1 and inositol depletion. We show that loss of Ino1 results in an inositol auxotrophy that can be rescued only partially by exogenous inositol. Removal of inositol supplementation from the ino1− mutant resulted in a rapid 56% reduction in inositol levels, triggering the induction of autophagy, reduced cytokinesis, and substrate adhesion. Inositol depletion also caused a dramatic generalized decrease in phosphoinositide levels that was rescued by inositol supplementation. However, loss of Ino1 triggered broad metabolic changes consistent with the induction of a catabolic state that was not rescued by inositol supplementation. These data suggest a metabolic role for Ino1 that is independent of inositol biosynthesis. To characterize this role, an Ino1 binding partner containing SEL1L1 domains (Q54IX5) and having homology to mammalian macromolecular complex adaptor proteins was identified. Our findings therefore identify a new role for Ino1, independent of inositol biosynthesis, with broad effects on cell metabolism. PMID:26951199

  15. Selectively blocked derivatives of muco-inositol and their conversion into derivatives of epi- and cis-inositol.

    PubMed

    Espelie, K E; Anderson, L

    1976-01-01

    Benzylation, and then hydrolysis, of 1, 2:4, 5-di-O-isopropylidene-muco-inositol (1) gave 3, 6-di-O-benzyl-muco-inositol (3). This was converted into a series of derivatives, including the 1, 5-di-O-benzoyl-3, 6-di-O-benzyl-2, 4-di-p-toluenesulfonate 7. The resistance to displacement of the sulfonate groups in 7 prevented conversion of 7 into an intermediate for the synthesis of aminoglycoside antibiotics. Monobenzylation of 1, followed by an oxidation-reduction cycle, yielded 6-O-benzyl-1, 2:4, 5-di-O-isopropylidene-epi-inositol (10). From this was synthesized a series of epi-inositol derivatives, analogous to the muco series but less complete. For derivatives of 1, 2:5, 6-di-O-isopropylidene-epi- and muco-inositol, the p. m. r. data indicate modified skew conformations. The reaction of the 3, 6-di-p-bromobenzenesulfonate (17) of 1 with anhydrous hydrazine proceeded in part by S-O cleavage to regenerate 1, and in part by displacement of both sulfonate groups by the same nitrogen atom. The resulting, novel 1, 4-epimino-cis-inositol was converted into further derivatives.

  16. myo-Inositol 1,3-acetals as early intermediates during the synthesis of cyclitol derivatives.

    PubMed

    Gurale, Bharat P; Sardessai, Richa S; Shashidhar, Mysore S

    2014-11-18

    Synthetic sequences starting from commercially available myo-inositol necessarily involve protection-deprotection strategies of its six hydroxyl groups. Several strategies have been developed/attempted over the last several decades leading to the synthesis of naturally occurring phosphoinositols, their analogs, and cyclitol derivatives. Of late, myo-inositol 1,3-acetals, which can be obtained by the reductive cleavage of myo-inositol orthoesters have emerged as early intermediates for the synthesis of phosphorylated and other inositol derivatives. This mini-review is an attempt to illustrate the economy and convenience of using myo-inositol 1,3-acetals as early intermediates during syntheses from myo-inositol.

  17. Synthesis of inositol phosphate-based competitive antagonists of inositol 1,4,5-trisphosphate receptors.

    PubMed

    Konieczny, Vera; Stefanakis, John G; Sitsanidis, Efstratios D; Ioannidou, Natalia-Anastasia T; Papadopoulos, Nikolaos V; Fylaktakidou, Konstantina C; Taylor, Colin W; Koumbis, Alexandros E

    2016-02-28

    Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular Ca(2+) channels that are widely expressed in animal cells, where they mediate the release of Ca(2+) from intracellular stores evoked by extracellular stimuli. A diverse array of synthetic agonists of IP3Rs has defined structure-activity relationships, but existing antagonists have severe limitations. We combined analyses of Ca(2+) release with equilibrium competition binding to IP3R to show that (1,3,4,6)IP4 is a full agonist of IP3R1 with lower affinity than (1,4,5)IP3. Systematic manipulation of this meso-compound via a versatile synthetic scheme provided a family of dimeric analogs of 2-O-butyryl-(1,3,4,6)IP4 and (1,3,4,5,6)IP5 that compete with (1,4,5)IP3 for binding to IP3R without evoking Ca(2+) release. These novel analogs are the first inositol phosphate-based competitive antagonists of IP3Rs with affinities comparable to that of the only commonly used competitive antagonist, heparin, the utility of which is limited by off-target effects.

  18. Reflections on inositol(s) for PCOS therapy: steps toward success.

    PubMed

    Nestler, John E; Unfer, Vittorio

    2015-07-01

    In polycystic ovary syndrome (PCOS) pathogenesis, both the insulin resistance and the related compensatory hyperinsulinemia are involved. Despite their similarities, Myo-inositol (MI) and d-chiro-inositol (DCI) play different roles in PCOS etiology and therapy. Indeed, in tissue such as the liver both molecules are involved in the insulin signaling, i.e. MI promotes glucose uptake and DCI glycogen synthesis. In reproductive tissue such as the ovary, MI regulates glucose uptake and follicle stimulating hormone (FSH) signaling, whereas DCI is devoted to the insulin-mediated androgen production. The new hypothesis on "DCI paradox" in the ovary has provided the key for a better understanding. Unlike other tissues, ovary is not insulin resistant, indeed because the epimerase enzyme, which converts MI to DCI, is insulin dependent, the "DCI paradox" hypothesis suggests that in the ovary of PCOS women, an increased epimerase activity leads to a DCI overproduction and MI depletion. This imbalance could be the cause of the poor oocyte quality and the impairment in the FSH signaling. Owing to this situation, the focal point is the administration of both MI and DCI in a proper ratio for treating PCOS. This topic, with several other "hot" issues, was the driving thread in the discussion between the two scientists.

  19. Serotonergic agonists stimulate inositol lipid metabolism in rabbit platelets

    SciTech Connect

    Schaechter, M.; Godfrey, P.P.; Minchin, M.C.W.; McClue, S.J.; Young, M.M.

    1985-10-28

    The metabolism of inositol phospholipids in response to serotonergic agonists was investigated in rabbit platelets. In platelets prelabelled with (/sup 3/H)-inositol, in a medium containing 10 mM LiCl which blocks the enzyme inositol-1-phosphatase, 5-hydroxytryptamine (5-HT) caused a dose-dependent accumulation of inositol phosphates (IP). This suggests a phospholipase-C-mediated breakdown of phosphoinositides. Ketanserin, a selective 5-HT/sub 2/ antagonist, was a potent inhibitor of the 5-HT response, with a Ki of 28 nM, indicating that 5-HT is activating receptors of the 5-HT/sub 2/ type in the platelet. Lysergic acid diethylamide (LSD) and quipazine also caused dose-related increases in inositol phosphate levels, though these were considerably less than those produced by 5-HT. These results show that relatively small changes in phosphoinositide metabolism induced by serotonergic agonists can be investigated in the rabbit platelet, and this cell may therefore be a useful model for the study of some 5-HT receptors. 30 references, 4 figures.

  20. The emerging roles of inositol pyrophosphates in eukaryotic cell physiology.

    PubMed

    Thota, Swarna Gowri; Bhandari, Rashna

    2015-09-01

    Inositol pyrophosphates are water soluble derivatives of inositol that contain pyrophosphate or diphosphate moieties in addition to monophosphates. The best characterised inositol pyrophosphates, are IP7 (diphosphoinositol pentakisphosphate or PP-IP5), and IP8 (bisdiphosphoinositol tetrakisphosphate or (PP)2-IP4). These energy-rich small molecules are present in all eukaryotic cells, from yeast to mammals, and are involved in a wide range of cellular functions including apoptosis, vesicle trafficking, DNA repair, osmoregulation, phosphate homeostasis, insulin sensitivity, immune signalling, cell cycle regulation, and ribosome synthesis. Identified more than 20 years ago, there is still only a rudimentary understanding of the mechanisms by which inositol pyrophosphates participate in these myriad pathways governing cell physiology and homeostasis. The unique stereochemical and bioenergetic properties these molecules possess as a consequence of the presence of one or two pyrophosphate moieties in the vicinity of densely packed monophosphates are likely to form the molecular basis for their participation in multiple signalling and metabolic pathways. The aim of this review is to provide first time researchers in this area with an introduction to inositol pyrophosphates and a comprehensive overview on their cellular functions.

  1. Inositol Treatment and ART Outcomes in Women with PCOS

    PubMed Central

    Garg, Deepika

    2016-01-01

    Polycystic ovary syndrome (PCOS) affects 5–10% of women in reproductive age and is characterized by oligo/amenorrhea, androgen excess, insulin resistance, and typical polycystic ovarian morphology. It is the most common cause of infertility secondary to ovulatory dysfunction. The underlying etiology is still unknown but is believed to be multifactorial. Insulin-sensitizing compounds such as inositol, a B-complex vitamin, and its stereoisomers (myo-inositol and D-chiro-inositol) have been studied as an effective treatment of PCOS. Administration of inositol in PCOS has been shown to improve not only the metabolic and hormonal parameters but also ovarian function and the response to assisted-reproductive technology (ART). Accumulating evidence suggests that it is also capable of improving folliculogenesis and embryo quality and increasing the mature oocyte yield following ovarian stimulation for ART in women with PCOS. In the current review, we collate the evidence and summarize our current knowledge on ovarian stimulation and ART outcomes following inositol treatment in women with PCOS undergoing in vitro fertilization (IVF) and/or intracytoplasmic sperm injection (ICSI). PMID:27795706

  2. Inositols affect the mating circadian rhythm of Drosophila melanogaster.

    PubMed

    Sakata, Kazuki; Kawasaki, Haruhisa; Suzuki, Takahiro; Ito, Kumpei; Negishi, Osamu; Tsuno, Takuo; Tsuno, Hiromi; Yamazaki, Youta; Ishida, Norio

    2015-01-01

    Accumulating evidence indicates that the molecular circadian clock underlies the mating behavior of Drosophila melanogaster. However, information about which food components affect circadian mating behavior is scant. The ice plant, Mesembryanthemum crystallinum has recently become a popular functional food. Here, we showed that the close-proximity (CP) rhythm of D. melanogaster courtship behavior was damped under low-nutrient conditions, but significantly enhanced by feeding the flies with powdered ice plant. Among various components of ice plants, we found that myo-inositol increased the amplitude and slightly shortened the period of the CP rhythm. Real-time reporter assays showed that myo-inositol and D-pinitol shortened the period of the circadian reporter gene Per2-luc in NIH 3T3 cells. These data suggest that the ice plant is a useful functional food and that the ability of inositols to shorten rhythms is a general phenomenon in insects as well as mammals.

  3. Inositol-Containing Lipids in Suspension-Cultured Plant Cells

    PubMed Central

    Drøbak, Bjørn K.; Ferguson, Ian B.; Dawson, Alan P.; Irvine, Robin F.

    1988-01-01

    Polar lipids were extracted from suspension-cultured tomato (Lycopersicon esculentum Mill.) cells and analyzed by thin layer chromatography. Four major inositol-containing compounds were found, and incorporation of [32P]orthosphosphate, [2-3H]glycerol, and myo-[2-3H]inositol was studied. Results showed that phosphatidylinositol-monophosphate is the phospholipid in these cells displaying the most rapid incorporation of [32P]orthophosphate. We suggest that the tracer is incorporated primarily into the phosphomonoester group. Two inositol-containing lipids showed chromatographic behavior similar to phosphatidylinositol-4,5-bisphosphate when using standard thin layer chromatography techniques. The labeling pattern of these compounds, however, reveals that it is unlikely that either of these is identical to phosphatidylinositol-4,5-bisphosphate. Should phosphatidylinositol-bisphosphate be present in suspension cultured plant cells, our data indicate chemical abundancies substantially lower than previously reported. Images Fig. 1 PMID:16666106

  4. Inositols affect the mating circadian rhythm of Drosophila melanogaster

    PubMed Central

    Sakata, Kazuki; Kawasaki, Haruhisa; Suzuki, Takahiro; Ito, Kumpei; Negishi, Osamu; Tsuno, Takuo; Tsuno, Hiromi; Yamazaki, Youta; Ishida, Norio

    2015-01-01

    Accumulating evidence indicates that the molecular circadian clock underlies the mating behavior of Drosophila melanogaster. However, information about which food components affect circadian mating behavior is scant. The ice plant, Mesembryanthemum crystallinum has recently become a popular functional food. Here, we showed that the close-proximity (CP) rhythm of D. melanogaster courtship behavior was damped under low-nutrient conditions, but significantly enhanced by feeding the flies with powdered ice plant. Among various components of ice plants, we found that myo-inositol increased the amplitude and slightly shortened the period of the CP rhythm. Real-time reporter assays showed that myo-inositol and D-pinitol shortened the period of the circadian reporter gene Per2-luc in NIH 3T3 cells. These data suggest that the ice plant is a useful functional food and that the ability of inositols to shorten rhythms is a general phenomenon in insects as well as mammals. PMID:26097456

  5. Synthesis of fagopyritols A1 and B1 from D-chiro-inositol.

    PubMed

    Cid, M Belén; Alfonso, Francisco; Martín-Lomas, Manuel

    2004-09-13

    Fagopyritol A1 (3-O-alpha-d-galactopyranosyl-d-chiro-inositol) and fagopyritol B1 (2-O-alpha-d-galactopyranosyl-d-chiro-inositol) have been synthesized by glycosylation of the diequatorial diol 1,4,5,6-tetra-O-benzoyl-d-chiro-inositol, readily obtained from d-chiro-inositol, with 2,3,4,6-tetra-O-benzyl-d-galactopyranosyl trichloroacetimidate. PMID:15337459

  6. [The role of inositol deficiency in the etiology of polycystic ovary syndrome disorders].

    PubMed

    Jakimiuk, Artur J; Szamatowicz, Jacek

    2014-01-01

    Inositol acts as a second messenger in insulin signaling pathway Literature data suggest inositol deficiency in insulin-resistant women with the polycystic ovary syndrome. Supplementation of myo-inisitol decreases insulin resistance as it works as an insulin sensitizing agent. The positive role of myo-inositol in the treatment of polycystic ovary syndrome has been of increased evidence recently The present review presents the effects of myo-inositol on the ovarian, hormonal and metabolic parameters in women with PCOS.

  7. Synthesis of fagopyritols A1 and B1 from D-chiro-inositol.

    PubMed

    Cid, M Belén; Alfonso, Francisco; Martín-Lomas, Manuel

    2004-09-13

    Fagopyritol A1 (3-O-alpha-d-galactopyranosyl-d-chiro-inositol) and fagopyritol B1 (2-O-alpha-d-galactopyranosyl-d-chiro-inositol) have been synthesized by glycosylation of the diequatorial diol 1,4,5,6-tetra-O-benzoyl-d-chiro-inositol, readily obtained from d-chiro-inositol, with 2,3,4,6-tetra-O-benzyl-d-galactopyranosyl trichloroacetimidate.

  8. A novel pathway for the synthesis of inositol phospholipids uses cytidine diphosphate (CDP)-inositol as donor of the polar head group.

    PubMed

    Jorge, Carla D; Borges, Nuno; Santos, Helena

    2015-07-01

    We describe a novel biosynthetic pathway for glycerophosphoinositides in Rhodothermus marinus in which inositol is activated by cytidine triphosphate (CTP); this is unlike all known pathways that involve activation of the lipid group instead. This work was motivated by the detection in the R. marinus genome of a gene with high similarity to CTP:L-myo-inositol-1-phosphate cytidylyltransferase, the enzyme that synthesizes cytidine diphosphate (CDP)-inositol, a metabolite only known in the synthesis of di-myo-inositol phosphate. However, this solute is absent in R. marinus. The fate of radiolabelled CDP-inositol was investigated in cell extracts to reveal that radioactive inositol was incorporated into the chloroform-soluble fraction. Mass spectrometry showed that the major lipid product has a molecular mass of 810 Da and contains inositol phosphate and alkyl chains attached to glycerol by ether bonds. The occurrence of ether-linked lipids is rare in bacteria and has not been described previously in R. marinus. The relevant synthase was identified by functional expression of the candidate gene in Escherichia coli. The enzyme catalyses the transfer of L-myo-inositol-1-phosphate from CDP-inositol to dialkylether glycerol yielding dialkylether glycerophosphoinositol. Database searching showed homologous proteins in two bacterial classes, Sphingobacteria and Alphaproteobacteria. This is the first report of the involvement of CDP-inositol in phospholipid synthesis.

  9. Inositol Hexaphosphate and Inositol Inhibit Colorectal Cancer Metastasis to the Liver in BALB/c Mice

    PubMed Central

    Fu, Min; Song, Yang; Wen, Zhaoxia; Lu, Xingyi; Cui, Lianhua

    2016-01-01

    Inositol hexaphosphate (IP6) and inositol (Ins), naturally occurring carbohydrates present in most mammals and plants, inhibit the growth of numerous cancers both in vitro and in vivo. In this study, we first examined the anti-metastatic effects of IP6 and Ins using a liver metastasis model of colorectal cancer (CRC) in BALB/c mice. CT-26 cells were injected into the splenic capsule of 48 BALB/c mice. The mice were then randomly divided into four groups: IP6, Ins, IP6 + Ins and normal saline control (n = 12 per group). IP6 and/or Ins (80 mg/kg each, 0.2 mL/day) were injected into the gastrointestinal tracts of the mice on the second day after surgery. All mice were sacrificed after 20 days, and the tumor inhibition rates were determined. The results demonstrated that the tumor weights of liver metastases and the tumor inhibition rates were reduced in the experimental groups compared to the control group and that treatment with the combination of IP6 and Ins resulted in greater inhibition of tumor growth than treatment with either compound alone. These findings suggest that IP6 and Ins prevent the development and metastatic progression of colorectal cancer to the liver in mice by altering expression of the extracellular matrix proteins collagen IV, fibronectin and laminin; the adhesion factor receptor integrin-β1; the proteolytic enzyme matrix metalloproteinase 9; and the angiogenic factors vascular endothelial growth factor, basic fibroblast growth factor, and transforming growth factor beta in the tumor metastasis microenvironment. In conclusion, IP6 and Ins inhibited the development and metastatic progression of colorectal cancer to the liver in BALB/c mice, and the effect of their combined application was significantly greater than the effect of either compound alone. This evidence supports further testing of the combined application of IP6 and Ins for the prevention of colorectal cancer metastasis to the liver in clinical studies. PMID:27187454

  10. The rationale of the myo-inositol and D-chiro-inositol combined treatment for polycystic ovary syndrome.

    PubMed

    Dinicola, Simona; Chiu, Tony T Y; Unfer, Vittorio; Carlomagno, Gianfranco; Bizzarri, Mariano

    2014-10-01

    PCOS is one of the most common endocrine disorders affecting women and it is characterized by a combination of hyper-androgenism, chronic anovulation, and insulin resistance. While a significant progress has recently been made in the diagnosis for PCOS, the optimal infertility treatment remains to be determined. Two inositol isomers, myo-inositol (MI) and D-chiro-inositol (DCI) have been proven to be effective in PCOS treatment, by improving insulin resistance, serum androgen levels and many features of the metabolic syndrome. However, DCI alone, mostly when it is administered at high dosage, negatively affects oocyte quality, whereas the association MI/DCI, in a combination reproducing the plasma physiological ratio (40:1), represents a promising alternative in achieving better clinical results, by counteracting PCOS at both systemic and ovary level.

  11. Functional properties of Drosophila inositol trisphosphate receptors.

    PubMed Central

    Swatton, J E; Morris, S A; Wissing, F; Taylor, C W

    2001-01-01

    The functional properties of the only inositol trisphosphate (IP(3)) receptor subtype expressed in Drosophila were examined in permeabilized S2 cells. The IP(3) receptors of S2 cells bound (1,4,5)IP(3) with high affinity (K(d)=8.5+/-1.1 nM), mediated positively co-operative Ca(2+) release from a thapsigargin-sensitive Ca(2+) store (EC(50)=75+/-4 nM, Hill coefficient=2.1+/-0.2), and they were recognized by an antiserum to a peptide conserved in all IP(3) receptor subtypes in the same way as mammalian IP(3) receptors. As with mammalian IP(3) receptors, (2,4,5)IP(3) (EC(50)=2.3+/-0.3 microM) and (4,5)IP(2) (EC(50) approx. 10 microM) were approx. 20- and 100-fold less potent than (1,4,5)IP(3). Adenophostin A, which is typically approx. 10-fold more potent than IP(3) at mammalian IP(3) receptors, was 46-fold more potent than IP(3) in S2 cells (EC(50)=1.67+/-0.07 nM). Responses to submaximal concentrations of IP(3) were quantal and IP(3)-evoked Ca(2+) release was biphasically regulated by cytosolic Ca(2+). Using rapid superfusion to examine the kinetics of IP(3)-evoked Ca(2+) release from S2 cells, we established that IP(3) (10 microM) maximally activated Drosophila IP(3) receptors within 400 ms. The activity of the receptors then slowly decayed (t(1/2)=2.03+/-0.07 s) to a stable state which had 47+/-1% of the activity of the maximally active state. We conclude that the single subtype of IP(3) receptor expressed in Drosophila has similar functional properties to mammalian IP(3) receptors and that analyses of IP(3) receptor function in this genetically tractable organism are therefore likely to contribute to understanding the roles of mammalian IP(3) receptors. PMID:11583592

  12. Using inositol as a biocompatible ligand for efficient transgene expression

    PubMed Central

    Zhang, Lei; Bellis, Susan L; Fan, Yiwen; Wu, Yunkun

    2015-01-01

    Transgene transfection techniques using cationic polymers such as polyethylenimines (PEIs) and PEI derivatives as gene vectors have shown efficacy, although they also have shortcomings. PEIs have decent DNA-binding capability and good cell internalization performance, but they cannot deliver gene payloads very efficiently to cell nuclei. In this study, three hyperbranched polyglycerol-polyethylenimine (PG6-PEI) polymers conjugated with myo-inositol (INO) molecules were developed. The three resulting PG6-PEI-INO polymers have an increased number of INO ligands per molecule. PG6-PEI-INO 1 had only 14 carboxymethyl INO (CMINO) units per molecule. PG6-PEI-INO 2 had approximately 130 CMINO units per molecule. PG6-PEI-INO 3 had as high as 415 CMINO units approximately. Mixing PG6-PEI-INO polymers with DNA produced compact nanocomposites. We then performed localization studies using fluorescent microscopy. As the number of conjugated inositol ligands increased in PG6-PEI-INO polymers, there was a corresponding increase in accumulation of the polymers within 293T cell nuclei. Transfection performed with spherical 293T cells yielded 82% of EGFP-positive cells when using PG6-PEI-INO 3 as the vehicle. Studies further revealed that extracellular adenosine triphosphate (eATP) can inhibit the transgene efficiency of PG6-PEI-INO polymers, as compared with PEI and PG6-PEI that were not conjugated with inositol. Our work unveiled the possibility of using inositol as an effective ligand for transgene expression. PMID:25926732

  13. Using inositol as a biocompatible ligand for efficient transgene expression.

    PubMed

    Zhang, Lei; Bellis, Susan L; Fan, Yiwen; Wu, Yunkun

    2015-01-01

    Transgene transfection techniques using cationic polymers such as polyethylenimines (PEIs) and PEI derivatives as gene vectors have shown efficacy, although they also have shortcomings. PEIs have decent DNA-binding capability and good cell internalization performance, but they cannot deliver gene payloads very efficiently to cell nuclei. In this study, three hyperbranched polyglycerol-polyethylenimine (PG6-PEI) polymers conjugated with myo-inositol (INO) molecules were developed. The three resulting PG6-PEI-INO polymers have an increased number of INO ligands per molecule. PG6-PEI-INO 1 had only 14 carboxymethyl INO (CMINO) units per molecule. PG6-PEI-INO 2 had approximately 130 CMINO units per molecule. PG6-PEI-INO 3 had as high as 415 CMINO units approximately. Mixing PG6-PEI-INO polymers with DNA produced compact nanocomposites. We then performed localization studies using fluorescent microscopy. As the number of conjugated inositol ligands increased in PG6-PEI-INO polymers, there was a corresponding increase in accumulation of the polymers within 293T cell nuclei. Transfection performed with spherical 293T cells yielded 82% of EGFP-positive cells when using PG6-PEI-INO 3 as the vehicle. Studies further revealed that extracellular adenosine triphosphate (eATP) can inhibit the transgene efficiency of PG6-PEI-INO polymers, as compared with PEI and PG6-PEI that were not conjugated with inositol. Our work unveiled the possibility of using inositol as an effective ligand for transgene expression.

  14. Lithium and valproate decrease inositol mass and increase expression of the yeast INO1 and INO2 genes for inositol biosynthesis.

    PubMed

    Vaden, D L; Ding, D; Peterson, B; Greenberg, M L

    2001-05-01

    Bipolar affective disorder (manic-depressive illness) is a chronic, severe, debilitating illness affecting 1-2% of the population. The Food and Drug Administration-approved drugs lithium and valproate are not completely effective in the treatment of this disorder, and the mechanisms underlying their therapeutic effects have not been established. We are employing genetic and molecular approaches to identify common targets of lithium and valproate in the yeast Saccharomyces cerevisiae. We show that both drugs affect molecular targets in the inositol metabolic pathway. Lithium and valproate cause a decrease in intracellular myo-inositol mass and an increase in expression of both a structural (INO1) and a regulatory (INO2) gene required for inositol biosynthesis. The opi1 mutant, which exhibits constitutive expression of INO1, is more resistant to inhibition of growth by lithium but not by valproate, suggesting that valproate may inhibit the Ino1p-catalyzed synthesis of inositol 1-phosphate. Consistent with this possibility, growth in valproate leads to decreased synthesis of inositol monophosphate. Thus, both lithium and valproate perturb regulation of the inositol biosynthetic pathway, albeit via different mechanisms. This is the first demonstration of increased expression of genes in the inositol biosynthetic pathway by both lithium and valproate. Because inositol is a key regulator of many cellular processes, the effects of lithium and valproate on inositol synthesis have far-reaching implications for predicting genetic determinants of responsiveness and resistance to these agents. PMID:11278273

  15. Comparison between effects of myo-inositol and D-chiro-inositol on ovarian function and metabolic factors in women with PCOS.

    PubMed

    Pizzo, Alfonsa; Laganà, Antonio Simone; Barbaro, Luisa

    2014-03-01

    Myo-inositol and D-chiro-inositol are capable of improving the ovarian function and metabolism of polycystic ovary syndrome (PCOS) patients. The aim of this work is to compare the effects of myo-inositol and D-chiro-inositol in PCOS. We enrolled 50 patients, with homogeneous bio-physical features, affected by PCOS and menstrual irregularities, and we randomly divided them into two groups: 25 were treated with 4 g of myo-inositol/die plus 400 mcg of folic acid/die orally for six months, 25 with 1 g of D-chiro-inositol/die plus 400 mcg of folic acid/die orally for six months. We analyzed in both groups pre-treatment and post-treatment BMI, systolic and diastolic blood pressure, Ferriman-Gallwey score, Cremoncini score, serum LH, LH/FSH ratio, total and free testosterone, dehydroepiandrosterone sulfate (DHEA-S), Δ-4-androstenedione, SHBG, prolactin, glucose/immunoreactive insulin (IRI) ratio, homeostatic model assessment (HOMA) index, and the resumption of regular menstrual cycles. Both the isoforms of inositol were effective in improving ovarian function and metabolism in patients with PCOS, although myo-inositol showed the most marked effect on the metabolic profile, whereas D-chiro-inositol reduced hyperandrogenism better.

  16. Myo-inositol oxygenase is important for the removal of excess myo-inositol from syncytia induced by Heterodera schachtii in Arabidopsis roots.

    PubMed

    Siddique, Shahid; Endres, Stefanie; Sobczak, Miroslaw; Radakovic, Zoran S; Fragner, Lena; Grundler, Florian M W; Weckwerth, Wolfram; Tenhaken, Raimund; Bohlmann, Holger

    2014-01-01

    The enzyme myo-inositol oxygenase is the key enzyme of a pathway leading from myo-inositol to UDP-glucuronic acid. In Arabidopsis, myo-inositol oxygenase is encoded by four genes. All genes are strongly expressed in syncytia induced by the beet cyst nematode Heterodera schachtii in Arabidopsis roots. Here, we studied the effect of a quadruple myo-inositol oxygenase mutant on nematode development. We performed metabolite profiling of syncytia induced in roots of the myo-inositol oxygenase quadruple mutant. The role of galactinol in syncytia was studied using Arabidopsis lines with elevated galactinol levels and by supplying galactinol to wild-type seedlings. The quadruple myo-inositol oxygenase mutant showed a significant reduction in susceptibility to H. schachtii, and syncytia had elevated myo-inositol and galactinol levels and an elevated expression level of the antimicrobial thionin gene Thi2.1. This reduction in susceptibility could also be achieved by exogenous application of galactinol to wild-type seedlings. The primary function of myo-inositol oxygenase for syncytium development is probably not the production of UDP-glucuronic acid as a precursor for cell wall polysaccharides, but the reduction of myo-inositol levels and thereby a reduction in the galactinol level to avoid the induction of defence-related genes.

  17. Myo-inositol oxygenase is important for the removal of excess myo-inositol from syncytia induced by Heterodera schachtii in Arabidopsis roots.

    PubMed

    Siddique, Shahid; Endres, Stefanie; Sobczak, Miroslaw; Radakovic, Zoran S; Fragner, Lena; Grundler, Florian M W; Weckwerth, Wolfram; Tenhaken, Raimund; Bohlmann, Holger

    2014-01-01

    The enzyme myo-inositol oxygenase is the key enzyme of a pathway leading from myo-inositol to UDP-glucuronic acid. In Arabidopsis, myo-inositol oxygenase is encoded by four genes. All genes are strongly expressed in syncytia induced by the beet cyst nematode Heterodera schachtii in Arabidopsis roots. Here, we studied the effect of a quadruple myo-inositol oxygenase mutant on nematode development. We performed metabolite profiling of syncytia induced in roots of the myo-inositol oxygenase quadruple mutant. The role of galactinol in syncytia was studied using Arabidopsis lines with elevated galactinol levels and by supplying galactinol to wild-type seedlings. The quadruple myo-inositol oxygenase mutant showed a significant reduction in susceptibility to H. schachtii, and syncytia had elevated myo-inositol and galactinol levels and an elevated expression level of the antimicrobial thionin gene Thi2.1. This reduction in susceptibility could also be achieved by exogenous application of galactinol to wild-type seedlings. The primary function of myo-inositol oxygenase for syncytium development is probably not the production of UDP-glucuronic acid as a precursor for cell wall polysaccharides, but the reduction of myo-inositol levels and thereby a reduction in the galactinol level to avoid the induction of defence-related genes. PMID:24117492

  18. Gonadotropin releasing hormone stimulates the formation of inositol phosphates in rat anterior pituitary tissue.

    PubMed Central

    Schrey, M P

    1985-01-01

    The production of inositol phosphates in response to gonadotropin releasing hormone (GnRH) was studied in rat anterior pituitary tissue preincubated with [3H]inositol. Prelabelled paired hemipituitaries from prepubertal female rats were incubated in the presence or absence of GnRH in medium containing 10 mM-Li+ X Li+, which inhibits myo-inositol-1-phosphatase, greatly amplified the stimulation of inositol phosphate production by GnRH (10(-7) M) to 159, 198 and 313% of paired control values for inositol 1-phosphate, inositol bisphosphate and inositol trisphosphate respectively after 20 min. The percentage distribution of [3H]inositol within the phosphoinositides was 91.3, 6.3 and 2.4 for phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate respectively and was unaffected by GnRH. The stimulation of inositol trisphosphate production by GnRH was evident after 5 min incubation, was dose-dependent with a half-maximal effect around 11 nM, and was not inhibited by removal of extracellular Ca2+. Elevation of cytosolic Ca2+ by membrane depolarization with 50 mM-K+ had no significant effect on inositol phosphate production. These findings are consistent with the hypothesis that GnRH action in the anterior pituitary involves the hydrolysis of phosphatidylinositol 4,5-bisphosphate. The resulting elevation of inositol trisphosphate may in turn lead to intracellular Ca2+ mobilization and subsequent stimulation of gonadotropin secretion. PMID:2986599

  19. Perturbation of the Vacuolar ATPase: A NOVEL CONSEQUENCE OF INOSITOL DEPLETION.

    PubMed

    Deranieh, Rania M; Shi, Yihui; Tarsio, Maureen; Chen, Yan; McCaffery, J Michael; Kane, Patricia M; Greenberg, Miriam L

    2015-11-13

    Depletion of inositol has profound effects on cell function and has been implicated in the therapeutic effects of drugs used to treat epilepsy and bipolar disorder. We have previously shown that the anticonvulsant drug valproate (VPA) depletes inositol by inhibiting myo-inositol-3-phosphate synthase, the enzyme that catalyzes the first and rate-limiting step of inositol biosynthesis. To elucidate the cellular consequences of inositol depletion, we screened the yeast deletion collection for VPA-sensitive mutants and identified mutants in vacuolar sorting and the vacuolar ATPase (V-ATPase). Inositol depletion caused by starvation of ino1Δ cells perturbed the vacuolar structure and decreased V-ATPase activity and proton pumping in isolated vacuolar vesicles. VPA compromised the dynamics of phosphatidylinositol 3,5-bisphosphate (PI3,5P2) and greatly reduced V-ATPase proton transport in inositol-deprived wild-type cells. Osmotic stress, known to increase PI3,5P2 levels, did not restore PI3,5P2 homeostasis nor did it induce vacuolar fragmentation in VPA-treated cells, suggesting that perturbation of the V-ATPase is a consequence of altered PI3,5P2 homeostasis under inositol-limiting conditions. This study is the first to demonstrate that inositol depletion caused by starvation of an inositol synthesis mutant or by the inositol-depleting drug VPA leads to perturbation of the V-ATPase.

  20. Updates on the myo-inositol plus D-chiro-inositol combined therapy in polycystic ovary syndrome.

    PubMed

    Unfer, Vittorio; Porcaro, Giuseppina

    2014-09-01

    Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders affecting women of reproductive age. It is characterized by chronic anovulation, hyperandrogenism, and insulin resistance. It is the main cause of infertility due to the menstrual dysfunction and metabolic disorders. Women with PCOS also have an increased cardiovascular risk because of dyslipidemia and insulin resistance. So far, we have a lot of information about the etiology of PCOS, and many steps forward have been made about the diagnosis of this syndrome, but there is still no certainty about the therapy. Myo-inositol (MI) and D-chiro-inositol, two inositol stereoisomers, have been proven to be effective in PCOS treatment. However, only MI has been shown to have beneficial effects on reproductive function, whereas the administration of MI/D-chiro-inositol, in the physiological plasma ratio (i.e., 40:1) ensures better clinical results, such as the reduction of insulin resistance, androgens' blood levels, cardiovascular risk and regularization of menstrual cycle with spontaneous ovulation.

  1. Inositol-phosphodihydroceramides in the periodontal pathogen Tannerella forsythia: Structural analysis and incorporation of exogenous myo-inositol

    PubMed Central

    Megson, Zoë Anne; Pittenauer, Ernst; Duda, Katarzyna Anna; Engel, Regina; Ortmayr, Karin; Koellensperger, Gunda; Mach, Lukas; Allmaier, Günter; Holst, Otto; Messner, Paul; Schäffer, Christina

    2015-01-01

    Background Unique phosphodihydroceramides containing phosphoethanolamine and glycerol have been previously described in Porphyromonas gingivalis. Importantly, they were shown to possess pro-inflammatory properties. Other common human bacteria were screened for the presence of these lipids, and they were found, amongst others, in the oral pathogen Tannerella forsythia. To date, no detailed study into the lipids of this organism has been performed. Methods Lipids were extracted, separated and purified by HPTLC, and analyzed using GC-MS, ESI–MS and NMR. Of special interest was how T. forsythia acquires the metabolic precursors for the lipids studied here. This was assayed by radioactive and stable isotope incorporation using carbon-14 and deuterium labeled myo-inositol, added to the growth medium. Results T. forsythia synthesizes two phosphodihydroceramides (Tf GL1, Tf GL2) which are constituted by phospho-myo-inositol linked to either a 17-, 18-, or 19-carbon sphinganine, N-linked to either a branched 17:0(3-OH) or a linear 16:0(3-OH) fatty acid which, in Tf GL2, is, in turn, ester-substituted with a branched 15:0 fatty acid. T. forsythia lacks the enzymatic machinery required for myo-inositol synthesis but was found to internalize inositol from the medium for the synthesis of both Tf GL1 and Tf GL2. Conclusion The study describes two novel glycolipids in T. forsythia which could be essential in this organism. Their synthesis could be reliant on an external source of myo-inositol. General significance The effects of these unique lipids on the immune system and their role in bacterial virulence could be relevant in the search for new drug targets. PMID:26277409

  2. Potential role and therapeutic interests of myo-inositol in metabolic diseases.

    PubMed

    Croze, Marine L; Soulage, Christophe O

    2013-10-01

    Several inositol isomers and in particular myo-inositol (MI) and D-chiro-inositol (DCI), were shown to possess insulin-mimetic properties and to be efficient in lowering post-prandial blood glucose. In addition, abnormalities in inositol metabolism are associated with insulin resistance and with long term microvascular complications of diabetes, supporting a role of inositol or its derivatives in glucose metabolism. The aim of this review is to focus on the potential benefits of a dietary supplement of myo-inositol, by far the most common inositol isomer in foodstuffs, in human disorders associated with insulin resistance (polycystic ovary syndrome, gestational diabetes mellitus or metabolic syndrome) or in prevention or treatment of some diabetic complications (neuropathy, nephropathy, cataract). The relevance of such a nutritional strategy will be discussed for each context on the basis of the clinical and/or animal studies. The dietary sources of myo-inositol and its metabolism from its dietary uptake to its renal excretion will be also covered in this review. Finally, the actual insights into inositol insulin-sensitizing effects will be addressed and in particular the possible role of inositol glycans as insulin second messengers. PMID:23764390

  3. Potential role and therapeutic interests of myo-inositol in metabolic diseases.

    PubMed

    Croze, Marine L; Soulage, Christophe O

    2013-10-01

    Several inositol isomers and in particular myo-inositol (MI) and D-chiro-inositol (DCI), were shown to possess insulin-mimetic properties and to be efficient in lowering post-prandial blood glucose. In addition, abnormalities in inositol metabolism are associated with insulin resistance and with long term microvascular complications of diabetes, supporting a role of inositol or its derivatives in glucose metabolism. The aim of this review is to focus on the potential benefits of a dietary supplement of myo-inositol, by far the most common inositol isomer in foodstuffs, in human disorders associated with insulin resistance (polycystic ovary syndrome, gestational diabetes mellitus or metabolic syndrome) or in prevention or treatment of some diabetic complications (neuropathy, nephropathy, cataract). The relevance of such a nutritional strategy will be discussed for each context on the basis of the clinical and/or animal studies. The dietary sources of myo-inositol and its metabolism from its dietary uptake to its renal excretion will be also covered in this review. Finally, the actual insights into inositol insulin-sensitizing effects will be addressed and in particular the possible role of inositol glycans as insulin second messengers.

  4. Metabolism of myo-[2-3H]Inositol and scyllo-[R-3H]Inositol in Ripening Wheat Kernels 1

    PubMed Central

    Sasaki, Ken; Loewus, Frank A.

    1980-01-01

    Injection of myo-[2-3H]inositol or scyllo-[R-3H]inositol into the peduncular cavity of wheat stalks about 2 to 4 weeks postanthesis led to rapid translocation into the spike and accumulation of label in developing kernels, especially the bran fraction. With myo-[2-3H]inositol, about 50 to 60% of the label was incorporated into high molecular weight cell wall substance in the region of the injection. That portion translocated to the kernels was utilized primarily for cell wall polysaccharide formation and phytate biosynthesis. A small amount was recovered as free myo-inositol and galactinol. When scyllo-[R-3H]inositol was supplied, most of the label was translocated into the developing kernels where it accumulated as free scyllo-inositol and O-α-d-galactopyranosyl-scyllo-inositol in approximately equal amount. None of the label from scyllo-[R-3H]inositol was utilized for either phytate biosynthesis or cell wall polysaccharide formation. PMID:16661513

  5. Decreased myo-inositol to chiro-inositol (M/C) ratios and increased M/C epimerase activity in PCOS theca cells demonstrate increased insulin sensitivity compared to controls.

    PubMed

    Heimark, Douglas; McAllister, Jan; Larner, Joseph

    2014-01-01

    Previous studies from our and other labs have shown that insulin resistance is associated with an inositol imbalance of excess myo-inositol and deficient chiro-inositol together with a deficiency of myo-inositol to chiro-inositol epimerase in vivo and in vitro. In this report, we utilized well characterized theca cells from normal cycling women, with normal insulin sensitivity, and theca cells from women with polycystic ovary syndrome (PCOS), with increased insulin sensitivity to examine the myo-inositol to chiro-inisitol (M/C) ratio and the myo-inositol to chiro-inositol epimerase activity. PCOS theca cells with increased insulin sensitivity were specifically used to investigate whether the inositol imbalance and myo-inositol to chiro-inositol epimerase are regulated in a similar or the opposite direction than that observed in insulin resistant cells. The results of these studies are the first to demonstrate that in insulin sensitive PCOS theca cells the inositol imbalance goes in the opposite direction to that observed in insulin resistant cells, and there is a decreased M/C ratio and an increased myo-inositol to chiro-inositol epimerase activity. Further biochemical and genetic studies will probe the mechanisms involved.

  6. Crystal Structures of Type-II Inositol Polyphosphate 5-Phosphatase INPP5B with Synthetic Inositol Polyphosphate Surrogates Reveal New Mechanistic Insights for the Inositol 5-Phosphatase Family

    PubMed Central

    2016-01-01

    The inositol polyphosphate 5-phosphatase INPP5B hydrolyzes the 5-phosphate group from water- and lipid-soluble signaling messengers. Two synthetic benzene and biphenyl polyphosphates (BzP/BiPhPs), simplified surrogates of inositol phosphates and phospholipid headgroups, were identified by thermodynamic studies as potent INPP5B ligands. The X-ray structure of the complex between INPP5B and biphenyl 3,3′,4,4′,5,5′-hexakisphosphate [BiPh(3,3′,4,4′,5,5′)P6, IC50 5.5 μM] was determined at 2.89 Å resolution. One inhibitor pole locates in the phospholipid headgroup binding site and the second solvent-exposed ring binds to the His-Tag of another INPP5B molecule, while a molecule of inorganic phosphate is also present in the active site. Benzene 1,2,3-trisphosphate [Bz(1,2,3)P3] [one ring of BiPh(3,3′,4,4′,5,5′)P6] inhibits INPP5B ca. 6-fold less potently. Co-crystallization with benzene 1,2,4,5-tetrakisphosphate [Bz(1,2,4,5)P4, IC50 = 6.3 μM] yielded a structure refined at 2.9 Å resolution. Conserved residues among the 5-phosphatase family mediate interactions with Bz(1,2,4,5)P4 and BiPh(3,3′,4,4′,5,5′)P6 similar to those with the polar groups present in positions 1, 4, 5, and 6 on the inositol ring of the substrate. 5-Phosphatase specificity most likely resides in the variable zone located close to the 2- and 3-positions of the inositol ring, offering insights to inhibitor design. We propose that the inorganic phosphate present in the INPP5B–BiPh(3,3′,4,4′,5,5′)P6 complex mimics the postcleavage substrate 5-phosphate released by INPP5B in the catalytic site, allowing elucidation of two new key features in the catalytic mechanism proposed for the family of phosphoinositide 5-phosphatases: first, the involvement of the conserved Arg-451 in the interaction with the 5-phosphate and second, identification of the water molecule that initiates 5-phosphate hydrolysis. Our model also has implications for the proposed “moving metal” mechanism

  7. Reduced parasitemia observed with erythrocytes containing inositol hexaphosphate.

    PubMed Central

    Mintzer, C L; Deloron, P; Rice-Ficht, A; Durica, D; Struck, D K; Roessner, C A; Nicolau, C; Ihler, G M

    1988-01-01

    Chemicals entrapped in erythrocytes by hypotonic hemolysis can be assessed for possible antiparasitic activity both in vivo and in vitro, regardless of whether they are able to diffuse into erythrocytes readily. Inositol hexaphosphate, a highly charged compound, produced a dramatic lowering of the percentage of cells infected by Babesia microti in vivo and both B. microti and Plasmodium falciparum in vitro. Several possible mechanisms for this observation are discussed. PMID:3364957

  8. Urinary Chiro- and Myo-Inositol Levels as a Biological Marker for Type 2 Diabetes Mellitus

    PubMed Central

    Hong, Jun Hwa; Jang, Hye Won; Kang, Yea Eun; Lee, Ju Hee; Kim, Koon Soon; Kim, Hyun Jin; Park, Kyu Ri; Ku, Bon Jeong

    2012-01-01

    Background: The aim of this study was to investigate the role of the urinary chiro- and myo-inositol levels in predicting type 2 diabetes mellitus (T2DM). Subjects and methods: A total of 212 normal controls and 101 type 2 diabetic patients were enrolled this study. The concentrations of urinary chiro- and myo-inositol were measured by high performance liquid chromatography/mass spectrometry. Results: The concentration of urinary chiro-inositol was significantly higher in the diabetic subjects (2.24 ± 5.18 ng/L) than those in the control group (0.38 ± 0.62 ng/L; p < 0.001). The urinary myo-inositol level of the diabetic subjects (36.95 ± 37.77 ng/L) was also significantly higher than that of the controls (8.17 ± 13.29 ng/L; p < 0.001). The urinary chiro-inositol multiplied by myo-inositol level of the diabetic subjects (148.10 ± 544.91) was significantly higher than in the controls (5.12 ± 24.15; p < 0.001). The area under the receiver operating characteristic curve for the urinary chiro-inositol multiplied by myo-inositol level to predict T2DM was 0.840 (confidence interval 0.789–0.891, p < 0.001). The cut-off value for the urinary chiro-inositol multiplied by myo-inositol level to predict T2DM was 2.20 (sensitivity 81.3%, specificity 70.3%). Conclusions: The urinary chiro- and myo-inositol concentrations were increased in the type 2 diabetic patients and the urinary chiro- times the myo-inositol was considered to be a sufficient marker in predicting T2DM. PMID:22960342

  9. The antidepressant activity of inositol in the forced swim test involves 5-HT(2) receptors.

    PubMed

    Einat, H; Clenet, F; Shaldubina, A; Belmaker, R H; Bourin, M

    2001-01-01

    The effect of inositol as an antidepressant was previously demonstrated in both animal models of depression-like behavior and in clinical trials. Unlike most antidepressant drugs, inositol does not have a clear target in the synapse and was not demonstrated to alter monoamine levels in the brain. The present study attempted to draw a psychopharmacological profile of inositol's behavioral effects by exploring the interactions between the drug and specific receptor agonists and antagonists in the forced swim test. Rats received inositol treatment (or control) in combination with the serotonergic metabolism inhibitor PCPA or with the noradrenergic neurotoxin DSP-4. Results indicated that PCPA but not DSP-4 abolished the ability of inositol to cause a reduction in immobility time in the forced swim test. In mice, the specific 5-HT(2A)/5-HT(2C) antagonist ritanserin, but not the 5-HT(1A)/5-HT(1B)/beta adrenergic antagonist pindolol, abolished inositol's effect in the forced swim test. The 5-HT(2A)/5-HT(2C) agonist DOI and the 5-HT(1A) agonist 8-OH-DPAT did not have any significant effects on inositol's activity. The present data indicates that the antidepressant effect of inositol may involve 5-HT(2) receptors. It is thus possible that the effects of reuptake antidepressant drugs and the effects of inositol may have a common final pathway.

  10. Regulation of cell-specific inositol metabolism and transport in plant salinity tolerance.

    PubMed Central

    Nelson, D E; Rammesmayer, G; Bohnert, H J

    1998-01-01

    myo-Inositol and its derivatives are commonly studied with respect to cell signaling and membrane biogenesis, but they also participate in responses to salinity in animals and plants. In this study, we focused on L-myo-inositol 1-phosphate synthase (INPS), which commits carbon to de novo synthesis, and myo-inositol O-methyltransferase (IMT), which uses myo-inositol for stress-induced accumulation of a methylinositol, D-ononitol. The Imt and Inps promoters are transcriptionally controlled. We determined that the transcription rates, transcript levels, and protein abundance are correlated. During normal growth, INPS is present in all cells, but IMT is repressed. After salinity stress, the amount of INPS was enhanced in leaves but repressed in roots. IMT was induced in all cell types. The absence of myo-inositol synthesis in roots is compensated by inositol/ononitol transport in the phloem. The mobilization of photosynthate through myo-inositol translocation links root metabolism to photosynthesis. Our model integrates the transcriptional control of a specialized metabolic pathway with physiological reactions in different tissues. The tissue-specific differential regulation of INPS, which leads to a gradient of myo-inositol synthesis, supports root growth and sodium uptake. By inducing expression of IMT and increasing myo-inositol synthesis, metabolic end products accumulate, facilitating sodium sequestration and protecting photosynthesis. PMID:9596634

  11. Results from the International Consensus Conference on Myo-inositol and d-chiro-inositol in Obstetrics and Gynecology: the link between metabolic syndrome and PCOS.

    PubMed

    Facchinetti, Fabio; Bizzarri, Mariano; Benvenga, Salvatore; D'Anna, Rosario; Lanzone, Antonio; Soulage, Christophe; Di Renzo, Gian Carlo; Hod, Moshe; Cavalli, Pietro; Chiu, Tony T; Kamenov, Zdravko A; Bevilacqua, Arturo; Carlomagno, Gianfranco; Gerli, Sandro; Oliva, Mario Montanino; Devroey, Paul

    2015-12-01

    In recent years, interest has been focused to the study of the two major inositol stereoisomers: myo-inositol (MI) and d-chiro-inositol (DCI), because of their involvement, as second messengers of insulin, in several insulin-dependent processes, such as metabolic syndrome and polycystic ovary syndrome. Although these molecules have different functions, very often their roles have been confused, while the meaning of several observations still needs to be interpreted under a more rigorous physiological framework. With the aim of clarifying this issue, the 2013 International Consensus Conference on MI and DCI in Obstetrics and Gynecology identified opinion leaders in all fields related to this area of research. They examined seminal experimental papers and randomized clinical trials reporting the role and the use of inositol(s) in clinical practice. The main topics were the relation between inositol(s) and metabolic syndrome, polycystic ovary syndrome (with a focus on both metabolic and reproductive aspects), congenital anomalies, gestational diabetes. Clinical trials demonstrated that inositol(s) supplementation could fruitfully affect different pathophysiological aspects of disorders pertaining Obstetrics and Gynecology. The treatment of PCOS women as well as the prevention of GDM seem those clinical conditions which take more advantages from MI supplementation, when used at a dose of 2g twice/day. The clinical experience with MI is largely superior to the one with DCI. However, the existence of tissue-specific ratios, namely in the ovary, has prompted researchers to recently develop a treatment based on both molecules in the proportion of 40 (MI) to 1 (DCI).

  12. Results from the International Consensus Conference on Myo-inositol and d-chiro-inositol in Obstetrics and Gynecology: the link between metabolic syndrome and PCOS.

    PubMed

    Facchinetti, Fabio; Bizzarri, Mariano; Benvenga, Salvatore; D'Anna, Rosario; Lanzone, Antonio; Soulage, Christophe; Di Renzo, Gian Carlo; Hod, Moshe; Cavalli, Pietro; Chiu, Tony T; Kamenov, Zdravko A; Bevilacqua, Arturo; Carlomagno, Gianfranco; Gerli, Sandro; Oliva, Mario Montanino; Devroey, Paul

    2015-12-01

    In recent years, interest has been focused to the study of the two major inositol stereoisomers: myo-inositol (MI) and d-chiro-inositol (DCI), because of their involvement, as second messengers of insulin, in several insulin-dependent processes, such as metabolic syndrome and polycystic ovary syndrome. Although these molecules have different functions, very often their roles have been confused, while the meaning of several observations still needs to be interpreted under a more rigorous physiological framework. With the aim of clarifying this issue, the 2013 International Consensus Conference on MI and DCI in Obstetrics and Gynecology identified opinion leaders in all fields related to this area of research. They examined seminal experimental papers and randomized clinical trials reporting the role and the use of inositol(s) in clinical practice. The main topics were the relation between inositol(s) and metabolic syndrome, polycystic ovary syndrome (with a focus on both metabolic and reproductive aspects), congenital anomalies, gestational diabetes. Clinical trials demonstrated that inositol(s) supplementation could fruitfully affect different pathophysiological aspects of disorders pertaining Obstetrics and Gynecology. The treatment of PCOS women as well as the prevention of GDM seem those clinical conditions which take more advantages from MI supplementation, when used at a dose of 2g twice/day. The clinical experience with MI is largely superior to the one with DCI. However, the existence of tissue-specific ratios, namely in the ovary, has prompted researchers to recently develop a treatment based on both molecules in the proportion of 40 (MI) to 1 (DCI). PMID:26479434

  13. Source of /sup 3/H-labeled inositol bis- and monophosphates in agonist-activated rat parotid acinar cells

    SciTech Connect

    Hughes, A.R.; Putney, J.W. Jr.

    1989-06-05

    The kinetics of (3H)inositol phosphate metabolism in agonist-activated rat parotid acinar cells were characterized in order to determine the sources of (3H)inositol monophosphates and (3H)inositol bisphosphates. The turnover rates of D-myo-inositol 1,4,5-trisphosphate and its metabolites, D-myo-inositol 1,4-bisphosphate and D-myo-inositol 1,3,4-trisphosphate, were examined following the addition of the muscarinic receptor antagonist, atropine, to cholinergically stimulated parotid cells. D-myo-Inositol 1,4,5-trisphosphate declined with a t1/2 of 7.6 +/- 0.7 s, D-myo-inositol 1,3,4-trisphosphate declined with a t1/2 of 8.6 +/- 1.2 min, and D-myo-inositol 1,4-bisphosphate was metabolized with a t1/2 of 6.0 +/- 0.7 min. The sum of the rates of flux through D-myo-inositol 1,4-bisphosphate and D-myo-inositol 1,3,4-trisphosphate (2.54% phosphatidylinositol/min) did not exceed the calculated rate of breakdown of D-myo-inositol 1,4,5-trisphosphate (2.76% phosphatidylinositol/min). Thus, there is no evidence for the direct hydrolysis of phosphatidylinositol 4-phosphate in intact cells since D-myo-inositol 1,4-bisphosphate formation can be attributed to the dephosphorylation of D-myo-inositol 1,4,5-trisphosphate. The source of the (3H)inositol monophosphates also was examined in cholinergically stimulated parotid cells. When parotid cells were stimulated with methacholine, D-myo-inositol 1,4,5-trisphosphate, D-myo-inositol 1,3,4,5-tetrakisphosphate, D-myo-inositol 1,4-bisphosphate, and D-myo-inositol 4-monophosphate levels increased within 2 s, whereas D-myo-inositol 1-monophosphate accumulation was delayed by several seconds. Rates of (3H)inositol monophosphate accumulation also were examined by the addition of LiCl to cells stimulated to steady state levels of (3H)inositol phosphates.

  14. GIPC: Glycosyl Inositol Phospho Ceramides, the major sphingolipids on earth

    PubMed Central

    Gronnier, Julien; Germain, Véronique; Gouguet, Paul; Cacas, Jean-Luc; Mongrand, Sébastien

    2016-01-01

    ABSTRACT What are the most abundant sphingolipids on earth? The answer is Glycosyl Inositol Phosphoryl Ceramides (GIPCs) present in fungi and the green lineage. In this review, we discuss the putative role of plant GIPCs in the lipid bilayer asymmetry, in the lateral organization of membrane rafts and in the very long chain fatty acid inter-leaflet coupling of lipids in the plant plasma membrane (PM). A special focus on the structural similarities -and putative functions- of GIPCs is discussed by comparison with animal gangliosides, structural homologs of plant GIPCs. PMID:27074617

  15. Myo-Inositol Supplementation to Prevent Gestational Diabetes Mellitus.

    PubMed

    Celentano, Claudio; Matarrelli, Barbara; Mattei, Peter A; Pavone, Giulia; Vitacolonna, Ester; Liberati, Marco

    2016-03-01

    Gestational diabetes mellitus (GDM) is a common complication characterized by increased insulin resistance, and by increased risk for adverse pregnancy outcomes affecting both the mother and the fetus. International guidelines describe optimal ways to recognize it, and the recommended treatment of patients affected to reduce adverse outcomes. Improving insulin resistance could reduce incidence of GDM and its complications. Recently, a few trials have been published on the possible prevention of GDM. Inositol has been proposed as a food supplement that might reduce gestational diabetes incidence in high-risk pregnant women. PMID:26898405

  16. Myo-Inositol Supplementation to Prevent Gestational Diabetes Mellitus.

    PubMed

    Celentano, Claudio; Matarrelli, Barbara; Mattei, Peter A; Pavone, Giulia; Vitacolonna, Ester; Liberati, Marco

    2016-03-01

    Gestational diabetes mellitus (GDM) is a common complication characterized by increased insulin resistance, and by increased risk for adverse pregnancy outcomes affecting both the mother and the fetus. International guidelines describe optimal ways to recognize it, and the recommended treatment of patients affected to reduce adverse outcomes. Improving insulin resistance could reduce incidence of GDM and its complications. Recently, a few trials have been published on the possible prevention of GDM. Inositol has been proposed as a food supplement that might reduce gestational diabetes incidence in high-risk pregnant women.

  17. Chloride secretagogues stimulate inositol phosphate formation in shark rectal gland tubules cultured in suspension

    SciTech Connect

    Ecay, T.W.; Valentich, J.D. )

    1991-03-01

    Neuroendocrine activation of transepithelial chloride secretion by shark rectal gland cells is associated with increases in cellular cAMP, cGMP, and free calcium concentrations. We report here on the effects of several chloride secretagogues on inositol phosphate formation in cultured rectal gland tubules. Vasoactive intestinal peptide (VIP), atriopeptin (AP), and ionomycin increase the total inositol phosphate levels of cultured tubules, as measured by ion exchange chromatography. Forskolin, a potent chloride secretagogue, has no effect on inositol phosphate formation. The uptake of {sup 3}H-myo-inositol into phospholipids is very slow, preventing the detection of increased levels of inositol trisphosphate. However, significant increases in inositol monophosphate (IP1) and inositol biphosphate (IP2) were measured. The time course of VIP- and AP-stimulated IP1 and IP2 formation is similar to the effects of these agents on the short-circuit current responses of rectal gland monolayer cultures. In addition, aluminum fluoride, an artificial activator of guanine nucleotide-binding proteins, stimulates IP1 and IP2 formation. We conclude that rectal gland cells contain VIP and AP receptors coupled to the activation of phospholipase C. Coupling may be mediated by G-proteins. Receptor-stimulated increases in inositol phospholipid metabolism is one mechanism leading to increased intracellular free calcium concentrations, an important regulatory event in the activation of transepithelial chloride secretion by shark rectal gland epithelial cells.

  18. Defective craniofacial development and brain function in a mouse model for depletion of intracellular inositol synthesis.

    PubMed

    Ohnishi, Tetsuo; Murata, Takuya; Watanabe, Akiko; Hida, Akiko; Ohba, Hisako; Iwayama, Yoshimi; Mishima, Kazuo; Gondo, Yoichi; Yoshikawa, Takeo

    2014-04-11

    myo-Inositol is an essential biomolecule that is synthesized by myo-inositol monophosphatase (IMPase) from inositol monophosphate species. The enzymatic activity of IMPase is inhibited by lithium, a drug used for the treatment of mood swings seen in bipolar disorder. Therefore, myo-inositol is thought to have an important role in the mechanism of bipolar disorder, although the details remain elusive. We screened an ethyl nitrosourea mutant mouse library for IMPase gene (Impa) mutations and identified an Impa1 T95K missense mutation. The mutant protein possessed undetectable enzymatic activity. Homozygotes died perinatally, and E18.5 embryos exhibited striking developmental defects, including hypoplasia of the mandible and asymmetric fusion of ribs to the sternum. Perinatal lethality and morphological defects in homozygotes were rescued by dietary myo-inositol. Rescued homozygotes raised on normal drinking water after weaning exhibited a hyper-locomotive trait and prolonged circadian periods, as reported in rodents treated with lithium. Our mice should be advantageous, compared with those generated by the conventional gene knock-out strategy, because they carry minimal genomic damage, e.g. a point mutation. In conclusion, our results reveal critical roles for intracellular myo-inositol synthesis in craniofacial development and the maintenance of proper brain function. Furthermore, this mouse model for cellular inositol depletion could be beneficial for understanding the molecular mechanisms underlying the clinical effect of lithium and myo-inositol-mediated skeletal development.

  19. Retinoic acid treatment of fibroblasts causes a rapid decrease in ( sup 3 H)inositol uptake

    SciTech Connect

    Sinha, R.; Creek, K.E.; Silverman-Jones, C.; de Luca, L.M. )

    1989-04-01

    NIH 3T3 fibroblasts treated with all-trans-retinoic acid (RA) showed a dramatic decrease in the uptake of ({sup 3}H)inositol compared to solvent-treated controls. The onset of RA-induced inhibition of ({sup 3}H)inositol uptake was rapid with a 10-15% decrease occurring after 2-3 h of RA exposure and 60-70% reduction after 16 h of RA treatment. A progressive dose-dependent decrease in inositol uptake was found as the concentration of RA increased from 10{sup {minus}8} to 10{sup {minus}5} M and the effect was fully reversible within 48 h after RA removal. RA inhibition of inositol uptake was also observed in 3T3-Swiss and Balb/3T3 cells but not in two virally transformed 3T3 cell lines. Phlorizin, amiloride, and monensin inhibited inositol uptake by 66, 74, and 58%, respectively, and this inhibition was additive when the cells were treated with RA as well as these inhibitors. A decreased incorporation of ({sup 3}H)inositol into polyphosphoinositides was also observed in RA-treated cells but not to the same extent as for ({sup 3}H)inositol uptake. In conclusion, RA treatment of 3T3 fibroblasts decreases the uptake of ({sup 3}H)inositol by up to 70% within 8 to 10 h at near physiological concentrations in a reversible and specific manner.

  20. Inositol is a constituent of detergent-solubilized immunoaffinity-purified rat liver 5'-nucleotidase.

    PubMed Central

    Bailyes, E M; Ferguson, M A; Colaco, C A; Luzio, J P

    1990-01-01

    myo-Inositol analysis of detergent-solubilized immunoaffinity-purified rat liver 5'-nucleotidase showed the presence of 1 mol of myo-inositol/mol of enzyme monomer. This provides unequivocal evidence that the ectoenzyme 5'-nucleotidase is attached to liver membranes by a glycosyl-phosphatidylinositol lipid anchor. PMID:2306224

  1. Abnormalities in myo-inositol metabolism associated with type 2 diabetes in mice fed a high-fat diet: benefits of a dietary myo-inositol supplementation.

    PubMed

    Croze, Marine L; Géloën, Alain; Soulage, Christophe O

    2015-06-28

    We previously reported that a chronic supplementation with myo-inositol (MI) improved insulin sensitivity and reduced fat accretion in mice. We then tested the potency of such dietary intervention in the prevention of insulin resistance in C57BL/6 male mouse fed a high-fat diet (HFD). In addition, some abnormalities in inositol metabolism were reported to be associated with insulin resistance in several animal and human studies. We then investigated the presence of such anomalies (i.e. inosituria and an inositol intra-tissue depletion) in this diet-induced obesity (DIO) mouse model, as well as the potential benefit of a MI supplementation for inositol intra-tissue deficiency correction. HFD (60 % energy from fat) feeding was associated with inosituria and inositol intra-tissue depletion in the liver and kidneys. MI supplementation (0·58 mg/g per d) restored inositol pools in kidneys (partially) and liver (fully). HFD feeding for 4 months induced ectopic lipid redistribution to liver and muscles, fasting hyperglycaemia and hyperinsulinaemia, insulin resistance and obesity that were not prevented by MI supplementation, despite a significant improvement in insulin sensitivity parameter K insulin tolerance test and a reduction in white adipose tissue (WAT) mass ( - 17 %, P< 0·05). MI supplementation significantly reduced fatty acid synthase activity in epididymal WAT, which might explain its beneficial, but modest, effect on WAT accretion in HFD-fed mice. Finally, we found some abnormalities in inositol metabolism in association with a diabetic phenotype (i.e. insulin resistance and fasting hyperglycaemia) in a DIO mouse model. Dietary MI supplementation was efficient in the prevention of inositol intra-tissue depletion, but did not prevent insulin resistance or obesity efficiently in this mouse model. PMID:25990651

  2. Abnormalities in myo-inositol metabolism associated with type 2 diabetes in mice fed a high-fat diet: benefits of a dietary myo-inositol supplementation.

    PubMed

    Croze, Marine L; Géloën, Alain; Soulage, Christophe O

    2015-06-28

    We previously reported that a chronic supplementation with myo-inositol (MI) improved insulin sensitivity and reduced fat accretion in mice. We then tested the potency of such dietary intervention in the prevention of insulin resistance in C57BL/6 male mouse fed a high-fat diet (HFD). In addition, some abnormalities in inositol metabolism were reported to be associated with insulin resistance in several animal and human studies. We then investigated the presence of such anomalies (i.e. inosituria and an inositol intra-tissue depletion) in this diet-induced obesity (DIO) mouse model, as well as the potential benefit of a MI supplementation for inositol intra-tissue deficiency correction. HFD (60 % energy from fat) feeding was associated with inosituria and inositol intra-tissue depletion in the liver and kidneys. MI supplementation (0·58 mg/g per d) restored inositol pools in kidneys (partially) and liver (fully). HFD feeding for 4 months induced ectopic lipid redistribution to liver and muscles, fasting hyperglycaemia and hyperinsulinaemia, insulin resistance and obesity that were not prevented by MI supplementation, despite a significant improvement in insulin sensitivity parameter K insulin tolerance test and a reduction in white adipose tissue (WAT) mass ( - 17 %, P< 0·05). MI supplementation significantly reduced fatty acid synthase activity in epididymal WAT, which might explain its beneficial, but modest, effect on WAT accretion in HFD-fed mice. Finally, we found some abnormalities in inositol metabolism in association with a diabetic phenotype (i.e. insulin resistance and fasting hyperglycaemia) in a DIO mouse model. Dietary MI supplementation was efficient in the prevention of inositol intra-tissue depletion, but did not prevent insulin resistance or obesity efficiently in this mouse model.

  3. Trypanosoma brucei Bloodstream Forms Depend upon Uptake of myo-Inositol for Golgi Complex Phosphatidylinositol Synthesis and Normal Cell Growth.

    PubMed

    González-Salgado, Amaia; Steinmann, Michael; Major, Louise L; Sigel, Erwin; Reymond, Jean-Louis; Smith, Terry K; Bütikofer, Peter

    2015-06-01

    myo-Inositol is a building block for all inositol-containing phospholipids in eukaryotes. It can be synthesized de novo from glucose-6-phosphate in the cytosol and endoplasmic reticulum. Alternatively, it can be taken up from the environment via Na(+)- or H(+)-linked myo-inositol transporters. While Na(+)-coupled myo-inositol transporters are found exclusively in the plasma membrane, H(+)-linked myo-inositol transporters are detected in intracellular organelles. In Trypanosoma brucei, the causative agent of human African sleeping sickness, myo-inositol metabolism is compartmentalized. De novo-synthesized myo-inositol is used for glycosylphosphatidylinositol production in the endoplasmic reticulum, whereas the myo-inositol taken up from the environment is used for bulk phosphatidylinositol synthesis in the Golgi complex. We now provide evidence that the Golgi complex-localized T. brucei H(+)-linked myo-inositol transporter (TbHMIT) is essential in bloodstream-form T. brucei. Downregulation of TbHMIT expression by RNA interference blocked phosphatidylinositol production and inhibited growth of parasites in culture. Characterization of the transporter in a heterologous expression system demonstrated a remarkable selectivity of TbHMIT for myo-inositol. It tolerates only a single modification on the inositol ring, such as the removal of a hydroxyl group or the inversion of stereochemistry at a single hydroxyl group relative to myo-inositol.

  4. A systematic approach to the amplified expression, functional characterization and purification of inositol transporters from Bacillus subtilis.

    PubMed

    Bettaney, Kim E; Sukumar, Preethi; Hussain, Rohanah; Siligardi, Giuliano; Henderson, Peter J F; Patching, Simon G

    2013-02-01

    Abstract A systematic approach was used for the cloning and amplified expression in Escherichia coli of the genes for each of three inositol transport proteins (IolF, IolT, YfiG) from Bacillus subtilis that are evolutionarily-related to human transporters. Inducible amplified expression of each was achieved to levels of ∼ 10-15% of total protein in E. coli inner membrane preparations. The functional integrity of each heterologously-expressed protein was demonstrated by measuring the kinetics of (3)H-myo-inositol transport into energized whole cells; this confirmed that IolT is the major inositol transporter, IolF is an inefficient transporter of this substrate and demonstrated that YfiG is an inositol transport protein for the first time. Competition for (3)H-myo-inositol transport by 17 unlabelled compounds revealed all three proteins to be highly specific in recognizing inositols over sugars. IolT was confirmed to be highly specific for both myo- and D-chiro-inositol and IolF was confirmed to prefer D-chiro-inositol over myo-inositol. YfiG selectively recognized myo-inositol, D-chiro-inositol and, uniquely, L-chiro-inositol. All three proteins were successfully solubilized and purified in milligram quantities from inner membrane preparations and their suitability for inclusion in crystallization trials was assessed by analysis of structural integrity and thermal stability using circular dichroism spectroscopy followed by examination for monodispersity using gel filtration chromatography.

  5. Trypanosoma brucei Bloodstream Forms Depend upon Uptake of myo-Inositol for Golgi Complex Phosphatidylinositol Synthesis and Normal Cell Growth

    PubMed Central

    González-Salgado, Amaia; Steinmann, Michael; Major, Louise L.; Sigel, Erwin; Reymond, Jean-Louis

    2015-01-01

    myo-Inositol is a building block for all inositol-containing phospholipids in eukaryotes. It can be synthesized de novo from glucose-6-phosphate in the cytosol and endoplasmic reticulum. Alternatively, it can be taken up from the environment via Na+- or H+-linked myo-inositol transporters. While Na+-coupled myo-inositol transporters are found exclusively in the plasma membrane, H+-linked myo-inositol transporters are detected in intracellular organelles. In Trypanosoma brucei, the causative agent of human African sleeping sickness, myo-inositol metabolism is compartmentalized. De novo-synthesized myo-inositol is used for glycosylphosphatidylinositol production in the endoplasmic reticulum, whereas the myo-inositol taken up from the environment is used for bulk phosphatidylinositol synthesis in the Golgi complex. We now provide evidence that the Golgi complex-localized T. brucei H+-linked myo-inositol transporter (TbHMIT) is essential in bloodstream-form T. brucei. Downregulation of TbHMIT expression by RNA interference blocked phosphatidylinositol production and inhibited growth of parasites in culture. Characterization of the transporter in a heterologous expression system demonstrated a remarkable selectivity of TbHMIT for myo-inositol. It tolerates only a single modification on the inositol ring, such as the removal of a hydroxyl group or the inversion of stereochemistry at a single hydroxyl group relative to myo-inositol. PMID:25888554

  6. Insights into the activation mechanism of class I HDAC complexes by inositol phosphates

    PubMed Central

    Watson, Peter J.; Millard, Christopher J.; Riley, Andrew M.; Robertson, Naomi S.; Wright, Lyndsey C.; Godage, Himali Y.; Cowley, Shaun M.; Jamieson, Andrew G.; Potter, Barry V. L.; Schwabe, John W. R.

    2016-01-01

    Histone deacetylases (HDACs) 1, 2 and 3 form the catalytic subunit of several large transcriptional repression complexes. Unexpectedly, the enzymatic activity of HDACs in these complexes has been shown to be regulated by inositol phosphates, which bind in a pocket sandwiched between the HDAC and co-repressor proteins. However, the actual mechanism of activation remains poorly understood. Here we have elucidated the stereochemical requirements for binding and activation by inositol phosphates, demonstrating that activation requires three adjacent phosphate groups and that other positions on the inositol ring can tolerate bulky substituents. We also demonstrate that there is allosteric communication between the inositol-binding site and the active site. The crystal structure of the HDAC1:MTA1 complex bound to a novel peptide-based inhibitor and to inositol hexaphosphate suggests a molecular basis of substrate recognition, and an entropically driven allosteric mechanism of activation. PMID:27109927

  7. Cholinesterase inhibitor soman increases inositol trisphosphate in rat brain. (Reannouncement with new availability information)

    SciTech Connect

    Mobley, P.L.

    1990-12-31

    Studies were conducted to determine the effect of the cholinesterase inhibitor soman on the amount of inositol trisphosphate in the neocortex, striatum, cerebellum, and medulla-pons regions of rat brain in vivo. The studies indicate that treatment with soman increase inositol trisphosphate in the neocortex and striatum, but not in the cerebellum or medulla-pons region. In the neocortex the most pronounced increases were observed in animals with severe poisoning symptoms; however, inositol trisphophate was also found to be elevated in animals with only mild poisoning symptoms. A variety of evidence suggests that the receptor-mediated hydrolysis of phosphatidyl inositol results in the formation of inositol trisphosphate (IP3) and diacylglycerol, both of which function as intracellular signal messengers, and that this mechanism represents a major signal transduction system through which extracellular signals can influence intracellular events.

  8. Results from the International Consensus Conference on myo-inositol and D-chiro-inositol in Obstetrics and Gynecology--assisted reproduction technology.

    PubMed

    Bevilacqua, Arturo; Carlomagno, Gianfranco; Gerli, Sandro; Montanino Oliva, Mario; Devroey, Paul; Lanzone, Antonio; Soulange, Christophe; Facchinetti, Fabio; Carlo Di Renzo, Gian; Bizzarri, Mariano; Hod, Moshe; Cavalli, Pietro; D'Anna, Rosario; Benvenga, Salvatore; Chiu, Tony T; Kamenov, Zdravko A

    2015-06-01

    A substantial body of research on mammalian gametogenesis and human reproduction has recently investigated the effect of myo-inositol (MyoIns) on oocyte and sperm cell quality, due to its possible application to medically assisted reproduction. With a growing number of both clinical and basic research papers, the meaning of several observations now needs to be interpreted under a solid and rigorous physiological framework. The 2013 Florence International Consensus Conference on Myo- and D-chiro-inositol in obstetrics and gynecology has answered a number of research questions concerning the use of the two stereoisomers in assisted reproductive technologies. Available clinical trials and studies on the physiological and pharmacological effects of these molecules have been surveyed. Specifically, the physiological involvement of MyoIns in oocyte maturation and sperm cell functions has been discussed, providing an answer to the following questions: (1) Are inositols physiologically involved in oocyte maturation? (2) Are inositols involved in the physiology of spermatozoa function? (3) Is treatment with inositols helpful within assisted reproduction technology cycles? (4) Are there any differences in clinical efficacy between MyoIns and D-chiro-inositol? The conclusions of this Conference, drawn depending on expert panel opinions and shared with all the participants, are summarized in this review paper.

  9. Purification and molecular structure of digalactosyl myo-inositol (DGMI), trigalactosyl myo-inositol (TGMI), and fagopyritol B3 from common buckwheat seeds by NMR.

    PubMed

    Gui, Wei; Lemley, Bethan A; Keresztes, Ivan; Condo, Anthony M; Steadman, Kathryn J; Obendorf, Ralph L

    2013-10-18

    Three galactosyl cyclitols, digalactosyl myo-inositol (assigned the trivial name DGMI), trigalactosyl myo-inositol (assigned the trivial name TGMI), and trigalactosyl d-chiro-inositol (fagopyritol B3), were isolated from common buckwheat (Fagopyrum esculentum Moench) seeds. Structures of the three compounds were determined by 2D NMR spectroscopy. DGMI is α-d-galactopyranosyl-(1→6)-α-d-galactopyranosyl-(1→1)-1l-myo-inositol, TGMI is α-d-galactopyranosyl-(1→6)-α-d-galactopyranosyl-(1→6)-α-d-galactopyranosyl-(1→1)-1l-myo-inositol, and fagopyritol B3 is α-d-galactopyranosyl-(1→6)-α-d-galactopyranosyl-(1→6)-α-d-galactopyranosyl-(1→2)-1d-chiro-inositol. DGMI and TGMI are higher oligomers of galactinol, a major galactosyl donor in plant seeds. Fagopyritol B3 is a higher oligomer of the fagopyritol B series and one of six fagopyritols found in buckwheat seeds and in the bran milling fraction. Samples of TGMI and fagopyritol B3 also contained the compounds N-(β-glucopyranosyl)-nicotinic acid and β-d-apiofuranosyl-(1→6)-β-d-(1-O-methyl)-glucopyranoside.

  10. myo-inositol and D-ribose ligand discrimination in an ABC periplasmic binding protein.

    PubMed

    Herrou, Julien; Crosson, Sean

    2013-05-01

    The periplasmic binding protein (PBP) IbpA mediates the uptake of myo-inositol by the IatP-IatA ATP-binding cassette transmembrane transporter. We report a crystal structure of Caulobacter crescentus IbpA bound to myo-inositol at 1.45 Å resolution. This constitutes the first structure of a PBP bound to inositol. IbpA adopts a type I PBP fold consisting of two α-β lobes that surround a central hinge. A pocket positioned between the lobes contains the myo-inositol ligand, which binds with submicromolar affinity (0.76 ± 0.08 μM). IbpA is homologous to ribose-binding proteins and binds D-ribose with low affinity (50.8 ± 3.4 μM). On the basis of IbpA and ribose-binding protein structures, we have designed variants of IbpA with inverted binding specificity for myo-inositol and D-ribose. Five mutations in the ligand-binding pocket are sufficient to increase the affinity of IbpA for D-ribose by 10-fold while completely abolishing binding to myo-inositol. Replacement of ibpA with these mutant alleles unable to bind myo-inositol abolishes C. crescentus growth in medium containing myo-inositol as the sole carbon source. Neither deletion of ibpA nor replacement of ibpA with the high-affinity ribose binding allele affected C. crescentus growth on D-ribose as a carbon source, providing evidence that the IatP-IatA transporter is specific for myo-inositol. This study outlines the evolutionary relationship between ribose- and inositol-binding proteins and provides insight into the molecular basis upon which these two related, but functionally distinct, classes of periplasmic proteins specifically bind carbohydrate ligands.

  11. Inositol Hexakisphosphate Kinase 3 Regulates Metabolism and Lifespan in Mice

    PubMed Central

    Moritoh, Yusuke; Oka, Masahiro; Yasuhara, Yoshitaka; Hozumi, Hiroyuki; Iwachidow, Kimihiko; Fuse, Hiromitsu; Tozawa, Ryuichi

    2016-01-01

    Inositol hexakisphosphate kinase 3 (IP6K3) generates inositol pyrophosphates, which regulate diverse cellular functions. However, little is known about its own physiological role. Here, we show the roles of IP6K3 in metabolic regulation. We detected high levels of both mouse and human IP6K3 mRNA in myotubes and muscle tissues. In human myotubes, IP6K3 was upregulated by dexamethasone treatment, which is known to inhibit glucose metabolism. Furthermore, Ip6k3 expression was elevated under diabetic, fasting, and disuse conditions in mouse skeletal muscles. Ip6k3−/− mice demonstrated lower blood glucose, reduced circulating insulin, deceased fat mass, lower body weight, increased plasma lactate, enhanced glucose tolerance, lower glucose during an insulin tolerance test, and reduced muscle Pdk4 expression under normal diet conditions. Notably, Ip6k3 deletion extended animal lifespan with concomitant reduced phosphorylation of S6 ribosomal protein in the heart. In contrast, Ip6k3−/− mice showed unchanged skeletal muscle mass and no resistance to the effects of high fat diet. The current observations suggest novel roles of IP6K3 in cellular regulation, which impact metabolic control and lifespan. PMID:27577108

  12. Inositol Hexakisphosphate Kinase 3 Regulates Metabolism and Lifespan in Mice.

    PubMed

    Moritoh, Yusuke; Oka, Masahiro; Yasuhara, Yoshitaka; Hozumi, Hiroyuki; Iwachidow, Kimihiko; Fuse, Hiromitsu; Tozawa, Ryuichi

    2016-01-01

    Inositol hexakisphosphate kinase 3 (IP6K3) generates inositol pyrophosphates, which regulate diverse cellular functions. However, little is known about its own physiological role. Here, we show the roles of IP6K3 in metabolic regulation. We detected high levels of both mouse and human IP6K3 mRNA in myotubes and muscle tissues. In human myotubes, IP6K3 was upregulated by dexamethasone treatment, which is known to inhibit glucose metabolism. Furthermore, Ip6k3 expression was elevated under diabetic, fasting, and disuse conditions in mouse skeletal muscles. Ip6k3(-/-) mice demonstrated lower blood glucose, reduced circulating insulin, deceased fat mass, lower body weight, increased plasma lactate, enhanced glucose tolerance, lower glucose during an insulin tolerance test, and reduced muscle Pdk4 expression under normal diet conditions. Notably, Ip6k3 deletion extended animal lifespan with concomitant reduced phosphorylation of S6 ribosomal protein in the heart. In contrast, Ip6k3(-/-) mice showed unchanged skeletal muscle mass and no resistance to the effects of high fat diet. The current observations suggest novel roles of IP6K3 in cellular regulation, which impact metabolic control and lifespan. PMID:27577108

  13. Changes in inositol phosphates in wild carrot cells upon initiation of cell wall digestion

    SciTech Connect

    Rincon, M.; Boss, W.F.

    1987-04-01

    Previous studies have shown that inositol trisphosphate (IP/sub 3/) stimulated /sup 45/Ca/sup +2/ efflux from fusogenic carrot protoplasts and it was suggested that IP/sub 3/ may serve as a second messenger for the mobilization of intracellular Ca/sup +2/ in higher plant cells. To determine whether or not inositol phosphate metabolism changes in response to external stimuli, the cells were labeled with myo-(2-/sup 3/H) inositol for 18 h and exposed to cell wall digestion enzymes, Driselase. The inositol phosphates were extracted with ice cold 10% TCA and separated by anion exchange chromatography. The radioactivity of the fraction that contained IP/sub 3/ increased 2-3.8 fold and that which contained inositol bisphosphate increased 1.9-2.6 fold within 1.5 min of exposure to Driselase. After 6 min, the radioactivity of both fractions increased 6-7.7 fold and an increase in inositol monophosphate was observed. These data indicate that inositol phosphate metabolism is stimulated by Driselase and suggest polyphosphoinositide hydrolysis occurs upon initiation of cell wall digestion.

  14. Effects of inositol supplementation in a cohort of mothers at risk of producing an NTD pregnancy.

    PubMed

    Cavalli, Pietro; Tonni, Gabriele; Grosso, Enrico; Poggiani, Carlo

    2011-11-01

    Neural tube defects (NTDs), most commonly spina bifida and anencephaly, can be prevented with periconceptional intake of folic acid in about 70% of cases. Recurrence of NTDs despite supplementation of high dose of folic acid further suggests that a proportion of NTD cases might be resistant to folic acid. Moreover, heterogeneity of NTDs has been suggested in animal studies, indicating that only some sub-type of NTDs should be considered sensitive to folate intake. Inositol isomers (particularly myo- and chiro-inositol) can prevent folate-resistant NTDs in the curly-tail mutant mouse, suggesting that some cases of human NTDs might benefit from inositol supplementation. In humans, lower inositol blood concentration was found in pregnant women carrying NTD fetuses, whereas a periconceptional combination therapy with folic acid associated with inositol has been linked to normal live births, despite high NTD recurrence risk. Fifteen pregnancies from 12 Caucasian women from different parts of Italy with at least one previous NTD-affected pregnancy underwent periconceptional combined myo-inositol and folic acid supplementation. Maternal serum α-feto-protein levels were found in the normal range, and normal results on ultrasound examination were found in all the pregnancies that followed. No collateral effects or intense uterine contractions were demonstrated in this pilot study in any of the pregnancies after inositol supplementation, and seventeen babies were born without any type of NTD.

  15. D-chiro-inositol--its functional role in insulin action and its deficit in insulin resistance.

    PubMed

    Larner, Joseph

    2002-01-01

    In this review we discuss the biological significance of D-chiro-inositol, originally discovered as a component of a putative mediator of intracellular insulin action, where as a putative mediator, it accelerates the dephosphorylation of glycogen synthase and pyruvate dehydrogenase, rate limiting enzymes of non-oxidative and oxidative glucose disposal. Early studies demonstrated a linear relationship between its decreased urinary excretion and the degree of insulin resistance present. When tissue contents, including muscle, of type 2 diabetic subjects were assayed, they demonstrated a more general body deficiency. Administration of D-chiro-inositol to diabetic rats, Rhesus monkeys and now to humans accelerated glucose disposal and sensitized insulin action. A defect in vivo in the epimerization of myo-inositol to chiro-inositol in insulin sensitive tissues of the GK type 2 diabetic rat has been elucidated. Thus, administered D-chiro-inositol may act to bypass a defective normal epimerization of myo-inositol to D-chiro-inositol associated with insulin resistance and act to at least partially restore insulin sensitivity and glucose disposal. PMID:11900279

  16. Transport and metabolism of indole-3-acetyl-myo-inositol-galactoside in seedlings of Zea mays

    NASA Technical Reports Server (NTRS)

    Komoszynski, M.; Bandurski, R. S.

    1986-01-01

    Indole-3-acetyl-myo-inositol galactoside labeled with 3H in the indole and 14C in the galactose moieties was applied to kernels of 5 day old germinating seedlings of Zea mays. Indole-3-acetyl-myo-inositol galactoside was not transported into either the shoot or root tissue as the intact molecule but was instead hydrolyzed to yield [3H]indole-3-acetyl-myo-inositol and [3H]indole-3-acetic acid which were then transported to the shoot with little radioactivity going to the root. With certain assumption concerning the equilibration of applied [3H]indole-3-acetyl-myo-inositol-[U-14C]galactose with the endogenous pool, it may be concluded that indole-3-acetyl-myo-inositol galactoside in the endosperm supplies about 2 picomoles per plant per hour of indole-3-acetyl-myo-inositol and 1 picomole per plant per hour of indole-3-acetic acid to the shoot and thus is comparable to indole-3-acetyl-myo-inositol as a source of indole-acetic acid for the shoot. Quantitative estimates of the amount of galactose in the kernels suggest that [3H]indole-3-acetyl-myo-inositol-[14C]galactose is hydrolyzed after the compound leaves the endosperm but before it reaches the shoot. In addition, [3H]indole-3-acetyl-myo-inositol-[14C]galactose supplies appreciable amounts of 14C to the shoot and both 14C and 3H to an uncharacterized insoluble fraction of the endosperm.

  17. Inositol for the prevention of neural tube defects: a pilot randomised controlled trial.

    PubMed

    Greene, Nicholas D E; Leung, Kit-Yi; Gay, Victoria; Burren, Katie; Mills, Kevin; Chitty, Lyn S; Copp, Andrew J

    2016-03-28

    Although peri-conceptional folic acid (FA) supplementation can prevent a proportion of neural tube defects (NTD), there is increasing evidence that many NTD are FA non-responsive. The vitamin-like molecule inositol may offer a novel approach to preventing FA-non-responsive NTD. Inositol prevented NTD in a genetic mouse model, and was well tolerated by women in a small study of NTD recurrence. In the present study, we report the Prevention of Neural Tube Defects by Inositol (PONTI) pilot study designed to gain further experience of inositol usage in human pregnancy as a preliminary trial to a future large-scale controlled trial to evaluate efficacy of inositol in NTD prevention. Study subjects were UK women with a previous NTD pregnancy who planned to become pregnant again. Of 117 women who made contact, ninety-nine proved eligible and forty-seven agreed to be randomised (double-blind) to peri-conceptional supplementation with inositol plus FA or placebo plus FA. In total, thirty-three randomised pregnancies produced one NTD recurrence in the placebo plus FA group (n 19) and no recurrences in the inositol plus FA group (n 14). Of fifty-two women who declined randomisation, the peri-conceptional supplementation regimen and outcomes of twenty-two further pregnancies were documented. Two NTD recurred, both in women who took only FA in their next pregnancy. No adverse pregnancy events were associated with inositol supplementation. The findings of the PONTI pilot study encourage a large-scale controlled trial of inositol for NTD prevention, but indicate the need for a careful study design in view of the unwillingness of many high-risk women to be randomised.

  18. The response to inositol: regulation of glycerolipid metabolism and stress response signaling in yeast

    PubMed Central

    Henry, Susan A.; Gaspar, Maria L.; Jesch, Stephen A.

    2014-01-01

    This article focuses on discoveries of the mechanisms governing the regulation of glycerolipid metabolism and stress response signaling in response to the phospholipid precursor, inositol. The regulation of glycerolipid lipid metabolism in yeast in response to inositol is highly complex, but increasingly well understood, and the roles of individual lipids in stress response are also increasingly well characterized. Discoveries that have emerged over several decades of genetic, molecular and biochemical analyses of metabolic, regulatory and signaling responses of yeast cells, both mutant and wild type, to the availability of the phospholipid precursor, inositol are discussed. PMID:24418527

  19. The response to inositol: regulation of glycerolipid metabolism and stress response signaling in yeast.

    PubMed

    Henry, Susan A; Gaspar, Maria L; Jesch, Stephen A

    2014-05-01

    This article focuses on discoveries of the mechanisms governing the regulation of glycerolipid metabolism and stress response signaling in response to the phospholipid precursor, inositol. The regulation of glycerolipid lipid metabolism in yeast in response to inositol is highly complex, but increasingly well understood, and the roles of individual lipids in stress response are also increasingly well characterized. Discoveries that have emerged over several decades of genetic, molecular and biochemical analyses of metabolic, regulatory and signaling responses of yeast cells, both mutant and wild type, to the availability of the phospholipid precursor, inositol are discussed.

  20. Measurement of Inositol Triphosphate Levels from Rat Hippocampal Slices

    PubMed Central

    Tabatadze, Nino; Woolley, Catherine

    2016-01-01

    Inositol triphosphate (IP3) is an important second messenger that participates in signal transduction pathways in diverse cell types including hippocampal neurons. Stimulation of phospholipase C in response to various stimuli (hormones, growth factors, neurotransmitters, neurotrophins, neuromodulators, odorants, light, etc) results in hydrolysis of phosphatidylinositol 4, 5-bisphosphate (PIP2), a phospholipid that is located in the plasma membrane, and leads to the production of IP3 and diacylglycerol. Binding of IP3 to the IP3 receptor (IP3R) induces Ca2+ release from intracellular stores and enables the initiation of intracellular Ca2+-dependent signaling. Here we describe a procedure for the measurement of cellular IP3 levels in tissue homogenates prepared from rat hippocampal slices. PMID:27468425

  1. Control of eukaryotic phosphate homeostasis by inositol polyphosphate sensor domains.

    PubMed

    Wild, Rebekka; Gerasimaite, Ruta; Jung, Ji-Yul; Truffault, Vincent; Pavlovic, Igor; Schmidt, Andrea; Saiardi, Adolfo; Jessen, Henning Jacob; Poirier, Yves; Hothorn, Michael; Mayer, Andreas

    2016-05-20

    Phosphorus is a macronutrient taken up by cells as inorganic phosphate (P(i)). How cells sense cellular P(i) levels is poorly characterized. Here, we report that SPX domains--which are found in eukaryotic phosphate transporters, signaling proteins, and inorganic polyphosphate polymerases--provide a basic binding surface for inositol polyphosphate signaling molecules (InsPs), the concentrations of which change in response to P(i) availability. Substitutions of critical binding surface residues impair InsP binding in vitro, inorganic polyphosphate synthesis in yeast, and P(i) transport in Arabidopsis In plants, InsPs trigger the association of SPX proteins with transcription factors to regulate P(i) starvation responses. We propose that InsPs communicate cytosolic P(i) levels to SPX domains and enable them to interact with a multitude of proteins to regulate P(i) uptake, transport, and storage in fungi, plants, and animals. PMID:27080106

  2. Nuclear inositol lipid metabolism: more than just second messenger generation?

    PubMed

    Martelli, Alberto M; Follo, Matilde Yung; Evangelisti, Camilla; Falà, Federica; Fiume, Roberta; Billi, Anna Maria; Cocco, Lucio

    2005-10-01

    A distinct polyphosphoinositide cycle is present in the nucleus, and growing evidence suggests its importance in DNA replication, gene transcription, and apoptosis. Even though it was initially thought that nuclear inositol lipids would function as a source for second messengers, recent findings strongly indicate that lipids present in the nucleus also fulfil other roles. The scope of this review is to highlight the most intriguing advances made in the field over the last few years, such as the possibility that nuclear phosphatidylinositol (4,5) bisphosphate is involved in maintaining chromatin in a transcriptionally active conformation, the new emerging roles for intranuclear phosphatidylinositol (3,4,5) trisphosphate and phosphoinositide 3-kinase, and the evidence which suggests a tight relationship between a decreased level of nuclear phosphoinositide specific phospholipase C-beta1 and the evolution of myelodisplastic syndrome into acute myeloid leukemia.

  3. Alterations in Lipid and Inositol Metabolisms in Two Dopaminergic Disorders

    PubMed Central

    Berger, Hannah S.; Do, Kieu Trinh; Kastenmüller, Gabi; Wahl, Simone; Adamski, Jerzy; Peters, Annette; Krumsiek, Jan; Suhre, Karsten; Haslinger, Bernhard; Ceballos-Baumann, Andres; Gieger, Christian; Winkelmann, Juliane

    2016-01-01

    Background Serum metabolite profiling can be used to identify pathways involved in the pathogenesis of and potential biomarkers for a given disease. Both restless legs syndrome (RLS) and Parkinson`s disease (PD) represent movement disorders for which currently no blood-based biomarkers are available and whose pathogenesis has not been uncovered conclusively. We performed unbiased serum metabolite profiling in search of signature metabolic changes for both diseases. Methods 456 metabolites were quantified in serum samples of 1272 general population controls belonging to the KORA cohort, 82 PD cases and 95 RLS cases by liquid-phase chromatography and gas chromatography separation coupled with tandem mass spectrometry. Genetically determined metabotypes were calculated using genome-wide genotyping data for the 1272 general population controls. Results After stringent quality control, we identified decreased levels of long-chain (polyunsaturated) fatty acids of individuals with PD compared to both RLS (PD vs. RLS: p = 0.0001 to 5.80x10-9) and general population controls (PD vs. KORA: p = 6.09x10-5 to 3.45x10-32). In RLS, inositol metabolites were increased specifically (RLS vs. KORA: p = 1.35x10-6 to 3.96x10-7). The impact of dopaminergic drugs was reflected in changes in the phenylalanine/tyrosine/dopamine metabolism observed in both individuals with RLS and PD. Conclusions A first discovery approach using serum metabolite profiling in two dopamine-related movement disorders compared to a large general population sample identified significant alterations in the polyunsaturated fatty acid metabolism in PD and implicated the inositol metabolism in RLS. These results provide a starting point for further studies investigating new perspectives on factors involved in the pathogenesis of the two diseases as well as possible points of therapeutic intervention. PMID:26808974

  4. The Arabidopsis thaliana Myo-Inositol 1-Phosphate Synthase1 Gene Is Required for Myo-inositol Synthesis and Suppression of Cell Death[W

    PubMed Central

    Donahue, Janet L.; Alford, Shannon R.; Torabinejad, Javad; Kerwin, Rachel E.; Nourbakhsh, Aida; Ray, W. Keith; Hernick, Marcy; Huang, Xinyi; Lyons, Blair M.; Hein, Pyae P.; Gillaspy, Glenda E.

    2010-01-01

    l-myo-inositol 1-phosphate synthase (MIPS; EC 5.5.1.4) catalyzes the rate-limiting step in the synthesis of myo-inositol, a critical compound in the cell. Plants contain multiple MIPS genes, which encode highly similar enzymes. We characterized the expression patterns of the three MIPS genes in Arabidopsis thaliana and found that MIPS1 is expressed in most cell types and developmental stages, while MIPS2 and MIPS3 are mainly restricted to vascular or related tissues. MIPS1, but not MIPS2 or MIPS3, is required for seed development, for physiological responses to salt and abscisic acid, and to suppress cell death. Specifically, a loss in MIPS1 resulted in smaller plants with curly leaves and spontaneous production of lesions. The mips1 mutants have lower myo-inositol, ascorbic acid, and phosphatidylinositol levels, while basal levels of inositol (1,4,5)P3 are not altered in mips1 mutants. Furthermore, mips1 mutants exhibited elevated levels of ceramides, sphingolipid precursors associated with cell death, and were complemented by a MIPS1-green fluorescent protein (GFP) fusion construct. MIPS1-, MIPS2-, and MIPS3-GFP each localized to the cytoplasm. Thus, MIPS1 has a significant impact on myo-inositol levels that is critical for maintaining levels of ascorbic acid, phosphatidylinositol, and ceramides that regulate growth, development, and cell death. PMID:20215587

  5. Improving D-glucaric acid production from myo-inositol in E. coli by increasing MIOX stability and myo-inositol transport.

    PubMed

    Shiue, Eric; Prather, Kristala L J

    2014-03-01

    D-glucaric acid has been explored for a myriad of potential uses, including biopolymer production and cancer treatment. A biosynthetic route to produce D-glucaric acid from glucose has been constructed in Escherichia coli (Moon et al., 2009b), and analysis of the pathway revealed myo-inositol oxygenase (MIOX) to be the least active enzyme. To increase pathway productivity, we explored protein fusion tags for increased MIOX solubility and directed evolution for increased MIOX activity. An N-terminal SUMO fusion to MIOX resulted in a 75% increase in D-glucaric acid production from myo-inositol. While our directed evolution efforts did not yield an improved MIOX variant, our screen isolated a 941 bp DNA fragment whose expression led to increased myo-inositol transport and a 65% increase in D-glucaric acid production from myo-inositol. Overall, we report the production of up to 4.85 g/L of D-glucaric acid from 10.8 g/L myo-inositol in recombinant E. coli.

  6. Inositol and IP3 levels regulate autophagy: biology and therapeutic speculations.

    PubMed

    Sarkar, Sovan; Rubinsztein, David C

    2006-01-01

    We recently showed that lithium induces autophagy via inositol monophosphatase (IMPase) inhibition, leading to free inositol depletion and reduced myo-inositol-1,4, 5-triphosphate (IP3) levels. This represents a novel way of regulating mammalian autophagy, independent of the mammalian target of rapamycin (mTOR). Induction of autophagy by lithium led to enhanced clearance of autophagy substrates, like mutant huntingtin fragments and mutant alpha-synucleins, associated with Huntington's disease (HD) and some autosomal dominant forms of Parkinson's disease (PD), respectively. Similar effects were observed with a specific IMPase inhibitor and mood-stabilizing drugs that decrease inositol levels. This may represent a new therapeutic strategy for upregulating autophagy in the treatment of neurodegenerative disorders, where the mutant protein is an autophagy substrate. In this Addendum, we review these findings, and some of the speculative possibilities they raise.

  7. Inositol polyphosphate multikinase is a physiologic PI3-kinase that activates Akt/PKB.

    PubMed

    Maag, David; Maxwell, Micah J; Hardesty, Douglas A; Boucher, Katie L; Choudhari, Namrata; Hanno, Adam G; Ma, Jenny F; Snowman, Adele S; Pietropaoli, Joseph W; Xu, Risheng; Storm, Phillip B; Saiardi, Adolfo; Snyder, Solomon H; Resnick, Adam C

    2011-01-25

    The second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)), formed by the p110 family of PI3-kinases, promotes cellular growth, proliferation, and survival, in large part by activating the protein kinase Akt/PKB. We show that inositol polyphosphate multikinase (IPMK) physiologically generates PIP(3) as well as water soluble inositol phosphates. IPMK deletion reduces growth factor-elicited Akt signaling and cell proliferation caused uniquely by loss of its PI3-kinase activity. Inhibition of p110 PI3-kinases by wortmannin prevents IPMK phosphorylation and activation. Thus, growth factor stimulation of Akt signaling involves PIP(3) generation through the sequential activations of the p110 PI3-kinases and IPMK. As inositol phosphates inhibit Akt signaling, IPMK appears to act as a molecular switch, inhibiting or stimulating Akt via its inositol phosphate kinase or PI3-kinase activities, respectively. Drugs regulating IPMK may have therapeutic relevance in influencing cell proliferation.

  8. Light induces a rapid and transient increase in inositol-trisphosphate in toad rod outer segments

    SciTech Connect

    Brown, J.E.; Blazynski, C.; Cohen, A.I.

    1987-08-14

    The sub-second time course of changes in the content of (/sup 3/H)inositol-1,4,5-trisphosphate was determined in rod outer segments from very rapidly frozen Bufo retinas that had been incubated with (/sup 3/H)inositol. Rod outer segments were cut off frozen specimens with a cryostat microtome and the water soluble extracts were analyzed. The content of (/sup 3/H)inositol-1,4,5-trisphosphate rose after approximately 250 msec of bright illumination, but returned to the unstimulated level after 1 sec, whether the stimulus remained on or not. That is, there was rapid but transient change in the content of (/sup 3/H)inositol-1,4,5-trisphosphate after the onset of stimulation.

  9. "Inosaminoacids": novel inositol-amino acid hybrid structures accessed by microbial arene oxidation.

    PubMed

    Pilgrim, Sarah; Kociok-Köhn, Gabriele; Lloyd, Matthew D; Lewis, Simon E

    2011-04-28

    Microbial 1,2-dihydroxylation of sodium benzoate permits the rapid construction of novel inositol-amino acid hybrid structures. Both β- and γ-amino acids are accessible by means of an acylnitroso Diels-Alder cycloaddition.

  10. Plasmodium falciparum is dependent on de novo myo-inositol biosynthesis for assembly of GPI glycolipids and infectivity.

    PubMed

    Macrae, James I; Lopaticki, Sash; Maier, Alexander G; Rupasinghe, Thusitha; Nahid, Amsha; Cowman, Alan F; McConville, Malcolm J

    2014-02-01

    Intra-erythrocytic stages of the malaria parasite, Plasmodium falciparum, are thought to be dependent on de novo synthesis of phosphatidylinositol, as red blood cells (RBC) lack the capacity to synthesize this phospholipid. The myo-inositol headgroup of PI can either be synthesized de novo or scavenged from the RBC. An untargeted metabolite profiling of P. falciparum infected RBC showed that trophozoite and schizont stages accumulate high levels of myo-inositol-3-phosphate, indicating increased de novo biosynthesis of myo-inositol from glucose 6-phosphate. Metabolic labelling studies with (13) C-U-glucose in the presence and absence of exogenous inositol confirmed that de novo myo-inositol synthesis occurs in parallel with myo-inositol salvage pathways. Unexpectedly, while both endogenous and scavenged myo-inositol was used to synthesize bulk PI, only de novo-synthesized myo-inositol was incorporated into GPI glycolipids. Moreover, gene disruption studies suggested that the INO1 gene, encoding myo-inositol 3-phosphate synthase, is essential in asexual parasite stages. Together these findings suggest that P. falciparum asexual stages are critically dependent on de novo myo-inositol biosynthesis for assembly of a sub-pool of PI species and GPI biosynthesis. These findings highlight unexpected complexity in phospholipid biosynthesis in P. falciparum and a lack of redundancy in some nutrient salvage versus endogenous biosynthesis pathways.

  11. Effects of formulation and process factors on the crystal structure of freeze-dried Myo-inositol.

    PubMed

    Izutsu, Ken-Ichi; Yomota, Chikako; Okuda, Haruhiro; Kawanishi, Toru; Yamaki, Takuya; Ohdate, Ryohei; Yu, Zhaokun; Yonemochi, Etsuo; Terada, Katsuhide

    2014-08-01

    The objective of this study was to elucidate effects of formulation and process variables on the physical forms of freeze-dried myo-inositol. Physical properties of myo-inositol in frozen solutions, freeze-dried solids, and cooled heat-melt solids were characterized by powder X-ray diffraction (PXRD), thermal analysis (differential scanning calorimetry [DSC] and thermogravimetric), and simultaneous PXRD-DSC analysis. Cooling of heat-melt myo-inositol produced two forms of metastable anhydrate crystals that change to stable form (melting point 225 °C-228 °C) with transition exotherms at around 123 °C and 181 °C, respectively. Freeze-drying of single-solute aqueous myo-inositol solutions after rapid cooling induced crystallization of myo-inositol as metastable anhydrate (transition at 80 °C-125 °C) during secondary drying segment. Contrarily, postfreeze heat treatment (i.e., annealing) induced crystallization of myo-inositol dihydrate. Removal of the crystallization water during the secondary drying produced the stable-form myo-inositol anhydrate crystal. Shelf-ramp slow cooling of myo-inositol solutions resulted in the stable and metastable anhydrous crystal solids depending on the solute concentrations and the solution volumes. Colyophilization with phosphate buffer retained myo-inositol in the amorphous state. Crystallization in different process segments varies crystal form of freeze-dried myo-inositol solids.

  12. Computational modeling and in silico analysis of differential regulation of myo-inositol catabolic enzymes in Cryptococcus neoformans

    PubMed Central

    Mackenzie, Emalee A; Klig, Lisa S

    2008-01-01

    Background Inositol is a key cellular metabolite for many organisms. Cryptococcus neoformans is an opportunistic pathogen which primarily infects the central nervous system, a region of high inositol concentration, of immunocompromised individuals. Through the use of myo-inositol oxygenase C. neoformans can catabolize inositol as a sole carbon source to support growth and viability. Results Three myo-inositol oxygenase gene sequences were identified in the C. neoformans genome. Differential regulation was suggested by computational analyses of the three gene sequences. This included examination of the upstream regulatory regions, identifying ORE/TonE and UASINO sequences, conserved introns/exons, and in frame termination sequences. Homology modeling of the proteins encoded by these genes revealed key differences in the myo-inositol active site. Conclusion The results suggest there are two functional copies of the myo-inositol oxygenase gene in the C. neoformans genome. The functional genes are differentially expressed in response to environmental inositol concentrations. Both the upstream regulatory regions of the genes and the structure of the specific proteins suggest that MIOX1 would function when inositol concentrations are low, whereas MIOX2 would function when inositol concentrations are high. PMID:18854045

  13. Modulation of hemodynamic and vascular filtration changes in diabetic rats by dietary myo-inositol

    SciTech Connect

    Pugliese, G.; Tilton, R.G.; Speedy, A.; Santarelli, E.; Eades, D.M.; Province, M.A.; Kilo, C.; Sherman, W.R.; Williamson, J.R. )

    1990-03-01

    To assess the potential of myo-inositol-supplemented diets to prevent diabetes-induced vascular functional changes, we examined the effects of diets supplemented with 0.5, 1, or 2% myo-inositol on blood flow and vascular filtration function in nondiabetic control rats and rats with streptozocin-induced diabetes (STZ-D). After 1 mo of diabetes and dietary myo-inositol supplementation, (1) 131I-labeled bovine serum albumin (BSA) permeation of vessels was assessed in multiple tissues, (2) glomerular filtration rate (GFR) was estimated as renal plasma clearance of 57Co-labeled EDTA, (3) regional blood flows were measured with 15-microns 85Sr-labeled microspheres, and (4) endogenous albumin and IgG urinary excretion rates were quantified by radial immunodiffusion assay. In STZ-D rats, 131I-BSA tissue clearance increased significantly (2- to 4-fold) in the anterior uvea, choroid-sclera, retina, sciatic nerve, aorta, new granulation tissue, diaphragm, and kidney but was unchanged in skin, forelimb muscle, and heart. myo-Inositol-supplemented diets reduced diabetes-induced increases in 131I-BSA clearance (in a dose-dependent manner) in all tissues; however, only in new granulation tissue and diaphragm did the 2% myo-inositol diet completely normalize vascular albumin permeation. Diabetes-induced increases in GFR and in urinary albumin and IgG excretion were also substantially reduced or normalized by dietary myo-inositol supplements. Increased blood flow in anterior uvea, choroid-sclera, kidney, new granulation tissue, and skeletal muscle in STZ-D rats also was substantially reduced or normalized by the 2% myo-inositol diet. myo-Inositol had minimal if any effects on the above parameters in control rats.

  14. Jasmonic acid perception by COI1 involves inositol polyphosphates in Arabidopsis thaliana.

    PubMed

    Mosblech, Alina; Thurow, Corinna; Gatz, Christiane; Feussner, Ivo; Heilmann, Ingo

    2011-03-01

    Plant responses to wounding are part of their defense responses against insects, and are tightly regulated. The isoleucin conjugate of jasmonic acid (JA-Ile) is a major regulatory molecule. We have previously shown that inositol polyphosphate signals are required for defense responses in Arabidopsis; however, the way in which inositol polyphosphates contribute to plant responses to wounding has so far remained unclear. Arabidopsis F-box proteins involved in the perception of JA-Ile (COI1) and auxin (TIR1) are structurally similar. Because TIR1 has recently been shown to contain inositol hexakisphosphate (InsP₆) as a co-factor of unknown function, here we explored the possibility that InsP₆ or another inositol polyphosphate is required for COI1 function. In support of this hypothesis, COI1 variants with changes in putative inositol polyphosphate coordinating residues exhibited a reduced interaction with the COI1 target, JAZ9, in yeast two-hybrid tests. The equivalent COI1 variants displayed a reduced capability to rescue jasmonate-mediated root growth inhibition or silique development in Arabidopsis coi1 mutants. Yeast two-hybrid tests using wild-type COI1 in an ipk1Δ yeast strain exhibiting increased levels of inositol pentakisphosphate (InsP₅) and reduced levels of InsP₆ indicate an enhanced COI1/JAZ9 interaction. Consistent with these findings, Arabidopsis ipk1-1 mutants, also with increased InsP₅ and reduced InsP₆ levels, showed increased defensive capabilities via COI1-mediated processes, including wound-induced gene expression, defense against caterpillars or root growth inhibition by jasmonate. The combined data from experiments using mutated COI1 variants, as well as yeast and Arabidopsis backgrounds altered in inositol polyphosphate metabolism, indicate that an inositol polyphosphate, and probably InsP₅, contributes to COI1 function.

  15. Inositol metabolism in WRK-1 cells. Relationship of hormone-sensitive to -insensitive pools of phosphoinositides

    SciTech Connect

    Monaco, M.E.

    1987-09-25

    Previous studies have indicated the existence of two separate pools of phosphoinositides in WRK-1 cells; one is labile and hormone-sensitive with respect to turnover, while the other is stable. Hormonal stimulation results in a rapid increase in /sup 32/Pi incorporation into the sensitive pool, while in the absence of hormone, incorporation of /sup 32/Pi into this pool is slow. Results are quite different when (/sup 3/H)inositol is the precursor utilized. Incorporation of (/sup 3/H)inositol into hormone-sensitive phosphoinositides is not stimulated in the presence of hormone, suggesting entry of this exogenous precursor into the cycle by a route other than the resynthetic phase of the cycle. Furthermore, failure of hormone to induce loss of (/sup 3/H)phosphoinositide in pulse-chase experiments in the absence of lithium suggests reutilization of the (/sup 3/H)inositol moiety generated by phosphodiesteratic cleavage of hormone-sensitive phosphoinositide. Time course studies indicate that the relative rates of incorporation of (/sup 3/H)inositol into sensitive and insensitive phosphoinositide remain constant from 2 to 24 h. Several factors are capable of increasing (/sup 3/H)inositol incorporation into hormone-insensitive phosphoinositide including vasopressin, calcium ionophores, and manganese. On the other hand, vasopressin treatment appears to decrease incorporation of (/sup 3/H)inositol into the hormone-sensitive pool, probably by shifting the equilibrium between phosphoinositides and inositol phosphates, since the decrease in radioactivity observed in the phosphoinositides is equaled by the increase observed in that in the inositol phosphates.

  16. Phosphatidic acid stimulates inositol 1,4,5-trisphosphate production in adult cardiac myocytes.

    PubMed

    Kurz, T; Wolf, R A; Corr, P B

    1993-03-01

    The cellular content of phosphatidic acid can increase in response to several agonists either by phosphorylation of diacylglycerol after phospholipase C-catalyzed hydrolysis of phospholipids or directly through activation of phospholipase D. Although previous findings indicated that the generation of phosphatidic acid was exclusively a means of regulation of the cellular concentration of diacylglycerol, more recent studies have indicated that phosphatidic acid may also directly regulate several cellular functions. Accordingly, the present study was performed to assess whether phosphatidic acid could stimulate cardiac phospholipase C in intact adult rabbit ventricular myocytes. The mass of inositol 1,4,5-trisphosphate [Ins (1,4,5)P3] was determined by a specific and sensitive binding protein assay and by direct mass measurement using anion exchange chromatography for separation of selected inositol phosphates and gas chromatography and mass spectrometry for quantification of inositol monophosphate (IP1), inositol bisphosphate (IP2), inositol trisphosphate (IP3), and inositol tetrakisphosphate (IP4). Phosphatidic acid (10(-9)-10(-6) M) elicited a rapid concentration-dependent increase in Ins (1,4,5)P3 accumulation, with the peak fourfold to fivefold increase at 30 seconds of stimulation; the concentration required for 50% of maximal stimulation was 4.4 x 10(-8) M. The time course of individual inositol phosphates indicated a successive increase in the mass of IP3, IP4, IP2, and IP1 in response to stimulation with phosphatidic acid. The production of Ins (1,4,5)P3 in response to phosphatidic acid was not altered in the absence of extracellular calcium or in the presence of extracellular EGTA (10(-3) M). Thus, these findings indicate that phosphatidic acid is a potent activator of inositol phosphate production in adult ventricular myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. New compatible solutes related to Di-myo-inositol-phosphate in members of the order Thermotogales.

    PubMed Central

    Martins, L O; Carreto, L S; Da Costa, M S; Santos, H

    1996-01-01

    The accumulation of intracellular organic solutes was examined in six species of the order Thermotogales by nuclear magnetic resonance spectroscopy. The newly discovered compounds di-2-O-beta-mannosyl-di-myo-inositol-1,1'(3,3')-phosphate and di-myo-inositol-1,3'-phosphate were identified in Thermotoga maritima and Thermotoga neapolitana. In the latter species, at the optimum temperature and salinity the organic solute pool was composed of di-myo-inositol-1,1'(3,3')-phosphate, beta-glutamate, and alpha-glutamate in addition to di-myo-inositol-1,3'-phosphate and di-2-O-beta-mannosyl-di-myo-inositol-1,1'(3,3')-phosphate. The concentrations of the last two solutes increased dramatically at supraoptimal growth temperatures, whereas beta-glutamate increased mainly in response to a salinity stress. Nevertheless, di-myo-inositol-1,1'(3,3')-phosphate was the major compatible solute at salinities above the optimum for growth. The amino acids alpha-glutamate and proline were identified under optimum growth conditions in Thermosipho africanus, and beta-mannosylglycerate, trehalose, and glycine betaine were detected in Petrotoga miotherma. Organic solutes were not detected, under optimum growth conditions, in Thermotoga thermarum and Fervidobacterium islandicum, which have a low salt requirement or none. PMID:8824608

  18. Does myo-inositol effect on PCOS follicles involve cytoskeleton regulation?

    PubMed

    Bizzarri, Mariano; Cucina, Alessandra; Dinicola, Simona; Harrath, Abdel Halim; Alwasel, Saleh H; Unfer, Vittorio; Bevilacqua, Arturo

    2016-06-01

    Inositol metabolism is severely impaired in follicles obtained from cystic ovaries, leading to deregulated insulin transduction and steroid synthesis. On the contrary, inositol administration to women suffering from polycystic ovary syndrome (PCOS) has been proven to efficiently counteract most of the clinical hallmarks displayed by PCOS patients, including insulin resistance, hyperandrogenism and oligo-amenorrhea. We have recently observed that myo-inositol induces significant changes in cytoskeletal architecture of breast cancer cells, by modulating different biochemical pathways, eventually modulating the epithelial-mesenchymal transition. We hypothesize that inositol and its monophosphate derivatives, besides their effects on insulin transduction, may efficiently revert histological and functional features of cystic ovary by inducing cytoskeleton rearrangements. We propose an experimental model that could address not only whether inositol modulates cytoskeleton dynamics in both normal and cystic ovary cells, but also whether this effect may interfere with ovarian steroidogenesis. A more compelling understanding of the mechanisms of action of inositol (and its derivatives) would greatly improve its therapeutic utilization, by conferring to current treatments a well-grounded scientific rationale. PMID:27142131

  19. Determination of phytic acid and inositol pentakisphosphates in foods by high-performance ion chromatography.

    PubMed

    Chen, Qingchuan

    2004-07-28

    A high-performance anion exchange chromatographic method was adapted for the quantitative determination of phytic acid and inositol pentakisphosphate isomers (excluding enantiomers) in foods. Because of the cost and limited availability of inositol phosphate standards, a phytic acid sodium salt standard was used for the calculation of an average relative response factor for the quantification of inositol pentakisphosphate isomers, and the purity of phytic acid sodium salt standard was also accurately established. The detection limits (S/N = 3) for phytic acid and inositol pentakisphosphates were in the range of 1.5-3.4 microM (0.1-0.2 microg/100 microL). This method has been successfully applied to the determination of phytic acid and inositol pentakisphosphates in a variety of beans and nuts after extraction with 0.5 M HCl and cleanup with solid phase extraction cartridges. The results demonstrated that there was a strong correlation between either the phytic acid content or the total content of phytic acid together with inositol pentakisphosphates and the total dietary fiber content in the group of all raw dry beans and in the group of raw dry black beans but not in the group of raw dry red kidney beans, which was probably due to the insufficient number of the raw dry red kidney bean samples. PMID:15264889

  20. Regioselective opening of myo-inositol orthoesters: mechanism and synthetic utility.

    PubMed

    Godage, Himali Y; Riley, Andrew M; Woodman, Timothy J; Thomas, Mark P; Mahon, Mary F; Potter, Barry V L

    2013-03-15

    Acid hydrolysis of myo-inositol 1,3,5-orthoesters, apart from orthoformates, exclusively affords the corresponding 2-O-acyl myo-inositol products via a 1,2-bridged five-membered ring dioxolanylium ion intermediate observed by NMR spectroscopy. These C-2-substituted inositol derivatives provide valuable precursors for rapid and highly efficient routes to 2-O-acyl inositol 1,3,4,5,6-pentakisphosphates and myo-inositol 1,3,4,5,6-pentakisphosphate with biologically interesting and anticancer properties. Deuterium incorporation into the α-methylene group of such alkyl ester products (2-O-C(O)CD2R), when the analogous alkyl orthoester is treated with deuterated acid, is established utilizing the novel orthoester myo-inositol 1,3,5-orthobutyrate as an example. Such deuterated ester products provide intermediates for deuterium-labeled synthetic analogues. Investigation into this selective formation of 2-O-ester products and the deuterium incorporation is presented with proposed mechanisms from NMR experiments.

  1. Fast separation and determination of free myo-inositol by hydrophilic liquid chromatography.

    PubMed

    Pazourek, Jiří

    2014-06-01

    A fast liquid chromatography method for separation and determination of myo-inositol is reported. Determination of the biologically important isomer of inositols, myo-inositol, was optimized to avoid overlapping to possible interferents according to European Pharmacopoeia (glycerol, d-mannitol) and saccharose. The method in HILIC mode is extremely selective to other carbohydrates which allows to separate myo-inositol from allo- and d-chiro-inositol with resolution 12.3 and 5.2, resp. and this way it enables to separate myo-inostiol from contingent carbohydrates present in a sample matrix. Retention time of myo-inositol was 12min at 10°C, though higher temperatures (25°C or 40°C) or higher water content in the mobile phase could speed up the separation and determination to four minutes. LOD of the method was 9mg/L at 10°C, and 5mg/L at 25°C, resp.

  2. D-chiro-inositol and pinitol extend the life span of Drosophila melanogaster.

    PubMed

    Hada, Binika; Yoo, Mi-Ra; Seong, Ki Moon; Jin, Young-Woo; Myeong, Hyeon-Koon; Min, Kyung-Jin

    2013-03-01

    D-chiro-inositol, a member of the inositol family, and pinitol, a 3-methoxy analogue of D-chiro-inositol, have been proposed to have antidiabetic, antiinflammatory, anticancer and stamina enhancing effects. We found that supplementing the diet of Drosophila with D-chiro-inositol and pinitol extended adult longevity in both male and female flies. Life span extension was accompanied by protection against oxidative and starvation stresses, improvement in health span, and no reduction in fecundity. Pinitol increased the fly life span, both in dietary restriction and in ad libitum conditions, suggesting that pinitol increased life span in a manner that was independent of the dietary restriction pathway. Nuclear localization of dFOXO increased in D-chiro-inositol and pinitol-fed flies when compared with controls. Pinitol treatment significantly activated JNK and S6K, but not AKT, indicating that the activation of dFOXO by pinitol is acquired by the activation of S6K and JNK signaling. Hence, our study indicated that D-chiro-inositol and pinitol could be novel food-derived antiaging compounds.

  3. Binding mechanism of inositol stereoisomers to monomers and aggregates of Aβ(16-22).

    PubMed

    Li, Grace; Pomès, Régis

    2013-06-01

    Alzheimer's disease (AD) is a severe neurodegenerative disease with no cure. A potential therapeutic approach is to prevent or reverse the amyloid formation of Aβ42, a key pathological hallmark of AD. We examine the molecular basis for stereochemistry-dependent inhibition of the formation of Aβ fibrils in vitro by a polyol, scyllo-inositol. We present molecular dynamics simulations of the monomeric, disordered aggregate, and protofibrillar states of Aβ(16-22), an amyloid-forming peptide fragment of full-length Aβ, successively with and without scyllo-inositol and its inactive stereoisomer chiro-inositol. Both stereoisomers bind monomers and disordered aggregates with similar affinities of 10-120 mM, whereas binding to β-sheet-containing protofibrils yields affinities of 0.2-0.5 mM commensurate with in vitro inhibitory concentrations of scyllo-inositol. Moreover, scyllo-inositol displays a higher binding specificity for phenylalanine-lined grooves on the protofibril surface, suggesting that scyllo-inositol coats the surface of Aβ protofibrils and disrupts their lateral stacking into amyloid fibrils.

  4. Inositol pyrophosphates regulate RNA polymerase I-mediated rRNA transcription in Saccharomyces cerevisiae.

    PubMed

    Thota, Swarna Gowri; Unnikannan, C P; Thampatty, Sitalakshmi R; Manorama, R; Bhandari, Rashna

    2015-02-15

    Ribosome biogenesis is an essential cellular process regulated by the metabolic state of a cell. We examined whether inositol pyrophosphates, energy-rich derivatives of inositol that act as metabolic messengers, play a role in ribosome synthesis in the budding yeast, Saccharomyces cerevisiae. Yeast strains lacking the inositol hexakisphosphate (IP6) kinase Kcs1, which is required for the synthesis of inositol pyrophosphates, display increased sensitivity to translation inhibitors and decreased protein synthesis. These phenotypes are reversed on expression of enzymatically active Kcs1, but not on expression of the inactive form. The kcs1Δ yeast cells exhibit reduced levels of ribosome subunits, suggesting that they are defective in ribosome biogenesis. The rate of rRNA synthesis, the first step of ribosome biogenesis, is decreased in kcs1Δ yeast strains, suggesting that RNA polymerase I (Pol I) activity may be reduced in these cells. We determined that the Pol I subunits, A190, A43 and A34.5, can accept a β-phosphate moiety from inositol pyrophosphates to undergo serine pyrophosphorylation. Although there is impaired rRNA synthesis in kcs1Δ yeast cells, we did not find any defect in recruitment of Pol I on rDNA, but observed that the rate of transcription elongation was compromised. Taken together, our findings highlight inositol pyrophosphates as novel regulators of rRNA transcription.

  5. Hydrolysis of inositol phospholipids precedes cellular proliferation in asbestos-stimulated tracheobronchial epithelial cells

    SciTech Connect

    Sesko, A.; Mossman, B. ); Cabot, M. )

    1990-10-01

    Metabolism of inositol phospholipids and phosphatidylcholine was investigated in tracheobronchial epithelial cells exposed to mitogenic concentrations of crocidolite asbestos. Alterations in levels of diacylglycerol, the endogenous activator of protein kinase C, and inositol polyphosphates, presumed mobilizers of intracellular calcium, were examined. Cultures labeled with ({sup 3}H)diacylglycerol. In contrast, crocidolite-exposed cells labeled with ({sup 3}H)myristic acid or ({sup 3}H)choline did not display elevated production of ({sup 3}H)diacylglycerol or release of ({sup 3}H)choline metabolites (i.e., evidence of phosphatidylcholine hydrolysis). The soluble tumor promoter phorbol 12-myristate 13-acetate catalyzed both of these changes. myo-({sup 3}H)Inositol-labeled cells exposed as briefly as 10 min to mitogenic concentrations of crocidolite demonstrated elevations in ({sup 3}H)inositol mono-, tris-, and terakisphosphates, phenomena indicating turnover of inositol phospholipids. The detection of diacylglycerol and inositol phosphates in crocidolite asbestos-exposed cells suggests that this fibrous tumor promoter activates phospholipase C as it stimulates cellular proliferation.

  6. Effects of inositol trisphosphate on calcium mobilization in high-voltage and saponin-permeabilized platelets

    SciTech Connect

    Gear, A.R.L.; Hallam, T.J.

    1986-03-01

    Interest in phosphatidylinositol metabolism has been greatly stimulated by the findings that diglyceride and inositol phosphates may serve as second messengers in modulating cellular function. Formation of 1,4,5-inositol trisphosphate (IP/sub 3/), in particular, has been linked to mobilization of intracellular calcium in a number of cell types. The authors have examined the ability of IP/sub 3/ to mobilize calcium in human platelets permeabilized by either saponin or high-voltage discharge. Saponin at 15 ..mu..g/ml effectively permeabilized platelets to exogenous inositol 1,4,5-trisphosphate which released bound (/sup 45/Ca) within 1 min and with a Ka of 7.4 +/- 4.1 ..mu..M. A small (25%) azide-sensitive pool was also responsive to inositol trisphosphate. The calcium pools were completely discharged by A-23187 and the ATP-dependent uptake was prevented by dinitrophenol. In contrast to the result with saponin, platelets accessed by high-voltage discharge were insensitive to challenge by inositol 1,4,5-trisphosphate. The data suggest that while inositol 1,4,5-trisphosphate can rapidly mobilize platelet calcium, the ability to demonstrate this depends on the method of permeabilization.

  7. Interconversion of inositol (1,4,5)-trisphosphate to inositol (1,3,4,5)-tetrakisphosphate and (1,3,4)-trisphosphate in permeabilized adrenal glomerulosa cells is calcium-sensitive and ATP-dependent

    SciTech Connect

    Rossier, M.F.; Dentand, I.A.; Lew, P.D.; Capponi, A.M.; Vallotton, M.B.

    1986-08-29

    The metabolism of (/sub 3/H)inositol (1,4,5)-trisphosphate was followed in permeabilized bovine adrenal glomerulosa cells. At low Ca++ concentration (pCa = 7.2), more than 90% of (/sub 3/H)inositol (1,4,5)-trisphosphate had disappeared within 2 min, while two other metabolites, (/sub 3/H)inositol (1,3,4)-trisphosphate and (/sub 3/H)inositol (1,3,4,5)-tetrakisphosphate appeared progressively. At higher Ca++ concentrations (pCa = 5.7 and 4.8), the formation of these two metabolites was markedly increased, but completely abolished if the medium was ATP-depleted. The peak levels for the generation of (/sub 3/H)inositol (1,3,4,5)-tetrakisphosphate (1 min) preceded those of (3H)inositol (1,3,4)-trisphosphate and were closely correlated. These results suggest that, in adrenal glomerulosa cells, the isomer inositol (1,3,4)-trisphosphate is generated from inositol (1,4,5)-trisphosphate via a calcium-sensitive and ATP-dependent phosphorylation/dephosphorylation pathway involving the formation of inositol (1,3,4,5)-tetrakisphosphate.

  8. The intracellular distribution of inositol polyphosphates in HL60 promyeloid cells.

    PubMed Central

    Stuart, J A; Anderson, K L; French, P J; Kirk, C J; Michell, R H

    1994-01-01

    1. HL60 promyeloid cells contain high intracellular concentrations of inositol polyphosphates, notably inositol 1,3,4,5,6-pentakisphosphate (InsP5) and inositol hexakisphosphate (InsP6). To determine their intracellular location(s), we studied the release of inositol (poly)phosphates, of ATP, and of cytosolic and granule-enclosed enzymes from cells permeabilized by four different methods. 2. When cells were treated with digitonin, all of the inositol phosphates were released in parallel with the cytosolic constituents. Most of the InsP5 and InsP6 was released before significant permeabilization of azurophil granules. 3. Similar results were obtained from cells preloaded with ethylene glycol and permeabilized by osmotic lysis. 4. Electroporation at approximately 500 V/cm caused rapid release of free inositol. Higher field strengths provoked release of most of the ATP, InsP5 and InsP6, but only slight release of the intracellular enzymes. Multiple discharges released approximately 80-90% of total InsP5 and InsP6. In the absence of bivalent-cation chelators, InsP5 and InsP6 were released less readily than ATP. 5. Treatment of cells with Staphylococcus aureus alpha-toxin caused quantitative release of inositol and ATP, without release of intracellular enzymes. However, inositol phosphates were released much less readily than inositol or ATP. Even after prolonged incubation with a high concentration of alpha-toxin, only approximately 50-70% of InsP2, InsP3 and InsP4 and < or = 20% of InsP5 and InsP6 were released, indicating that the high charge or large hydrated radius of InsP5 and InsP6 might limit their release through small toxin-induced pores. 6. These results indicate that most intracellular inositol metabolites are either in, or in rapid exchange with, the cytosolic compartment of HL60 cells. However, they leave open the possibility that a small proportion of cellular InsP5 and InsP6 (< or = 10-20%) might be in some intracellular bound form. Images Figure 2 PMID

  9. Determining the effects of inositol supplementation and the opi1 mutation on ethanol tolerance of Saccharomyces cerevisiae

    PubMed Central

    Krause, Erin L.; Villa-García, Manuel J.; Henry, Susan A.

    2009-01-01

    The yeast Saccharomyces cerevisiae is an important microorganism for the ethanol fuel industry. As with many microorganisms, the production and accumulation of certain metabolites, such as ethanol, can have a detrimental effect on cell growth and productivity. Yeast cells containing a higher concentration of phosphatidylinositol (PI) in the cellular membrane, due to inositol supplementation in the growth media, have been shown to tolerate and produce higher concentrations of ethanol. The specific goal of our research was to assess the effects of inositol supplementation in the growth media as well as to compare the ethanol tolerance of the wild-type S. cerevisiae to a mutant, the opi1 strain (opi=overproduction of inositol). The OPI1 gene product is a negative regulatory factor that controls the transcription of the INO1 structural gene, which encodes the enzyme catalyzing the limiting step in the biosynthesis of inositol, that is, the conversion of glucose-6-phosphate to inositol-3-phosphate. Upon the deletion of the OPI1 gene, the cell will constitutively produce inositol, regardless of the extracellular inositol concentration. Inositol supplementation in cultures of wild-type cells increased ethanol tolerance in terms of cell viability. Cells grown in −I media had a 20% higher specific death rate than cells grown in +I media when exposed to 15% ethanol. The opi1 strain, with the ability to constitutively produce inositol regardless of media composition, showed less inhibition of cell growth in the presence of ethanol than did the wild-type strain, particularly in inositol-free media. We conclude that the introduction of an opi1 mutation in yeast results in an inherent increase in PI levels and constitutive biosynthesis of inositol that, in turn, will reduce the cost of supplementing inositol into the media to achieve a higher ethanol tolerance. PMID:19812714

  10. Crystal Structure and Product Analysis of an Archaeal myo-Inositol Kinase Reveal Substrate Recognition Mode and 3-OH Phosphorylation.

    PubMed

    Nagata, Ryuhei; Fujihashi, Masahiro; Sato, Takaaki; Atomi, Haruyuki; Miki, Kunio

    2015-06-01

    The TK2285 protein from Thermococcus kodakarensis was recently characterized as an enzyme catalyzing the phosphorylation of myo-inositol. Only two myo-inositol kinases have been identified so far, the TK2285 protein and Lpa3 from Zea mays, both of which belong to the ribokinase family. In either case, which of the six hydroxyl groups of myo-inositol is phosphorylated is still unknown. In addition, little is known about the myo-inositol binding mechanism of these enzymes. In this work, we determined two crystal structures: those of the TK2285 protein complexed with the substrates (ATP analogue and myo-inositol) or the reaction products formed by the enzyme. Analysis of the ternary substrates-complex structure and site-directed mutagenesis showed that five residues were involved in the interaction with myo-inositol. Structural comparison with other ribokinase family enzymes indicated that two of the five residues, Q136 and R140, are characteristic of myo-inositol kinase. The crystal structure of the ternary products-complex, which was prepared by incubating the TK2285 protein with myo-inositol and ATP, holds 1d-myo-inositol 3-phosphate (Ins(3)P) in the active site. NMR and HPLC analyses with a chiral column also indicated that the TK2285 reaction product was Ins(3)P. The results obtained here showed that the TK2285 protein specifically catalyzes the phosphorylation of the 3-OH of myo-inositol. We thus designated TK2285 as myo-inositol 3-kinase (MI3K). The precise identification of the reaction product should provide a sound basis to further explore inositol metabolism in Archaea.

  11. Cellular Cations Control Conformational Switching of Inositol Pyrophosphate Analogues.

    PubMed

    Hager, Anastasia; Wu, Mingxuan; Wang, Huanchen; Brown, Nathaniel W; Shears, Stephen B; Veiga, Nicolás; Fiedler, Dorothea

    2016-08-22

    The inositol pyrophosphate messengers (PP-InsPs) are emerging as an important class of cellular regulators. These molecules have been linked to numerous biological processes, including insulin secretion and cancer cell migration, but how they trigger such a wide range of cellular responses has remained unanswered in many cases. Here, we show that the PP-InsPs exhibit complex speciation behaviour and propose that a unique conformational switching mechanism could contribute to their multifunctional effects. We synthesised non-hydrolysable bisphosphonate analogues and crystallised the analogues in complex with mammalian PPIP5K2 kinase. Subsequently, the bisphosphonate analogues were used to investigate the protonation sequence, metal-coordination properties, and conformation in solution. Remarkably, the presence of potassium and magnesium ions enabled the analogues to adopt two different conformations near physiological pH. Understanding how the intrinsic chemical properties of the PP-InsPs can contribute to their complex signalling outputs will be essential to elucidate their regulatory functions. PMID:27460418

  12. Enigmatic ion-exchange behavior of myo-inositol phosphates.

    PubMed

    Shelor, C Phillip; Liao, Hongzhu; Kadjo, Akinde Florence; Dasgupta, Purnendu K

    2015-05-01

    The separation of myo-inositol mono-, di-, tri-, tetra-, pentakis-, and hexakisphosphate (InsP1, InsP2, InsP3, InsP4, InsP5, InsP6) was carried out using hydroxide eluent ion chromatography. Acid hydrolysis of InsP6 (phytate) was used to prepare a distribution of InsPs, ranging from InsP1 to InsP5's and including unhydrolyzed InsP6. Counting all possible positional isomers (many of which have stereoisomers that will not be separable by conventional ion exchange), 40 chromatographically separable peaks are possible; up to 22 were separated and identified by mass spectrometry. InsPs show unusual ion-exchange behavior in two respects: (a) the retention order is not monotonically related with the charge on the ion and (b) at the same hydroxide eluent concentration, retention is greatly dependent on the eluent metal cation. The retention of InsP3-InsP6 was determined to be controlled by steric factors while elution was influenced by eluent cation complexation. These highly phosphorylated InsPs have a much greater affinity for alkali metals (Li(+) > Na(+) > K(+)) than quaternary ammonium ions. This difference in cation affinity was exploited to improve separation through the use of a tetramethylammonium hydroxide-sodium hydroxide gradient.

  13. Prometabolites of 5-Diphospho-myo-inositol Pentakisphosphate.

    PubMed

    Pavlovic, Igor; Thakor, Divyeshsinh T; Bigler, Laurent; Wilson, Miranda S C; Laha, Debabrata; Schaaf, Gabriel; Saiardi, Adolfo; Jessen, Henning J

    2015-08-10

    Diphospho-myo-inositol phosphates (PP-InsP(y)) are an important class of cellular messengers. Thus far, no method for the transport of PP-InsP(y) into living cells is available. Owing to their high negative charge density, PP-InsP(y) will not cross the cell membrane. A strategy to circumvent this issue involves the generation of precursors in which the negative charges are masked with biolabile groups. A PP-InsP(y) prometabolite would require twelve to thirteen biolabile groups, which need to be cleaved by cellular enzymes to release the parent molecules. Such densely modified prometabolites of phosphate esters and anhydrides have never been reported to date. This study discloses the synthesis of such agents and an analysis of their metabolism in tissue homogenates by gel electrophoresis. The acetoxybenzyl-protected system is capable of releasing 5-PP-InsP5 in mammalian cell/tissue homogenates within a few minutes and can be used to release 5-PP-InsP5 inside cells. These molecules will serve as a platform for the development of fundamental tools required to study PP-InsP(y) physiology.

  14. Metabolism of the phospholipid precursor inositol and its relationship to growth and viability in the natural auxotroph Schizosaccharomyces pombe.

    PubMed Central

    Fernandez, S; Homann, M J; Henry, S A; Carman, G M

    1986-01-01

    Phospholipid metabolism in the fission yeast Schizosaccharomyces pombe was examined. Three enzymes of phospholipid biosynthesis, cytidine diphosphate diacylglycerol synthase (CDP-DG), phosphatidylinositol (PI) synthase, and phosphatidylserine (PS) synthase, were characterized in extracts of S. pombe cells. Contrary to an earlier report, we were able to demonstrate that CDP-DG served as a precursor for PI and PS biosynthesis in S. pombe. S. pombe is naturally auxotrophic for the phospholipid precursor inositol. We found that S. pombe was much more resistant to loss of viability during inositol starvation than artificially generated inositol auxotrophs of Saccharomyces cerevisiae. The phospholipid composition of S. pombe cells grown in inositol-rich medium (50 microM) was similar to that of S. cerevisiae cells grown under similar conditions. However, growth of S. pombe at low inositol concentrations (below 30 microM) affected the ratio of the anionic phospholipids PI and PS, while the relative proportions of other glycerophospholipids remained unchanged. During inositol starvation, the rate of PI synthesis decreased rapidly, and there was a concomitant increase in the rate of PS synthesis. Phosphatidic acid and CDP-DG, which are precursors to these phospholipids, also increased when PI synthesis was blocked by lack of exogenous inositol. The major product of turnover of inositol-containing phospholipids in S. pombe was found to be free inositol, which accumulated in the medium and could be reused by the cell. Images PMID:3011744

  15. Efficient desymmetrization of 4,6-di-O-benzyl-myo-inositol by Lipozyme TL-IM.

    PubMed

    Vasconcelos, Marcela G; Briggs, Raissa H C; Aguiar, Lucia C S; Freire, Denise M G; Simas, Alessandro B C

    2014-03-11

    The enantioselective enzymatic desymmetrization of 4,6-di-O-benzyl-myo-inositol, a myo-inositol derivative, was effectively catalyzed by Thermomyces lanuginosus lipase (TL-IM). The product 1D-1-O-acetyl-4,6-di-O-benzyl-myo-inositol, a useful precursor to inositol phosphates, was obtained in excellent yield and enantiomeric excess. Through the investigation of the effects of solvent, biocatalyst load, and temperature, a more economical procedure resulted. The feasibility of biocatalyst reuse was also shown.

  16. Early effects of Escherichia coli endotoxin infusion on vasopressin-stimulated breakdown and metabolism of inositol lipids in rat hepatocytes

    SciTech Connect

    Rodriguez de Turco, E.B.; Spitzer, J.A.

    1988-08-30

    The turnover of vasopressin-stimulated 32P-phosphoinositides and 32P-phosphatidic acid and accumulation of (2-3H)-inositol phosphates were examined in hepatocytes from rats infused i.v. with saline and E. coli endotoxin for 3 hrs. Within 60s of VP stimulation the decrease in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate labeling as well as the increased uptake of 32P into phosphatidic acid were similar in both groups. However, at a later time (300s) the 32P-phosphatidylinositol turnover was greatly decreased concomitantly with a higher labeling of phosphatidic acid. The accumulation of (2-3H)-inositol phosphates in ET-cells was significantly decreased both at 30s and 600s after VP addition. The distribution of (2-3H)-inositol labeling accumulated in the different inositol phosphate fractions over the first 30s of VP stimulation showed a tendency to lower accumulation of inositol trisphosphate, and a significantly lower accumulation of inositol bisphosphate simultaneously with a higher labeling of the inositol tetrakisphosphate fraction. These observations reflect an early effect of ET-infusion on VP-stimulated inositol lipid turnover and on the subsequent metabolism of the released inositol phosphates.

  17. The diabetic rat kidney mediates inosituria and selective urinary partitioning of D-chiro-inositol

    PubMed Central

    Chang, Hao-Han; Choong, Bernard; Phillips, Anthony RJ

    2015-01-01

    Diabetic nephropathy is a serious complication of diabetes mellitus with a pressing need for effective metabolic markers to detect renal impairment. Of potential significance are the inositol compounds, myo-inositol (MI), and the less abundant stereoisomer, D-chiro-inositol (DCI), which are excreted at increased levels in the urine in diabetes mellitus, a phenomenon known as inosituria. There is also a selective urinary excretion of DCI compared to MI. As the biological origins of altered inositol metabolism in diabetes mellitus are unknown, the aim of this study was to determine whether the diabetic kidney was directly responsible. Kidneys isolated from four-week streptozotocin-induced diabetic rats were characterized by a 3-fold reduction in glomerular filtration rate (GFR) compared to matched non-diabetic kidneys. When perfused with fixed quantities of MI (50 µM) and DCI (5 µM) under normoglycemic conditions (5 mM glucose), GFR-normalized urinary excretion of MI was increased by 1.7-fold in diabetic vs. non-diabetic kidneys. By comparison, GFR-normalized urinary excretion of DCI was increased by 4-fold. Perfusion conditions replicating hyperglycemia (20 mM glucose) potentiated DCI but not MI urinary excretion in both non-diabetic and diabetic kidneys. Overall, there was a 2.4-fold increase in DCI urinary excretion compared to MI in diabetic kidneys that was independent of glucose ambience. This increased urinary excretion of DCI and MI in diabetic kidneys occurred despite increased renal expression of the inositol transporters, sodium myo-inositol transporter subtype 1 and 2 (SMIT1 and SMIT2). These findings show that the diabetic kidney primarily mediates inosituria and altered urinary partitioning of MI and DCI. Urinary inositol levels might therefore serve as an indicator of impaired renal function in diabetes mellitus with wider implications for monitoring chronic kidney disease. PMID:25060739

  18. Inositol Pyrophosphates Modulate S Phase Progression after Pheromone-induced Arrest in Saccharomyces cerevisiae*

    PubMed Central

    Banfic, Hrvoje; Bedalov, Antonio; York, John D.; Visnjic, Dora

    2013-01-01

    Several studies have demonstrated the activation of phosphoinositide-specific phospholipase C (Plc) in nuclei of mammalian cells during synchronous progression through the cell cycle, but the downstream targets of Plc-generated inositol 1,4,5-trisphosphate are poorly described. Phospholipid signaling in the budding yeast Saccharomyces cerevisiae shares similarities with endonuclear phospholipid signaling in mammals, and many recent studies point to a role for inositol phosphates, including InsP5, InsP6, and inositol pyrophosphates, in mediating the action of Plc. In this study, we investigated the changes in inositol phosphate levels in α-factor-treated S. cerevisiae, which allows cells to progress synchronously through the cell cycle after release from a G1 block. We found an increase in the activity of Plc1 early after release from the block with a concomitant increase in the levels of InsP7 and InsP8. Treatment of cells with the Plc inhibitor U73122 prevented increases in inositol phosphate levels and blocked progression of cells through S phase after pheromone arrest. The enzymatic activity of Kcs1 in vitro and HPLC analysis of [3H]inositol-labeled kcs1Δ cells confirmed that Kcs1 is the principal kinase responsible for generation of pyrophosphates in synchronously progressing cells. Analysis of plc1Δ, kcs1Δ, and ddp1Δ yeast mutants further confirmed the role that a Plc1- and Kcs1-mediated increase in pyrophosphates may have in progression through S phase. Our data provide genetic, metabolic, and biochemical evidence that synthesis of inositol pyrophosphates through activation of Plc1 and Kcs1 plays an important role in the signaling response required for cell cycle progression after mating pheromone arrest. PMID:23179856

  19. Dictyostelium discoideum contains three inositol monophosphatase activities with different substrate specificities and sensitivities to lithium.

    PubMed Central

    Van Dijken, P; Bergsma, J C; Hiemstra, H S; De Vries, B; Van Der Kaay, J; Van Haastert, P J

    1996-01-01

    The small ion lithium, a very effective agent in the treatment of manic depressive patients, inhibits the mammalian enzyme inositol monophosphatase, which is proposed as the biological target for the effects of lithium. In this study we investigated Dictyostelium discoideum inositol monophosphatase activity. Partial purification of the proteins in the soluble cell fraction using anion-exchange chromatography revealed the presence of at least three enzyme activities capable of degrading inositol monophosphate isomers. The first activity was similar to the monophosphatase found in mammalian cells, as it degraded Ins(4)P, Ins(1)P and to a lesser extent Ins(3)P, was dependent on MgCl2 and inhibited by LiCl in a uncompetitive [corrected] manner. The second enzyme activity was specific for Ins(4)P; the enzyme activity was not dependent on MgCl2 and not inhibited by LiCl. The third monophosphatase activity degraded especially Ins(3)P, but also Ins(4)P and Ins(1)P; increasing concentrations of MgCl2 inhibited this enzyme activity, whereas LiCl had no effect. In vivo, LiCl induces a reduction of inositol levels by about 20%. In [3H]inositol-labelled cells LiCl causes a 6-fold increase in the radioactivity of [3H]Ins(1)P, a doubling of [3H]Ins(4)P and a slight decrease in the radioactivity in [3H]Ins(3)P. These data indicate that the biological effects of lithium in Dictyostelium are not due to depletion of the inositol pool by inhibition of inositol monophosphatase activity. PMID:8670062

  20. The diabetic rat kidney mediates inosituria and selective urinary partitioning of D-chiro-inositol.

    PubMed

    Chang, Hao-Han; Choong, Bernard; Phillips, Anthony R J; Loomes, Kerry M

    2015-01-01

    Diabetic nephropathy is a serious complication of diabetes mellitus with a pressing need for effective metabolic markers to detect renal impairment. Of potential significance are the inositol compounds, myo-inositol (MI), and the less abundant stereoisomer, D-chiro-inositol (DCI), which are excreted at increased levels in the urine in diabetes mellitus, a phenomenon known as inosituria. There is also a selective urinary excretion of DCI compared to MI. As the biological origins of altered inositol metabolism in diabetes mellitus are unknown, the aim of this study was to determine whether the diabetic kidney was directly responsible. Kidneys isolated from four-week streptozotocin-induced diabetic rats were characterized by a 3-fold reduction in glomerular filtration rate (GFR) compared to matched non-diabetic kidneys. When perfused with fixed quantities of MI (50 µM) and DCI (5 µM) under normoglycemic conditions (5 mM glucose), GFR-normalized urinary excretion of MI was increased by 1.7-fold in diabetic vs. non-diabetic kidneys. By comparison, GFR-normalized urinary excretion of DCI was increased by 4-fold. Perfusion conditions replicating hyperglycemia (20 mM glucose) potentiated DCI but not MI urinary excretion in both non-diabetic and diabetic kidneys. Overall, there was a 2.4-fold increase in DCI urinary excretion compared to MI in diabetic kidneys that was independent of glucose ambience. This increased urinary excretion of DCI and MI in diabetic kidneys occurred despite increased renal expression of the inositol transporters, sodium myo-inositol transporter subtype 1 and 2 (SMIT1 and SMIT2). These findings show that the diabetic kidney primarily mediates inosituria and altered urinary partitioning of MI and DCI. Urinary inositol levels might therefore serve as an indicator of impaired renal function in diabetes mellitus with wider implications for monitoring chronic kidney disease.

  1. Tilapia (Oreochromis mossambicus) brain cells respond to hyperosmotic challenge by inducing myo-inositol biosynthesis.

    PubMed

    Gardell, Alison M; Yang, Jun; Sacchi, Romina; Fangue, Nann A; Hammock, Bruce D; Kültz, Dietmar

    2013-12-15

    This study aimed to determine the regulation of the de novo myo-inositol biosynthetic (MIB) pathway in Mozambique tilapia (Oreochromis mossambicus) brain following acute (25 ppt) and chronic (30, 60 and 90 ppt) salinity acclimations. The MIB pathway plays an important role in accumulating the compatible osmolyte, myo-inositol, in cells in response to hyperosmotic challenge and consists of two enzymes, myo-inositol phosphate synthase and inositol monophosphatase. In tilapia brain, MIB enzyme transcriptional regulation was found to robustly increase in a time (acute acclimation) or dose (chronic acclimation) dependent manner. Blood plasma osmolality and Na(+) and Cl(-) concentrations were also measured and significantly increased in response to both acute and chronic salinity challenges. Interestingly, highly significant positive correlations were found between MIB enzyme mRNA and blood plasma osmolality in both acute and chronic salinity acclimations. Additionally, a mass spectrometry assay was established and used to quantify total myo-inositol concentration in tilapia brain, which closely mirrored the hyperosmotic MIB pathway induction. Thus, myo-inositol is a major compatible osmolyte that is accumulated in brain cells when exposed to acute and chronic hyperosmotic challenge. These data show that the MIB pathway is highly induced in response to environmental salinity challenge in tilapia brain and that this induction is likely prompted by increases in blood plasma osmolality. Because the MIB pathway uses glucose-6-phosphate as a substrate and large amounts of myo-inositol are being synthesized, our data also illustrate that the MIB pathway likely contributes to the high energetic demand posed by salinity challenge.

  2. Evaluation of Myo-Inositol as a Potential Biomarker for Depression in Schizophrenia.

    PubMed

    Chiappelli, Joshua; Rowland, Laura M; Wijtenburg, S Andrea; Muellerklein, Florian; Tagamets, Malle; McMahon, Robert P; Gaston, Frank; Kochunov, Peter; Hong, L Elliot

    2015-08-01

    Depression is highly prevalent in patients with schizophrenia and is associated with significant clinical consequences, but there is no known biomarker for depression in schizophrenia. One of the putative neurochemical biomarkers for depression in major depressive disorder (MDD) is reduced cerebral concentration of myo-Inositol. We examined whether myo-Inositol levels provide a potential marker for depressive symptoms in schizophrenia similar to that in MDD and are informative regarding causal biological pathways underlying both depression and schizophrenia. We used proton magnetic resonance spectroscopy to examine myo-Inositol levels in the anterior cingulate cortex (ACC) in 59 schizophrenia spectrum disorder (SSD) patients and 69 matched community comparison participants. Participants completed the Maryland Trait and State Depression (MTSD) scale to measure symptoms of depression experienced around time of assessment ('State' subscale) and longitudinally ('Trait' subscale). Myo-Inositol in the ACC was negatively correlated with MTSD-Trait scores in both patients (ρ=-0.336, p=0.009) and community comparison samples (ρ=-0.328, p=0.006). Furthermore, patients with a diagnosis of schizoaffective disorder or a history of at least one major depressive episode had lower levels of myo-Inositol compared with schizophrenia patients without a current or past affective diagnosis (p=0.012). Since reduced brain myo-Inositol is associated with MDD, myo-Inositol may be a biochemical marker of depressive mood symptoms across diagnostic boundaries. If confirmed, this finding may aid investigation of the pathophysiology and therapeutics of depression common between depression, schizophrenia and other psychiatric diagnoses.

  3. Evaluation of Myo-Inositol as a Potential Biomarker for Depression in Schizophrenia

    PubMed Central

    Chiappelli, Joshua; Rowland, Laura M; Wijtenburg, S Andrea; Muellerklein, Florian; Tagamets, Malle; McMahon, Robert P; Gaston, Frank; Kochunov, Peter; Hong, L Elliot

    2015-01-01

    Depression is highly prevalent in patients with schizophrenia and is associated with significant clinical consequences, but there is no known biomarker for depression in schizophrenia. One of the putative neurochemical biomarkers for depression in major depressive disorder (MDD) is reduced cerebral concentration of myo-Inositol. We examined whether myo-Inositol levels provide a potential marker for depressive symptoms in schizophrenia similar to that in MDD and are informative regarding causal biological pathways underlying both depression and schizophrenia. We used proton magnetic resonance spectroscopy to examine myo-Inositol levels in the anterior cingulate cortex (ACC) in 59 schizophrenia spectrum disorder (SSD) patients and 69 matched community comparison participants. Participants completed the Maryland Trait and State Depression (MTSD) scale to measure symptoms of depression experienced around time of assessment (‘State' subscale) and longitudinally (‘Trait' subscale). Myo-Inositol in the ACC was negatively correlated with MTSD-Trait scores in both patients (ρ=−0.336, p=0.009) and community comparison samples (ρ=−0.328, p=0.006). Furthermore, patients with a diagnosis of schizoaffective disorder or a history of at least one major depressive episode had lower levels of myo-Inositol compared with schizophrenia patients without a current or past affective diagnosis (p=0.012). Since reduced brain myo-Inositol is associated with MDD, myo-Inositol may be a biochemical marker of depressive mood symptoms across diagnostic boundaries. If confirmed, this finding may aid investigation of the pathophysiology and therapeutics of depression common between depression, schizophrenia and other psychiatric diagnoses. PMID:25722115

  4. Asymmetric distribution of glucose and indole-3-acetyl-myo-inositol in geostimulated Zea mays seedlings

    NASA Technical Reports Server (NTRS)

    Momonoki, Y. S.; Bandurski, R. S. (Principal Investigator)

    1988-01-01

    Indole-3-acetyl-myo-inositol occurs in both the kernel and vegetative shoot of germinating Zea mays seedlings. The effect of a gravitational stimulus on the transport of [3H]-5-indole-3-acetyl-myo-inositol and [U-14C]-D-glucose from the kernel to the seedling shoot was studied. Both labeled glucose and labeled indole-3-acetyl-myo-inositol become asymmetrically distributed in the mesocotyl cortex of the shoot with more radioactivity occurring in the bottom half of a horizontally placed seedling. Asymmetric distribution of [3H]indole-3-acetic acid, derived from the applied [3H]indole-3-acetyl-myo-inositol, occurred more rapidly than distribution of total 3H-radioactivity. These findings demonstrate that the gravitational stimulus can induce an asymmetric distribution of substances being transported from kernel to shoot. They also indicate that, in addition to the transport asymmetry, gravity affects the steady state amount of indole-3-acetic acid derived from indole-3-acetyl-myo-inositol.

  5. Inositol lipid metabolism in vasopressin stimulated hepatocytes from rats infused with tumor necrosis factor

    SciTech Connect

    Spitzer, J.A.; Rodriguez de Turco, E.B. )

    1989-05-30

    We studied the effect of i.v. infusion of human recombinant tumor necrosis factor alpha (rHuTNF alpha, Cetus, 15 micrograms/100 g bw over 3 h) on vasopressin (VP)-stimulated {sup 32}P-inositol lipid turnover and the release of {sup 3}H-inositol phosphates in isolated rat hepatocytes. The early VP-induced decrease (within 30 s) in {sup 32}P-phosphatidylinositol 4-phosphate and {sup 32}P-phosphatidylinositol 4,5-bisphosphate labeling was significantly reduced (-40%) and at the same time the uptake of {sup 32}P into phosphatidic acid was 50% lower than in saline-infused (matched control) rats. Within 5 min of VP-stimulation, lower {sup 32}P phosphatidylinositol (-40%) and higher {sup 32}P-phosphatidic acid (+30%) labeling were observed in rHuTNF alpha-infused rats. Infusion of rHuTNF alpha also affected the VP-induced release of {sup 3}H-inositol phosphates. The accumulation of {sup 3}H-inositol-labeled water soluble products was decreased by 25% and 17% at 30 s and 10 min, respectively. These data show that rHuTNF alpha mimics early perturbations induced by Escherichia coli endotoxin infusion in VP-stimulated inositol lipid metabolism in rat hepatocytes.

  6. Metabolism and Ovarian Function in PCOS Women: A Therapeutic Approach with Inositols.

    PubMed

    Laganà, Antonio Simone; Rossetti, Paola; Buscema, Massimo; La Vignera, Sandro; Condorelli, Rosita Angela; Gullo, Giuseppe; Granese, Roberta; Triolo, Onofrio

    2016-01-01

    Polycystic ovary syndrome (PCOS) is characterized by chronical anovulation and hyperandrogenism which may be present in a different degree of severity. Insulin-resistance and hyperinsulinemia are the main physiopathological basis of this syndrome and the failure of inositol-mediated signaling may concur to them. Myo (MI) and D-chiro-inositol (DCI), the most studied inositol isoforms, are classified as insulin sensitizers. In form of glycans, DCI-phosphoglycan and MI-phosphoglycan control key enzymes were involved in glucose and lipid metabolism. In form of phosphoinositides, they play an important role as second messengers in several cellular biological functions. Considering the key role played by insulin-resistance and androgen excess in PCOS patients, the insulin-sensitizing effects of both MI and DCI were tested in order to ameliorate symptoms and signs of this syndrome, including the possibility to restore patients' fertility. Accumulating evidence suggests that both isoforms of inositol are effective in improving ovarian function and metabolism in patients with PCOS, although MI showed the most marked effect on the metabolic profile, whereas DCI reduced hyperandrogenism better. The purpose of this review is to provide an update on inositol signaling and correlate data on biological functions of these multifaceted molecules, in view of a rational use for the therapy in women with PCOS. PMID:27579037

  7. Metabolism and Ovarian Function in PCOS Women: A Therapeutic Approach with Inositols

    PubMed Central

    Rossetti, Paola; Buscema, Massimo; Condorelli, Rosita Angela; Gullo, Giuseppe; Triolo, Onofrio

    2016-01-01

    Polycystic ovary syndrome (PCOS) is characterized by chronical anovulation and hyperandrogenism which may be present in a different degree of severity. Insulin-resistance and hyperinsulinemia are the main physiopathological basis of this syndrome and the failure of inositol-mediated signaling may concur to them. Myo (MI) and D-chiro-inositol (DCI), the most studied inositol isoforms, are classified as insulin sensitizers. In form of glycans, DCI-phosphoglycan and MI-phosphoglycan control key enzymes were involved in glucose and lipid metabolism. In form of phosphoinositides, they play an important role as second messengers in several cellular biological functions. Considering the key role played by insulin-resistance and androgen excess in PCOS patients, the insulin-sensitizing effects of both MI and DCI were tested in order to ameliorate symptoms and signs of this syndrome, including the possibility to restore patients' fertility. Accumulating evidence suggests that both isoforms of inositol are effective in improving ovarian function and metabolism in patients with PCOS, although MI showed the most marked effect on the metabolic profile, whereas DCI reduced hyperandrogenism better. The purpose of this review is to provide an update on inositol signaling and correlate data on biological functions of these multifaceted molecules, in view of a rational use for the therapy in women with PCOS. PMID:27579037

  8. Purification and properties of myo-inositol-1-phosphate dehydrogenase from germinating mung bean seeds.

    PubMed

    Ghosh, B; De, B P; Biswas, B B

    1984-01-01

    A novel enzyme, myo-inositol-1-phosphate dehydrogenase, which catalyzes the conversion of myo-inositol 1-phosphate to ribulose 5-phosphate has been purified 84-fold from mung bean seedling employing several common techniques. The molecular weight of this purified enzyme has been recorded as 88,500 by Sephadex G-200 column chromatography, and in sodium dodecyl sulfate-polyacrylamide gel electrophoresis one protein band containing three subunits of Mr 32,000 each was discernible. Km values for NAD+ and myo-inositol 1-phosphate have been recorded as 2.8 X 10(-4) and 5.0 X 10(-4) M, respectively. Production of NADH in myo-inositol-1-phosphate dehydrogenase reaction has also been evidenced by measurement of NADH fluorescence. Dehydrogenation and decarboxylation of myo-inositol 1-phosphate are mediated by the same enzyme. In fact, the rate of dehydrogenation corroborates with that of decarboxylation. Stoichiometry of this reaction suggests that for the production of 1 mol of ribulose 5-phosphate 2 mol of NAD+ are reduced.

  9. Inositol trisphosphate metabolism in carrot (Daucus carota L. ) cells

    SciTech Connect

    Memon, A.R.; Rincon, M.; Boss, W.F. )

    1989-10-01

    The metabolism of exogenously added D-myo-(1-{sup 3}H)inositol 1,4,5-trisphosphate (IP{sub 3}) has been examined in microsomal membrane and soluble fractions of carrot cells grown in suspension culture. When ({sup 3}H)IP{sub 3} was added to a microsomal membrane fraction, ({sup 3}H)IP{sub 2} was the primary metabolite consisting of approximately 83% of the total recovered ({sup 3}H) by electrophoresis. ({sup 3}H)IP was only 6% of the ({sup 3}H) recovered, and 10% of the ({sup 3}H)IP{sub 3} was not further metabolized. In contrast, when ({sup 3}H)IP{sub 3} was added to the soluble fraction, approximately equal amounts of ({sup 3}H)IP{sub 2} and ({sup 3}H)IP were recovered. Ca{sup 2+} (100 micromolar) tended to enhance IP{sub 3} dephosphorylation but inhibited the IP{sub 2} dephosphorylation in the soluble fraction by about 20%. MoO{sub 4}{sup 2{minus}} (1 millimolar) inhibited the dephosphorylation of IP{sub 3} by the microsomal fraction and the dephosphorylation of IP{sub 2} by the soluble fraction. MoO{sub 4}{sup 2{minus}}, however, did not inhibit the dephosphorylation of IP{sub 3} by the soluble fraction. Li{sup +} (10 and 50 millimolar) had no effect on IP{sub 3} metabolism in either the soluble or membrane fraction; however, Li{sup +} (50 millimolar) inhibited IP{sub 2} dephosphorylation in the soluble fraction about 25%.

  10. Immunomodulating effect of inositol hexaphosphate against Aeromonas hydrophila-endotoxin.

    PubMed

    Abu-El-Saad, Abdel-Aziz S A

    2007-01-01

    The present study was carried out to evaluate the effect of inositol hexaphosphate (IP6) administration on endotoxemia as an example of the systemic inflammatory response. Mice were divided into three groups as follows: First group, remained as a naive group injected intraperitoneally (i.p.) with PBS (pH 7.4; 0.2 ml/mice) at intervals parallel to the treated groups. The second group was injected i.p. with the lipopolysaccharide (LPS) of Aeromonas hydrophila once a week for four weeks at a dose of LPS suspension: 20 mg/kg mice/week. The third group was injected with the same LPS dose and synergistically intubated with IP6 three times a week for four weeks at a total dose of 4 0mg/kg. At different experimental periods (1, 2, 3 and 4 weeks), six animals from each group were sacrificed under mild diethyl ether anesthesia. Blood and sera were taken for the estimation of phagocytic activity, electrophoretic pattern of proteins and immunoglobulin levels. Also, a slice of liver was homogenized to estimate the respiratory burst enzymes activities and nitric acid synthesis. Histopathological changes of hepatic tissues were investigated. In the LPS-treated group, marked increase in the phagocytic activities and nitric oxide synthesis, and a decrease in hepatocyte catalase, total peroxidase and superoxide dismutase activities were observed. The histopathological features revealed a degeneration and highly mitotic division within the hepatic nuclei in addition to some karyomegaly and nuclear pyknosis. During the treatment period, liver sections of the LPS+IP6 group showed somewhat regenerative features. Reduction in the toxicity of free radicals by IP6 was observed and the IP6 effect seemed to be responsible for the observed ameliorative influence.

  11. A second-generation Bacillus cell factory for rare inositol production.

    PubMed

    Tanaka, Kosei; Takanaka, Shinji; Yoshida, Ken-ichi

    2014-01-01

    Some rare inositol stereoisomers are known to exert specific health-promoting effects, including scyllo-inositol (SI), which is a promising therapeutic agent for Alzheimer disease. We recently reported a Bacillus subtilis cell factory that performed the efficient production of SI from the cheapest and most abundant isomer myo-inositol (MI). In the cell factory all "useless" genes involved in MI and SI metabolism were deleted and overexpression of the key enzymes, IolG and IolW, was appended. It converted 10 g/L MI into the same amount of SI in 48 h of cultivation. In this addendum, we discuss further improvement in the cell factory and its possible applications.

  12. Vasopressin induced production of inositol trisphosphate and calcium efflux in a smooth muscle cell line

    SciTech Connect

    Doyle, V.M.; Rueegg, U.T.

    1985-08-30

    Phosphatidylinositol metabolism and /sup 45/Ca/sup 2 +/ efflux were examined in a vascular smooth muscle cell line (A7r5). (Arg 8)Vasopressin stimulated the rapid formation (measurable at 1 sec) of inositol phosphates in a concentration-dependent manner. The time course for formation of inositol phosphates was similar to that for /sup 45/Ca/sup 2 +/ efflux from preloaded cells. The efflux of /sup 45/Ca/sup 2 +/ in response to (Arg8)vasopressin could be inhibited by a vasopressin antagonist. This supports the hypothesis that inositol 1,4,5-trisphosphate plays a role in vasopressin stimulated calcium mobilization from an intracellular source in cultured vascular smooth muscle cells.

  13. Chronic treatment with myo-inositol reduces white adipose tissue accretion and improves insulin sensitivity in female mice.

    PubMed

    Croze, Marine L; Vella, Roxane E; Pillon, Nicolas J; Soula, Hédi A; Hadji, Lilas; Guichardant, Michel; Soulage, Christophe O

    2013-02-01

    Type 2 diabetes is a complex disease characterized by a state of insulin resistance in peripheral tissues such as skeletal muscle, adipose tissue or liver. Some inositol isomers have been reported to possess insulin-mimetic activity and to be efficient in lowering blood glucose level. The aim of the present study was to assess in mice the metabolic effects of a chronic treatment with myo-inositol, the most common stereoisomer of inositol. Mice given myo-inositol treatment (0.9 or 1.2 mg g(-1) day(-1), 15 days, orally or intraperitoneally) exhibited an improved glucose tolerance due to a greater insulin sensitivity. Mice treated with myo-inositol exhibited a decreased white adipose tissue accretion (-33%, P<.005) compared with controls. The decrease in white adipose tissue deposition was due to a decrease in adipose cell volume (-33%, P<.05), while no change was noticed in total adipocyte number. In skeletal muscle, in vivo as well as ex vivo myo-inositol treatment increased protein kinase B/Akt phosphorylation under baseline and insulin-stimulated conditions, suggesting a synergistic action of myo-inositol treatment and insulin on proteins of the insulin signalling pathway. Myo-inositol could therefore constitute a viable nutritional strategy for the prevention and/or treatment of insulin resistance and type 2 diabetes.

  14. Inositol Pentakisphosphate Isomers Bind PH Domains with Varying Specificity and Inhibit Phosphoinositide Interactions

    SciTech Connect

    S Jackson; S Al-Saigh; C Schultz; M Junop

    2011-12-31

    PH domains represent one of the most common domains in the human proteome. These domains are recognized as important mediators of protein-phosphoinositide and protein-protein interactions. Phosphoinositides are lipid components of the membrane that function as signaling molecules by targeting proteins to their sites of action. Phosphoinositide based signaling pathways govern a diverse range of important cellular processes including membrane remodeling, differentiation, proliferation and survival. Myo-Inositol phosphates are soluble signaling molecules that are structurally similar to the head groups of phosphoinositides. These molecules have been proposed to function, at least in part, by regulating PH domain-phosphoinositide interactions. Given the structural similarity of inositol phosphates we were interested in examining the specificity of PH domains towards the family of myo-inositol pentakisphosphate isomers. In work reported here we demonstrate that the C-terminal PH domain of pleckstrin possesses the specificity required to discriminate between different myo-inositol pentakisphosphate isomers. The structural basis for this specificity was determined using high-resolution crystal structures. Moreover, we show that while the PH domain of Grp1 does not possess this high degree of specificity, the PH domain of protein kinase B does. These results demonstrate that some PH domains possess enough specificity to discriminate between myo-inositol pentakisphosphate isomers allowing for these molecules to differentially regulate interactions with phosphoinositides. Furthermore, this work contributes to the growing body of evidence supporting myo-inositol phosphates as regulators of important PH domain-phosphoinositide interactions. Finally, in addition to expanding our knowledge of cellular signaling, these results provide a basis for developing tools to probe biological pathway.

  15. Anti-inflammatory Inositol Derivatives from the Whole Plant of Inula cappa.

    PubMed

    Wu, Jiewei; Tang, Chunping; Yao, Sheng; Zhang, Lei; Ke, Changqiang; Feng, Linyin; Lin, Ge; Ye, Yang

    2015-10-23

    Twelve new inositol derivatives, classified into myoinositol (1-6) and l-inositol (10-15) types, along with five known analogues were isolated from the whole plant of Inula cappa. The structures of the new compounds were established by extensive analysis of mass spectrometric and 1D and 2D NMR spectroscopic data. All the tested compounds showed anti-inflammatory activities against the production of NO in RAW264.7 macrophages stimulated by lipopolysaccharide, with IC50 values ranging from 7 to 23 μM.

  16. Putative Key Role of Inositol Messengers in Endothelial Cells in Preeclampsia

    PubMed Central

    Kunjara, Sirilaksana; McLean, Patricia; Rademacher, Laurens; Rademacher, Thomas W.; Fascilla, Fabiana; Bettocchi, Stefano

    2016-01-01

    Immunological alterations, endothelial dysfunction, and insulin resistance characterize preeclampsia. Endothelial cells hold the key role in the pathogenesis of this disease. The signaling pathways mediating these biological abnormalities converge on PKB/Akt, an intracellular kinase regulating cell survival, proliferation, and metabolism. Inositol second messengers are involved in metabolic and cell signaling pathways and are highly expressed during preeclampsia. Intracellular action of these molecules is deeply affected by zinc, manganese, and calcium. To evaluate the pathophysiological significance, we present the response of the intracellular pathways of inositol phosphoglycans involved in cellular metabolism and propose a link with the disease. PMID:27738431

  17. Regulation of myo-inositol catabolism by a GntR-type repressor SCO6974 in Streptomyces coelicolor.

    PubMed

    Yu, Lingjun; Li, Shuxian; Gao, Wenyan; Pan, Yuanyuan; Tan, Huarong; Liu, Gang

    2015-04-01

    Myo-inositol is important for Streptomyces growth and morphological differentiation. Genomic sequence analysis revealed a myo-inositol catabolic gene cluster in Streptomyces coelicolor. Disruption of the corresponding genes in this cluster abolished the bacterial growth on myo-inositol as a single carbon source. The transcriptions of these genes were remarkably enhanced by addition of myo-inositol in minimal medium. A putative regulatory gene SCO6974, encoding a GntR family protein, is situated in the cluster. Disruption of SCO6974 significantly enhanced the transcription of myo-inositol catabolic genes. SCO6974 was shown to interact with the promoter regions of myo-inositol catabolic genes using electrophoretic mobility shift assays. DNase I footprinting assays demonstrated that SCO6974 recognized a conserved palindromic sequence (A/T)TGT(A/C)N(G/T)(G/T)ACA(A/T). Base substitution of the conserved sequence completely abolished the binding of SCO6974 to the targets demonstrating that SCO6974 directly represses the transcriptions of myo-inositol catabolic genes. Furthermore, the disruption of SCO6974 was correlated with a reduced sporulation of S. coelicolor in mannitol soya flour medium and with the overproduction of actinorhodin and calcium-dependent antibiotic. The addition of myo-inositol suppressed the sporulation deficiency of the mutant, indicating that the effect could be related to a shortage in myo-inositol due to its enhanced catabolism in this strain. This enhanced myo-inositol catabolism likely yields dihydroxyacetone phosphate and acetyl-CoA that are indirect or direct precursors of the overproduced antibiotics.

  18. Phytic acid and myo-inositol support adipocyte differentiation and improve insulin sensitivity in 3T3-L1 cells.

    PubMed

    Kim, Jin Nam; Han, Sung Nim; Kim, Hye-Kyeong

    2014-08-01

    Phytic acid, also known as myo-inositol hexaphosphate, has been shown to lower blood glucose levels and to improve insulin sensitivity in rodents. We investigated the effects of phytic acid and myo-inositol on differentiation, insulin-stimulated glucose uptake, and lipolysis of adipocytes to test the hypothesis that the antidiabetic properties of phytic acid and myo-inositol are mediated directly through adipocytes. 3T3-L1 cells were treated with 10, 50, or 200 μmol/L of phytic acid or myo-inositol. Oil Red O staining and an intracellular triacylglycerol assay were used to determine lipid accumulation during adipocyte differentiation. Immunoblotting and real-time polymerase chain reaction (PCR) were performed to evaluate expression of transcription factors, a target protein, and insulin signaling molecules. Phytic acid and myo-inositol exposures increased lipid accumulation in a dose-dependent manner (P < .01). The expression of key transcription factors associated with adipocyte differentiation, such as peroxisome proliferator-activated receptor γ (PPARγ) and sterol regulatory element-binding protein 1c, and the expression of fatty acid synthase increased upon treatments with phytic acid and myo-inositol (P < .05). Insulin-stimulated glucose uptake in mature adipocytes increased with phytic acid and myo-inositol treatments (P < .01). In addition, mRNA levels of insulin receptor substrate 1 (IRS1), mRNA levels of glucose transporter 4, and phosphorylation of tyrosine in IRS1 increased upon phytic acid and myo-inositol treatments. In fully differentiated adipocytes, phytic acid and myo-inositol reduced basal lipolysis dose dependently (P < .01). These results suggest that phytic acid and myo-inositol increase insulin sensitivity in adipocytes by increasing lipid storage capacity, improving glucose uptake, and inhibiting lipolysis.

  19. Metabolism of extracellular inositol hexaphosphate (phytate) by Saccharomyces cerevisiae.

    PubMed

    Andlid, Thomas A; Veide, Jenny; Sandberg, Ann-Sofie

    2004-12-15

    Iron and zinc deficiencies are global problems, frequently leading to severe illness in vulnerable human populations. Addition of phytases can improve the bioavailability of iron and zinc in food. Saccharomyces cerevisiae would be an ideal candidate as a bioavailability improving food additive if it demonstrates significant phytase activity. The purpose of the paper was to study yeast phytase activity to obtain information required to improve strains. All yeasts tested readily degraded extracellular inositol hexaphosphate (phytate; IP6) in media with IP6 as the sole phosphorous source. Phosphate (Pi) addition yielded repression consistent with the PHO system. However, repression of IP6-degrading enzymes was not only dependent on level of Pi, but also on pH and medium composition. In complex medium, containing Pi at a concentration previously suggested to yield full repression of the secretory acid phosphatases (SAPs; e.g., [Mol. Biol. Cell 11 (2000) 4309]), and at relatively high pH, repression of phytate-degrading enzymes was weak. The capacity to degrade phytate, irrespective of Pi addition or not, was highest at the pH most distant from the pH optimum of the SAPs [Microbiol. Res. 151 (1996) 291], suggesting that expression rather than enzyme activity was affected by pH. In synthetic medium, repression was strong and pH-independent (no IP6 degradation within the range tested). The distinct difference between media shows that, in addition to known regulatory role of Pi for the PHO system, additional factors may be involved. Using a deletion strain, we further demonstrate that the main secretory acid phosphatase Pho5p is not essential for intact phytate-degrading capacity and growth without Pi, neither is Pho3p. However, when constitutively overexpressing PHO5 an increased net phytase activity was obtained, in repressing and non-repressing conditions. This proves that, although redundant in a wild type, Pho5p can catalyze hydrolysis of IP6 and that at least one

  20. Inositol phosphate-induced stabilization of inositol 1,3,4,5,6-pentakisphosphate 2-kinase and its role in substrate specificity

    PubMed Central

    Gosein, Varin; Leung, Ting-Fung; Krajden, Oren; Miller, Gregory J

    2012-01-01

    Inositol phosphate kinases (IPKs) sequentially phosphorylate inositol phosphates (IPs) on their inositol rings to yield an array of signaling molecules. IPKs must possess the ability to recognize their physiological substrates from among a pool of over 30 cellular IPs that differ in numbers and positions of phosphates. Crystal structures from IPK subfamilies have revealed structural determinants for IP discrimination, which vary considerably between IPKs. However, recent structures of inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) did not reveal how IPK1 selectively recognizes its physiological substrate, IP5, while excluding others. Here, we report that limited proteolysis has revealed the presence of multiple conformational states in the IPK1 catalytic cycle, with notable protection from protease only in the presence of IP. Further, a 3.1-Å crystal structure of IPK1 bound to ADP in the absence of IP revealed decreased order in residues 110–140 within the N-lobe of the kinase compared with structures in which IP is bound. Using this solution and crystallographic data, we propose a model for recognition of IP substrate by IPK1 wherein phosphate groups at the 4-, 5-, and 6-positions are recognized initially by the C-lobe with subsequent interaction of the 1-position phosphate by Arg130 that stabilizes this residue and the N-lobe. This model explains how IPK1 can be highly specific for a single IP substrate by linking its interactions with substrate phosphate groups to the stabilization of the N- and C-lobes and kinase activation. PMID:22362712

  1. Novel compound heterozygous mutations in inositol polyphosphate phosphatase-like 1 in a family with severe opsismodysplasia.

    PubMed

    Feist, Cori; Holden, Paul; Fitzgerald, Jamie

    2016-10-01

    This study aimed to identify the genetic basis of a severe skeletal lethal dysplasia. The main clinical features of two affected fetuses included short limbs with flared metaphyses, bowed radii, femora and tibiae, irregular ossification of hands and feet, and marked platyspondyly. Affected and nonaffected family members were subjected to whole-exome sequencing, followed by immunoblot analysis on amniocytes isolated from one of the affected individuals. Unique compound heterozygous variants in the inositol polyphosphate phosphatase-like 1 (INPPL1) gene encoding the SHIP2 protein were identified in both affected individuals. One variant was inherited from each unaffected parent. Both allelic variants, c.(2327-1G>C);(1150_1151delGA), are predicted to result in premature stop codons leading to nonsense-mediated mRNA decay of the mutant alleles and no production of SHIP2. The absence of SHIP2 was confirmed by immunoblot analysis of proband amniocytes. This skeletal disorder is caused by the complete absence of the SHIP2 protein. INPPL1 mutations have been reported in opsismodysplasia, an autosomal recessive skeletal dysplasias with significant delayed bone formation. Our finding highlights the critical role that INPPL1/SHIP2 plays in skeletal development. PMID:27233067

  2. Lactic acid and thermal treatments trigger the hydrolysis of myo-inositol hexakisphosphate and modify the abundance of lower myo-inositol phosphates in barley (Hordeum vulgare L.).

    PubMed

    Metzler-Zebeli, Barbara U; Deckardt, Kathrin; Schollenberger, Margit; Rodehutscord, Markus; Zebeli, Qendrim

    2014-01-01

    Barley is an important source of dietary minerals, but it also contains myo-inositol hexakisphosphate (InsP6) that lowers their absorption. This study evaluated the effects of increasing concentrations (0.5, 1, and 5%, vol/vol) of lactic acid (LA), without or with an additional thermal treatment at 55°C (LA-H), on InsP6 hydrolysis, formation of lower phosphorylated myo-inositol phosphates, and changes in chemical composition of barley grain. Increasing LA concentrations and thermal treatment linearly reduced (P<0.001) InsP6-phosphate (InsP6-P) by 0.5 to 1 g compared to the native barley. In particular, treating barley with 5% LA-H was the most efficient treatment to reduce the concentrations of InsP6-P, and stimulate the formation of lower phosphorylated myo-inositol phosphates such as myo-inositol tetraphosphate (InsP4) and myo-inositol pentaphosphates (InsP5). Also, LA and thermal treatment changed the abundance of InsP4 and InsP5 isomers with Ins(1,2,5,6)P4 and Ins(1,2,3,4,5)P5 as the dominating isomers with 5% LA, 1% LA-H and 5% LA-H treatment of barley, resembling to profiles found when microbial 6-phytase is applied. Treating barley with LA at room temperature (22°C) increased the concentration of resistant starch and dietary fiber but lowered those of total starch and crude ash. Interestingly, total phosphorus (P) was only reduced (P<0.05) in barley treated with LA-H but not after processing of barley with LA at room temperature. In conclusion, LA and LA-H treatment may be effective processing techniques to reduce InsP6 in cereals used in animal feeding with the highest degradation of InsP6 at 5% LA-H. Further in vivo studies are warranted to determine the actual intestinal P availability and to assess the impact of changes in nutrient composition of LA treated barley on animal performance.

  3. Lactic Acid and Thermal Treatments Trigger the Hydrolysis of Myo-Inositol Hexakisphosphate and Modify the Abundance of Lower Myo-Inositol Phosphates in Barley (Hordeum vulgare L.)

    PubMed Central

    Metzler-Zebeli, Barbara U.; Deckardt, Kathrin; Schollenberger, Margit; Rodehutscord, Markus; Zebeli, Qendrim

    2014-01-01

    Barley is an important source of dietary minerals, but it also contains myo-inositol hexakisphosphate (InsP6) that lowers their absorption. This study evaluated the effects of increasing concentrations (0.5, 1, and 5%, vol/vol) of lactic acid (LA), without or with an additional thermal treatment at 55°C (LA-H), on InsP6 hydrolysis, formation of lower phosphorylated myo-inositol phosphates, and changes in chemical composition of barley grain. Increasing LA concentrations and thermal treatment linearly reduced (P<0.001) InsP6-phosphate (InsP6-P) by 0.5 to 1 g compared to the native barley. In particular, treating barley with 5% LA-H was the most efficient treatment to reduce the concentrations of InsP6-P, and stimulate the formation of lower phosphorylated myo-inositol phosphates such as myo-inositol tetraphosphate (InsP4) and myo-inositol pentaphosphates (InsP5). Also, LA and thermal treatment changed the abundance of InsP4 and InsP5 isomers with Ins(1,2,5,6)P4 and Ins(1,2,3,4,5)P5 as the dominating isomers with 5% LA, 1% LA-H and 5% LA-H treatment of barley, resembling to profiles found when microbial 6-phytase is applied. Treating barley with LA at room temperature (22°C) increased the concentration of resistant starch and dietary fiber but lowered those of total starch and crude ash. Interestingly, total phosphorus (P) was only reduced (P<0.05) in barley treated with LA-H but not after processing of barley with LA at room temperature. In conclusion, LA and LA-H treatment may be effective processing techniques to reduce InsP6 in cereals used in animal feeding with the highest degradation of InsP6 at 5% LA-H. Further in vivo studies are warranted to determine the actual intestinal P availability and to assess the impact of changes in nutrient composition of LA treated barley on animal performance. PMID:24967651

  4. Extraction and analysis of inositols and other carbohydrates from soybean plant tissues

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An outstanding characteristic of soybean plants is their ability to produce large amounts of the carbohydrate pinitol. Pinitol and the closely related inositols are currently undergoing widespread investigation for their biological and nutritional value. These and all the carbohydrates are typical...

  5. Production of glucaric acid from myo-inositol in engineered Pichia pastoris.

    PubMed

    Liu, Ye; Gong, Xu; Wang, Cui; Du, Guocheng; Chen, Jian; Kang, Zhen

    2016-09-01

    A potential myo-inositol oxygenase (ppMIOX) was identified as a functional enzyme and a glucaric acid synthetic pathway was firstly constructed in Pichia pastoris. Coexpression of the native ppMIOX and the urinate dehydrogenase (Udh) from Pseudomonas putida KT2440 led to obvious accumulation of glucaric acid (90.46±0.04mg/L) from myo-inositol whereas no glucaric acid was detected from glucose. In comparison, coexpression of the heterologous mouse MIOX (mMIOX) and Udh resulted in higher titers of glucaric acid from glucose and myo-inositol, 107.19±11.91mg/L and 785.4±1.41mg/L, respectively. By applying a fusion expression strategy with flexible peptides, the mMIOX specific activity and the glucaric acid concentration were significantly increased. Using glucose and myo-inositol as carbon substrates, the production of glucaric acid was substantially enhanced to 6.61±0.30g/L in fed-batch cultures. To the best of our knowledge, this is the highest reported value to date. PMID:27444324

  6. Rat brain myo-inositol 3-phosphate synthase is a phosphoprotein.

    PubMed

    Parthasarathy, R N; Lakshmanan, J; Thangavel, M; Seelan, R S; Stagner, J I; Janckila, A J; Vadnal, R E; Casanova, M F; Parthasarathy, L K

    2013-06-01

    The therapeutic effects of lithium in bipolar disorder are poorly understood. Lithium decreases free inositol levels by inhibiting inositol monophosphatase 1 and myo-inositol 3-phosphate synthase (IPS). In this study, we demonstrate for the first time that IPS can be phosphorylated. This was evident when purified rat IPS was dephosphorylated by lambda protein phosphatase and analyzed by phospho-specific ProQ-Diamond staining and Western blot analysis. These techniques demonstrated a mobility shift consistent with IPS being phosphorylated. Mass spectral analysis revealed that Serine-524 (S524), which resides in the hinge region derived from exon 11 of the gene, is the site for phosphorylation. Further, an antibody generated against a synthetic peptide of IPS containing monophosphorylated-S524, was able to discriminate the phosphorylated and non-phosphorylated forms of IPS. The phosphoprotein is found in the brain and testis, but not in the intestine. The intestinal IPS isoform lacks the peptide bearing S524, and hence, cannot be phosphorylated. Evidences suggest that IPS is monophosphorylated at S524 and that the removal of this phosphate does not alter its enzymatic activity. These observations suggest a novel function for IPS in brain and other tissues. Future studies should resolve the functional role of phospho-IPS in brain inositol signaling.

  7. myo-Inositol and Phytate Are Toxic to Formosan Subterranean Termites (Isoptera: Rhinotermitidae).

    PubMed

    Veillon, Lucas; Bourgeois, Jared; Leblanc, Amanda; Henderson, Gregg; Marx, Brian D; Muniruzzaman, Syed; Laine, Roger A

    2014-10-01

    Several rare and common monosaccharides were screened for toxic effects on the Formosan subterranean termite, Coptotermes formosanus Shiraki, with the aim of identifying environmentally friendly termiticides. myo-Inositol and phytic acid, which are nontoxic to mammals, were identified as potential termite control compounds. Feeding bioassays with termite workers, where both compounds were supplied on filter paper in concentrations from 160.2 to 1,281.7 μg/mm(3), showed concentration-dependent toxicity within 2 wk. Interestingly myo-inositol was nontoxic when administered to termites in agar (40 mg/ml) in the absence of a cellulosic food source, an unexplained phenomenon. In addition, decreased populations of termite hindgut protozoa were observed upon feeding on myo-inositol but not phytate-spiked filter paper. Radiotracer feeding studies using myo-inositol-[2-(3)H] with worker termites showed no metabolism after ingestion over a 2-d feeding period, ruling out metabolites responsible for the selective toxicity.

  8. How to Achieve High-Quality Oocytes? The Key Role of Myo-Inositol and Melatonin

    PubMed Central

    Rossetti, Paola; Corrado, Francesco; Rapisarda, Agnese Maria Chiara; Condorelli, Rosita Angela; Valenti, Gaetano; Sapia, Fabrizio; Buscema, Massimo

    2016-01-01

    Assisted reproductive technologies (ART) have experienced growing interest from infertile patients seeking to become pregnant. The quality of oocytes plays a pivotal role in determining ART outcomes. Although many authors have studied how supplementation therapy may affect this important parameter for both in vivo and in vitro models, data are not yet robust enough to support firm conclusions. Regarding this last point, in this review our objective has been to evaluate the state of the art regarding supplementation with melatonin and myo-inositol in order to improve oocyte quality during ART. On the one hand, the antioxidant effect of melatonin is well known as being useful during ovulation and oocyte incubation, two occasions with a high level of oxidative stress. On the other hand, myo-inositol is important in cellular structure and in cellular signaling pathways. Our analysis suggests that the use of these two molecules may significantly improve the quality of oocytes and the quality of embryos: melatonin seems to raise the fertilization rate, and myo-inositol improves the pregnancy rate, although all published studies do not fully agree with these conclusions. However, previous studies have demonstrated that cotreatment improves these results compared with melatonin alone or myo-inositol alone. We recommend that further studies be performed in order to confirm these positive outcomes in routine ART treatment.

  9. On the correlation between hydrogen bonding and melting points in the inositols.

    PubMed

    Bekö, Sándor L; Alig, Edith; Schmidt, Martin U; van de Streek, Jacco

    2014-01-01

    Inositol, 1,2,3,4,5,6-hexahydroxycyclohexane, exists in nine stereoisomers with different crystal structures and melting points. In a previous paper on the relationship between the melting points of the inositols and the hydrogen-bonding patterns in their crystal structures [Simperler et al. (2006 ▶). CrystEngComm 8, 589], it was noted that although all inositol crystal structures known at that time contained 12 hydrogen bonds per molecule, their melting points span a large range of about 170 °C. Our preliminary investigations suggested that the highest melting point must be corrected for the effect of molecular symmetry, and that the three lowest melting points may need to be revised. This prompted a full investigation, with additional experiments on six of the nine inositols. Thirteen new phases were discovered; for all of these their crystal structures were examined. The crystal structures of eight ordered phases could be determined, of which seven were obtained from laboratory X-ray powder diffraction data. Five additional phases turned out to be rotator phases and only their unit cells could be determined. Two previously unknown melting points were measured, as well as most enthalpies of melting. Several previously reported melting points were shown to be solid-to-solid phase transitions or decomposition points. Our experiments have revealed a complex picture of phases, rotator phases and phase transitions, in which a simple correlation between melting points and hydrogen-bonding patterns is not feasible.

  10. Blast neurotrauma impairs working memory and disrupts prefrontal myo-inositol levels in rats.

    PubMed

    Sajja, Venkata Siva Sai Sujith; Perrine, Shane A; Ghoddoussi, Farhad; Hall, Christina S; Galloway, Matthew P; VandeVord, Pamela J

    2014-03-01

    Working memory, which is dependent on higher-order executive function in the prefrontal cortex, is often disrupted in patients exposed to blast overpressure. In this study, we evaluated working memory and medial prefrontal neurochemical status in a rat model of blast neurotrauma. Adult male Sprague-Dawley rats were anesthetized with 3% isoflurane and exposed to calibrated blast overpressure (17 psi, 117 kPa) while sham animals received only anesthesia. Early neurochemical effects in the prefrontal cortex included a significant decrease in betaine (trimethylglycine) and an increase in GABA at 24 h, and significant increases in glycerophosphorylcholine, phosphorylethanolamine, as well as glutamate/creatine and lactate/creatine ratios at 48 h. Seven days after blast, only myo-inositol levels were altered showing a 15% increase. Compared to controls, short-term memory in the novel object recognition task was significantly impaired in animals exposed to blast overpressure. Working memory in control animals was negatively correlated with myo-inositol levels (r=-.759, p<0.05), an association that was absent in blast exposed animals. Increased myo-inositol may represent tardive glial scarring in the prefrontal cortex, a notion supported by GFAP changes in this region after blast overexposure as well as clinical reports of increased myo-inositol in disorders of memory.

  11. Myo-inositol improves the host's ability to eliminate balofloxacin-resistant Escherichia coli.

    PubMed

    Chen, Xin-Hai; Zhang, Bing-Wen; Li, Hui; Peng, Xuan-Xian

    2015-06-01

    Antibiotic-resistant mechanisms are associated with fitness costs. However, why antibiotic-resistant bacteria usually show increasing adaptation to hosts is largely unknown, especially from the host's perspective. The present study reveals the host's varied response to balofloxacin-resistant Escherichia coli (BLFX-R) using an integrated proteome and metabolome approach and identifies myo-inositol and phagocytosis-related proteins as crucial biomarkers. Originally, macrophages have an optimal attractive preference to BLFX-S due to more polarization of BLFX-S than BLFX-R, which renders faster elimination to BLFX-S than BLFX-R. The slower elimination to BLFX-R may be reversed by exogenous myo-inositol. Primarily, myo-inositol depolarizes macrophages, elevating adherence to both BLFX-S and BLFX-R. Since the altered adherence is equal to both strains, the myo-inositol-treated macrophages are free of the barrier to BLFX-R and thereby promote phagocytosis of BLFX-R. This work provides a novel strategy based on metabolic modulation for eliminating antibiotic-resistant bacteria with a high degree of host adaptation.

  12. On the correlation between hydrogen bonding and melting points in the inositols

    PubMed Central

    Bekö, Sándor L.; Alig, Edith; Schmidt, Martin U.; van de Streek, Jacco

    2014-01-01

    Inositol, 1,2,3,4,5,6-hexahydroxycyclohexane, exists in nine stereoisomers with different crystal structures and melting points. In a previous paper on the relationship between the melting points of the inositols and the hydrogen-bonding patterns in their crystal structures [Simperler et al. (2006 ▶). CrystEngComm 8, 589], it was noted that although all inositol crystal structures known at that time contained 12 hydrogen bonds per molecule, their melting points span a large range of about 170 °C. Our preliminary investigations suggested that the highest melting point must be corrected for the effect of molecular symmetry, and that the three lowest melting points may need to be revised. This prompted a full investigation, with additional experiments on six of the nine inositols. Thirteen new phases were discovered; for all of these their crystal structures were examined. The crystal structures of eight ordered phases could be determined, of which seven were obtained from laboratory X-ray powder diffraction data. Five additional phases turned out to be rotator phases and only their unit cells could be determined. Two previously unknown melting points were measured, as well as most enthalpies of melting. Several previously reported melting points were shown to be solid-to-solid phase transitions or decomposition points. Our experiments have revealed a complex picture of phases, rotator phases and phase transitions, in which a simple correlation between melting points and hydrogen-bonding patterns is not feasible. PMID:25075320

  13. How to Achieve High-Quality Oocytes? The Key Role of Myo-Inositol and Melatonin.

    PubMed

    Vitale, Salvatore Giovanni; Rossetti, Paola; Corrado, Francesco; Rapisarda, Agnese Maria Chiara; La Vignera, Sandro; Condorelli, Rosita Angela; Valenti, Gaetano; Sapia, Fabrizio; Laganà, Antonio Simone; Buscema, Massimo

    2016-01-01

    Assisted reproductive technologies (ART) have experienced growing interest from infertile patients seeking to become pregnant. The quality of oocytes plays a pivotal role in determining ART outcomes. Although many authors have studied how supplementation therapy may affect this important parameter for both in vivo and in vitro models, data are not yet robust enough to support firm conclusions. Regarding this last point, in this review our objective has been to evaluate the state of the art regarding supplementation with melatonin and myo-inositol in order to improve oocyte quality during ART. On the one hand, the antioxidant effect of melatonin is well known as being useful during ovulation and oocyte incubation, two occasions with a high level of oxidative stress. On the other hand, myo-inositol is important in cellular structure and in cellular signaling pathways. Our analysis suggests that the use of these two molecules may significantly improve the quality of oocytes and the quality of embryos: melatonin seems to raise the fertilization rate, and myo-inositol improves the pregnancy rate, although all published studies do not fully agree with these conclusions. However, previous studies have demonstrated that cotreatment improves these results compared with melatonin alone or myo-inositol alone. We recommend that further studies be performed in order to confirm these positive outcomes in routine ART treatment. PMID:27651794

  14. How to Achieve High-Quality Oocytes? The Key Role of Myo-Inositol and Melatonin

    PubMed Central

    Rossetti, Paola; Corrado, Francesco; Rapisarda, Agnese Maria Chiara; Condorelli, Rosita Angela; Valenti, Gaetano; Sapia, Fabrizio; Buscema, Massimo

    2016-01-01

    Assisted reproductive technologies (ART) have experienced growing interest from infertile patients seeking to become pregnant. The quality of oocytes plays a pivotal role in determining ART outcomes. Although many authors have studied how supplementation therapy may affect this important parameter for both in vivo and in vitro models, data are not yet robust enough to support firm conclusions. Regarding this last point, in this review our objective has been to evaluate the state of the art regarding supplementation with melatonin and myo-inositol in order to improve oocyte quality during ART. On the one hand, the antioxidant effect of melatonin is well known as being useful during ovulation and oocyte incubation, two occasions with a high level of oxidative stress. On the other hand, myo-inositol is important in cellular structure and in cellular signaling pathways. Our analysis suggests that the use of these two molecules may significantly improve the quality of oocytes and the quality of embryos: melatonin seems to raise the fertilization rate, and myo-inositol improves the pregnancy rate, although all published studies do not fully agree with these conclusions. However, previous studies have demonstrated that cotreatment improves these results compared with melatonin alone or myo-inositol alone. We recommend that further studies be performed in order to confirm these positive outcomes in routine ART treatment. PMID:27651794

  15. Muscarinic receptor-mediated inositol tetrakisphosphate response in bovine adrenal chromaffin cells

    SciTech Connect

    Sanborn, B.B.; Schneider, A.S. )

    1990-01-01

    Inositol trisphosphate (IP{sub 3}), a product of the phosphoinositide cycle, mobilizes intracellular Ca{sup 2+} in many cell types. New evidence suggests that inositol tetrakisphosphate (IP{sub 4}), an IP{sub 3} derivative, may act as another second messenger to further alter calcium homeostasis. However, the function and mechanism of action of IP{sub 4} are presently unresolved. We now report evidence of muscarinic receptor-mediated accumulation of IP{sub 4} in bovine adrenal chromaffin cells, a classic neurosecretory system in which calcium movements have been well studied. Muscarine stimulated an increase in ({sup 3}H)IP{sub 4} and ({sup 3}H)IP{sub 3} accumulation in chromaffin cells and this effect was completely blocked by atropine. ({sup 3}H)IP{sub 4} accumulation was detectable within 15 sec, increased to a maximum by 30 sec and thereafter declined. 2,3-diphosphoglycerate, an inhibitor of IP{sub 3} and IP{sub 4} hydrolysis, enhanced accumulation of these inositol polyphosphates. The results provide the first evidence of a rapid inositol tetrakisphosphate response in adrenal chromaffin cells, which should facilitate the future resolution of the relationship between IP{sub 4} and calcium homeostasis.

  16. Characterization of a tetrameric inositol monophosphatase from the hyperthermophilic bacterium Thermotoga maritima

    SciTech Connect

    Chen, L.; Roberts, M.F.

    1999-10-01

    Inositol monophosphatase (I-1-Pase) catalyzes the dephosphorylation step in the de novo biosynthetic pathway of inositol and is crucial for all inositol-dependent processes. An extremely heat-stable tetrameric form of I-1-Pase from the hyperthermophilic bacterium Thermotoga maritima was overexpressed in Escherichia coli. In addition to its different quaternary structure, this enzyme displayed a 20-fold higher rate of hydrolysis of D-inositol 1-phosphate than of the L isomer. The homogeneous recombinant T. maritima I-1-Pase possessed an unusually high V{sub max} that was much higher than the V{sub max} of the same enzyme from another hyperthermophile, Methanococcus jannaschii. Although T. maritima is a eubacterium, its I-1-Pase is more similar to archaeal I-1-Pases than to the other known bacterial or mammalian I-1-Pases with respect to substrate specificity, Li{sup +} inhibition, inhibition by high Mg{sup 2+} concentrations, metal ion activation, heat stability, and activation energy. Possible reasons for the observed kinetic differences are discussed based on an active site sequence alignment of the human and T. maritima I-1-Pases.

  17. GATA4-mediated cardiac hypertrophy induced by D-myo-inositol 1,4,5-tris-phosphate

    SciTech Connect

    Zhu Zhiming . E-mail: zhuzming@mail.dph-fsi.com; Zhu Shanjun; Liu Daoyan; Yu Zengping; Yang Yongjian; Giet, Markus van der; Tepel, Martin . E-mail: Martin.Tepel@charite.de

    2005-12-16

    We evaluated the effects of D-myo-inositol 1,4,5-tris-phosphate on cardiac hypertrophy. D-myo-inositol 1,4,5-tris-phosphate augmented cardiac hypertrophy as evidenced by its effects on DNA synthesis, protein synthesis, and expression of immediate-early genes c-myc and c-fos, {beta}-myosin heavy chain, and {alpha}-actin. The administration of D-myo-inositol 1,4,5-tris-phosphate increased the expression of nuclear factor of activated T-cells and cardiac-restricted zinc finger transcription factor (GATA4). Real-time quantitative RT-PCR showed that D-myo-inositol 1,4,5-tris-phosphate-induced GATA4 mRNA was significantly enhanced even in the presence of the calcineurin inhibitor, cyclosporine A. The effect of D-myo-inositol 1,4,5-tris-phosphate was blocked after inhibition of inositol-trisphosphate receptors but not after inhibition of c-Raf/mitogen-activated protein kinase kinase (MEK)/mitogen-activated protein kinase (ERK) or p38 mitogen-activated protein kinase pathways. The study shows that D-myo-inositol 1,4,5-tris-phosphate-induced cardiac hypertrophy is mediated by GATA4 but independent from the calcineurin pathway.

  18. Finding the sweet spot: how human fungal pathogens acquire and turn the sugar inositol against their hosts.

    PubMed

    Xue, Chaoyang

    2015-03-03

    Inositol is an essential nutrient with important structural and signaling functions in eukaryotes. Its role in microbial pathogenesis has been reported in fungi, protozoans, and eubacteria. In a recent article, Porollo et al. [mBio 5(6):e01834-14, 2014, doi:10.1128/mBio.01834-14] demonstrated the importance of inositol metabolism in the development and viability of Pneumocystis species--obligate fungal pathogens that remain unculturable in vitro. To understand their obligate nature, the authors used innovative comparative genomic approaches and discovered that Pneumocystis spp. are inositol auxotrophs due to the lack of inositol biosynthetic enzymes and that inositol insufficiency is a contributing factor preventing fungal growth in vitro. This work is in accord with other studies suggesting that inositol plays a conserved role in microbial pathogenesis. Inositol uptake and metabolism therefore may represent novel antimicrobial drug targets. Using comparative genomics to analyze metabolic pathways offers a powerful tool to gain new insights into nutrient utilization in microbes, especially obligate pathogens.

  19. Inositol hexakisphosphate kinase 1 (IP6K1) activity is required for cytoplasmic dynein-driven transport.

    PubMed

    Chanduri, Manasa; Rai, Ashim; Malla, Aushaq Bashir; Wu, Mingxuan; Fiedler, Dorothea; Mallik, Roop; Bhandari, Rashna

    2016-10-01

    Inositol pyrophosphates, such as diphosphoinositol pentakisphosphate (IP7), are conserved eukaryotic signaling molecules that possess pyrophosphate and monophosphate moieties. Generated predominantly by inositol hexakisphosphate kinases (IP6Ks), inositol pyrophosphates can modulate protein function by posttranslational serine pyrophosphorylation. Here, we report inositol pyrophosphates as novel regulators of cytoplasmic dynein-driven vesicle transport. Mammalian cells lacking IP6K1 display defects in dynein-dependent trafficking pathways, including endosomal sorting, vesicle movement, and Golgi maintenance. Expression of catalytically active but not inactive IP6K1 reverses these defects, suggesting a role for inositol pyrophosphates in these processes. Endosomes derived from slime mold lacking inositol pyrophosphates also display reduced dynein-directed microtubule transport. We demonstrate that Ser51 in the dynein intermediate chain (IC) is a target for pyrophosphorylation by IP7, and this modification promotes the interaction of the IC N-terminus with the p150(Glued) subunit of dynactin. IC-p150(Glued) interaction is decreased, and IC recruitment to membranes is reduced in cells lacking IP6K1. Our study provides the first evidence for the involvement of IP6Ks in dynein function and proposes that inositol pyrophosphate-mediated pyrophosphorylation may act as a regulatory signal to enhance dynein-driven transport. PMID:27474409

  20. PTH (parathyroid hormone) elevates inositol polyphosphates and diacylglycerol in a rat osteoblast-like cell line

    SciTech Connect

    Civitelli, R.; Reid, I.R.; Westbrook, S.; Avioli, L.V.; Hruska, K.A. )

    1988-11-01

    Parathyroid hormone (PTH)-stimulated signal transduction through mechanisms alternate to adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP) production were studied in UMR 106-01 cells, a cell line with an osteoblastic phenotype. PTH produced transient, dose-related increases in cytosolic calcium ((Ca{sup 2+}){sub i}), inositol trisphosphates, and diacylglycerol (DAG). Both inositol 1,4,5-trisphosphate (Ins-1,4,5P{sub 3}) and inositol 1,3,4-trisphosphate (Ins-1,3,4P{sub 3}) production were rapidly stimulated by PTH. Consistent with the production of Ins-1,3,4P{sub 3}, rapid stimulation of late eluting inositol tetrakisphosphate was observed. The effects on the inositol phosphates were induced rapidly, consistent with roles as signals for changes in (Ca{sup 2+}){sub i}. In saponin-permeabilized UMR 106-01 cells, Ins-1,4,5P{sub 3} stimulated {sup 45}Ca release from a nonmitochondrial intracellular pool. Thus the hypothesis that PTH-stimulated Ins-1,4,5P{sub 3} production initiates Ca{sup 2+} release and contributes to transient elevations of (Ca{sup 2+}){sub i} is supported. These data suggest that stimulation of cAMP production during PTH stimulation may negatively affect production of rises in (Ca{sup 2+}){sub i} during PTH stimulation. The inactivation of the inhibitory G protein of adenylate cyclase by pertussis toxin could explain its action similar to cAMP analogues. Cyclci nucleotides diminish the effects of PTH on (Ca{sup 2+}){sub i}, probably interacting on a biochemical step subsequent to or independent of Ins-1,4,5P{sub 3} release.

  1. Determination of Myo-Inositol in Infant, Pediatric, and Adult Formulas by Liquid Chromatography-Pulsed Amperometric Detection with Column Switching: Collaborative Study, Final Action 2011.18.

    PubMed

    Butler-Thompson, Linda D; Jacobs, Wesley A; Schimpf, Karen J

    2015-01-01

    AOAC First Action Method 2011.18, Myo-Inositol (Free and Bound as Phosphatidylinositol) in Infant and Pediatric Formulas and Adult Nutritionals, was collaboratively studied. With this method free myo-inositol and phosphatidylinositol bound myo-inositol are extracted using two different sample preparation procedures, separated by ion chromatography using a combination of Dionex Carbo Pac PA1 and MA1 columns with column switching, and detected with pulsed amperometry using a gold electrode. Free myo-inositol is extracted from samples with dilute hydrochloric acid and water. Phosphatidylinositol is extracted from samples with chloroform and separated from other fats with silica SPE cartridges. Myo-inositol is then released from the glycerol backbone with concentrated acetic and hydrochloric acids at 120°C. During this collaborative study, nine laboratories from five different countries analyzed blind duplicates of nine infant and pediatric nutritional formulas for both free and phosphatidylinositol bound myo-inositol, and one additional laboratory only completed the free myo-inositol analyses. The method demonstrated acceptable repeatability and reproducibility and met the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) Standard Method Performance Requirements (SMPRs®) for free myo-inositol plus phosphatidylinositol bound myo-inositol for all the matrixes analyzed. SMPRs for repeatability were ≤5% RSD at myo-inositol concentrations of 2-68 mg/100 g ready-to-feed (RTF) liquid. SMPRs for reproducibility were ≤8% RSD in products with myo-inositol concentrations ranging from 2 to 68 mg/100 g RTF liquid. During this collaborative study, repeatability RSDs ranged from 0.51 to 3.22%, and RSDs ranged from 2.66 to 7.55% for free myo-inositol plus phosphatidylinositol bound myo-inositol.

  2. The RpiR-Like Repressor IolR Regulates Inositol Catabolism in Sinorhizobium meliloti▿†

    PubMed Central

    Kohler, Petra R. A.; Choong, Ee-Leng; Rossbach, Silvia

    2011-01-01

    Sinorhizobium meliloti, the nitrogen-fixing symbiont of alfalfa, has the ability to catabolize myo-, scyllo-, and d-chiro-inositol. Functional inositol catabolism (iol) genes are required for growth on these inositol isomers, and they play a role during plant-bacterium interactions. The inositol catabolism genes comprise the chromosomally encoded iolA (mmsA) and the iolY(smc01163)RCDEB genes, as well as the idhA gene located on the pSymB plasmid. Reverse transcriptase assays showed that the iolYRCDEB genes are transcribed as one operon. The iol genes were weakly expressed without induction, but their expression was strongly induced by myo-inositol. The putative transcriptional regulator of the iol genes, IolR, belongs to the RpiR-like repressor family. Electrophoretic mobility shift assays demonstrated that IolR recognized a conserved palindromic sequence (5′-GGAA-N6-TTCC-3′) in the upstream regions of the idhA, iolY, iolR, and iolC genes. Complementation assays found IolR to be required for the repression of its own gene and for the downregulation of the idhA-encoded myo-inositol dehydrogenase activity in the presence and absence of inositol. Further expression studies indicated that the late pathway intermediate 2-keto-5-deoxy-d-gluconic acid 6-phosphate (KDGP) functions as the true inducer of the iol genes. The iolA (mmsA) gene encoding methylmalonate semialdehyde dehydrogenase was not regulated by IolR. The S. meliloti iolA (mmsA) gene product seems to be involved in more than only the inositol catabolic pathway, since it was also found to be essential for valine catabolism, supporting its more recent annotation as mmsA. PMID:21784930

  3. Synthesis of 2-diphospho-myo-inositol 1,3,4,5,6-pentakisphosphate and a photocaged analogue.

    PubMed

    Pavlovic, I; Thakor, D T; Jessen, H J

    2016-06-15

    Diphosphoinositol polyphosphates (inositol pyrophosphates, X-InsP7) are a family of second messengers with important roles in eukaryotic biology. Their chemical synthesis and modification remains a challenging task due to the high density of phosphate groups arranged around the myo-inositol core. Here, a novel approach is presented that facilitates the incorporation of the diphosphate in the 2-position (2-InsP7) and that enables the introduction of a photocage subunit. PMID:26923707

  4. Osmoregulatory inositol transporter SMIT1 modulates electrical activity by adjusting PI(4,5)P2 levels.

    PubMed

    Dai, Gucan; Yu, Haijie; Kruse, Martin; Traynor-Kaplan, Alexis; Hille, Bertil

    2016-06-01

    Myo-inositol is an important cellular osmolyte in autoregulation of cell volume and fluid balance, particularly for mammalian brain and kidney cells. We find it also regulates excitability. Myo-inositol is the precursor of phosphoinositides, key signaling lipids including phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. However, whether myo-inositol accumulation during osmoregulation affects signaling and excitability has not been fully explored. We found that overexpression of the Na(+)/myo-inositol cotransporter (SMIT1) and myo-inositol supplementation enlarged intracellular PI(4,5)P2 pools, modulated several PI(4,5)P2-dependent ion channels including KCNQ2/3 channels, and attenuated the action potential firing of superior cervical ganglion neurons. Further experiments using the rapamycin-recruitable phosphatase Sac1 to hydrolyze PI(4)P and the P4M probe to visualize PI(4)P suggested that PI(4)P levels increased after myo-inositol supplementation with SMIT1 expression. Elevated relative levels of PIP and PIP2 were directly confirmed using mass spectrometry. Inositol trisphosphate production and release of calcium from intracellular stores also were augmented after myo-inositol supplementation. Finally, we found that treatment with a hypertonic solution mimicked the effect we observed with SMIT1 overexpression, whereas silencing tonicity-responsive enhancer binding protein prevented these effects. These results show that ion channel function and cellular excitability are under regulation by several "physiological" manipulations that alter the PI(4,5)P2 setpoint. We demonstrate a previously unrecognized linkage between extracellular osmotic changes and the electrical properties of excitable cells. PMID:27217553

  5. Characterization of a ligand-gated cation channel based on an inositol receptor in the silkworm, Bombyx mori.

    PubMed

    Kikuta, Shingo; Endo, Haruka; Tomita, Natsuo; Takada, Tomoyuki; Morita, Chiharu; Asaoka, Kiyoshi; Sato, Ryoichi

    2016-07-01

    Insect herbivores recognize non-volatile compounds in plants to direct their feeding behavior. Gustatory receptors (Gr) appear to be required for nutrient recognition by gustatory organs in the mouthparts of insects. Gr10 is expressed in Bombyx mori (BmGr10) mouthparts such as maxillary galea, maxillary palp, and labrum. BmGr10 is predicted to function in sugar recognition; however, the precise biochemical function remains obscure. Larvae of B. mori are monophagous feeders able to find and feed on mulberry leaves. Soluble mulberry leaf extract contains sucrose, glucose, fructose, and myo-inositol. In this study, we identified BmGr10 as an inositol receptor using electrophysiological analysis with the Xenopus oocyte expression system and Ca(2+) imaging techniques using mammalian cells. These results demonstrated that Xenopus oocytes or HEK293T cells expressing BmGr10 specifically respond to myo-inositol and epi-inositol but do not respond to any mono-, di-, or tri-saccharides or to some sugar alcohols. These inositols caused Ca(2+) and Na(+) influxes into the cytoplasm independently of a G protein-mediated signaling cascade, indicating that BmGr10 is a ligand-gated cation channel. Overall, BmGr10 plays an important role in the myo-inositol recognition required for B. mori larval feeding behavior. PMID:27132146

  6. myo-Inositol synthesis from (1-/sup 3/H)glucose in Phaseolus vulgaris L. during early stages of germination

    SciTech Connect

    Sasaki, K.; Taylor, I.E.P.

    1986-06-01

    Radiolabeled D-(1-/sup 3/H)glucose was fed by imbibition under sterile conditions to bean (Phaseolus vulgaris L.) seeds. After 72 and 96 hours of feeding, the /sup 3/H was located in uronic acid and pentose residues as well as hexose residues of cell wall polysaccharides in growing hypocotyl and root. Free myo-inositol present in cotyledons, hypocotyl, and root also contained /sup 3/H, showing that de novo synthesis of myo-inositol from (1-/sup 3/H)glucose did occur during the first 72 hours of germination. More than 90% of the labeled, free myo-inositol was present in the cotyledons. The /sup 3/H percentage in trifluoroacetic acid-soluble arabinaose residues of cell wall polysaccharides from 72-hour-old bean hypocotyls was only half of their mole percentage. On the other hand, /sup 3/H percentages in hexose residues were higher than their mole percentages. The results suggest that myo-inositol is synthesized from reserve sugars during the very early stages of germination, and that the newly synthesized myo-inositol, as well as that stored in cotyledons, can be used for the construction of new hypocotyl and root cell wall polysaccharides after conversion into uronic acids and pentoses via the myo-inositol oxidation pathway.

  7. Bst1 is required for Candida albicans infecting host via facilitating cell wall anchorage of Glycosylphosphatidyl inositol anchored proteins

    PubMed Central

    Liu, Wei; Zou, Zui; Huang, Xin; Shen, Hui; He, Li Juan; Chen, Si Min; Li, Li Ping; Yan, Lan; Zhang, Shi Qun; Zhang, Jun Dong; Xu, Zheng; Xu, Guo Tong; An, Mao Mao; Jiang, Yuan Ying

    2016-01-01

    Glycosylphosphatidyl inositol anchored proteins (GPI-APs) on fungal cell wall are essential for invasive infections. While the function of inositol deacylation of GPI-APs in mammalian cells has been previously characterized the impact of inositol deacylation in fungi and implications to host infection remains largely unexplored. Herein we describe our identification of BST1, an inositol deacylase of GPI-Aps in Candida albicans, was critical for GPI-APs cell wall attachment and host infection. BST1-deficient C. albicans (bst1Δ/Δ) was associated with severely impaired cell wall anchorage of GPI-APs and subsequen unmasked β-(1,3)-glucan. Consistent with the aberrant cell wall structures, bst1Δ/Δ strain did not display an invasive ability and could be recognized more efficiently by host immune systems. Moreover, BST1 null mutants or those expressing Bst1 variants did not display inositol deacylation activity and exhibited severely attenuated virulence and reduced organic colonization in a murine systemic candidiasis model. Thus, Bst1 can facilitate cell wall anchorage of GPI-APs in C. albicans by inositol deacylation, and is critical for host invasion and immune escape. PMID:27708385

  8. PtdIns(3,4,5)P(3) and inositol depletion as a cellular target of mood stabilizers.

    PubMed

    Teo, Regina; King, Jason; Dalton, Emma; Ryves, Jonathan; Williams, Robin S B; Harwood, Adrian J

    2009-10-01

    Lithium (Li(+)) is the mood stabilizer most frequently used in the treatment of bipolar mood disorder; however, its therapeutic mechanism is unknown. In the 1980s, Berridge and colleagues proposed that Li(+) treatment acts via inhibition of IMPase (inositol monophosphatase) to deplete the cellular concentration of myo-inositol. Inositol depletion is also seen with the alternative mood stabilizers VPA (valproic acid) and CBZ (carbamazepine), suggesting a common therapeutic action. All three drugs cause changes in neuronal cell morphology and cell chemotaxis; however, it is unclear how reduced cellular inositol modulates these changes in cell behaviour. It is often assumed that reduced inositol suppresses Ins(1,4,5)P(3), a major intracellular signal molecule, but there are other important phosphoinostide-based signal molecules in the cell. In the present paper, we discuss evidence that Li(+) has a substantial effect on PtdIns(3,4,5)P(3), an important signal molecule within the nervous system. As seen for Ins(1,4,5)P(3) signalling, suppression of PtdIns(3,4,5)P(3) signalling also occurs via an inositol-depletion mechanism. This has implications for the cellular mechanisms controlling phosphoinositide signalling, and offers insight into the genetics underlying risk of bipolar mood disorder.

  9. Pharmacokinetics and Safety of a Single Intravenous Dose of myo-Inositol in Preterm Infants of 23 to 29 weeks

    PubMed Central

    Phelps, Dale L.; Ward, Robert M.; Williams, Rick L.; Watterberg, Kristi L.; Laptook, Abbot R.; Wrage, Lisa A.; Nolen, Tracy L.; Fennell, Timothy R.; Ehrenkranz, Richard A.; Poindexter, Brenda B.; Cotten, C. Michael; Hallman, Mikko K.; Frantz, Ivan D.; Faix, Roger G.; Zaterka-Baxter, Kristin M.; Das, Abhik; Ball, M. Bethany; O’Shea, T. Michael; Lacy, Conra Backstrom; Walsh, Michele C.; Shankaran, Seetha; Sánchez, Pablo J.; Bell, Edward F.; Higgins, Rosemary D.

    2014-01-01

    Background Myo-inositol given to preterm infants with respiratory distress has reduced death, increased survival without bronchopulmonary dysplasia (BPD) and reduced severe retinopathy of prematurity (ROP) in 2 randomized trials. Pharmacokinetic (PK) studies in extremely preterm infants are needed prior to efficacy trials. Methods Infants of 23–29 weeks gestation were randomized to a single intravenous (IV) dose of inositol at 60 or 120 mg/kg or placebo. Over 96 h, serum levels (sparse sampling population PK) and urine inositol excretion were determined. Population PK models were fit using a nonlinear mixed effects approach. Safety outcomes were recorded. Results A 1-compartment model that included factors for endogenous inositol production, allometric size based on weight, gestational age (GA) strata and creatinine clearance fit the data best. The central volume of distribution was 0.5115 l/kg, the clearance 0.0679 l/kg/h, endogenous production 2.67 mg/kg/h and the half life 5.22 h when modeled without the covariates. During the first 12 h renal inositol excretion quadrupled in the 120 mg/kg group, returning to near baseline after 48 h. There was no diuretic side-effect. No significant differences in adverse events occurred between the 3 groups (p > 0.05). Conclusions A single compartment model accounting for endogenous production satisfactorily described the PK of IV inositol. PMID:24067395

  10. Myo-Inositol Esters of Indole-3-acetic Acid as Seed Auxin Precursors of Zea mays L. 1

    PubMed Central

    Nowacki, Janusz; Bandurski, Robert S.

    1980-01-01

    Indole-3-acetyl-myo-inositol esters constitute 30% of the low molecular weight derivatives of indole-3-acetic acid (IAA) in seeds of Zea mays. [14C]Indole-3-acetyl-myo-inositol was applied to a cut in the endosperm of the seed and found to be transported from endosperm to shoot at 400 times the rate of transport of free IAA. The rate of transport of indole-3-acetyl-myo-inositol from endosperm to shoot was 6.3 picomoles per shoot per hour and thus adequate to serve as the seed auxin precursor for the free IAA diffusing downward from the shoot tip. Indole-3-acetyl-myo-inositol is the first seed auxin precursor to be identified. Application of either [14C]IAA or 14C-indole-3-acetyl-myo-inositol ester to the endosperm results in both free and esterified [14C]IAA in the seedling shoot. Esterification of free IAA and hydrolysis of indole-3-acetyl-myo-inositol occurred in the shoot and not the endosperm yielding ratios of ester to free IAA which approximate the ratios of ester to free IAA normally found in corn shoot tissue. This proves, for the first time, that esterified IAA and free IAA are interconvertible in the growing shoot. Since free IAA may be limiting for plant growth, knowledge that the free hormone is in “equilibrium” with its conjugates suggests new methods for the control of plant growth. PMID:16661205

  11. Huntington’s disease: Neural dysfunction linked to inositol polyphosphate multikinase

    PubMed Central

    Ahmed, Ishrat; Sbodio, Juan I.; Harraz, Maged M.; Tyagi, Richa; Grima, Jonathan C.; Albacarys, Lauren K.; Hubbi, Maimon E.; Xu, Risheng; Kim, Seyun; Paul, Bindu D.; Snyder, Solomon H.

    2015-01-01

    Huntington’s disease (HD) is a progressive neurodegenerative disease caused by a glutamine repeat expansion in mutant huntingtin (mHtt). Despite the known genetic cause of HD, the pathophysiology of this disease remains to be elucidated. Inositol polyphosphate multikinase (IPMK) is an enzyme that displays soluble inositol phosphate kinase activity, lipid kinase activity, and various noncatalytic interactions. We report a severe loss of IPMK in the striatum of HD patients and in several cellular and animal models of the disease. This depletion reflects mHtt-induced impairment of COUP-TF-interacting protein 2 (Ctip2), a striatal-enriched transcription factor for IPMK, as well as alterations in IPMK protein stability. IPMK overexpression reverses the metabolic activity deficit in a cell model of HD. IPMK depletion appears to mediate neural dysfunction, because intrastriatal delivery of IPMK abates the progression of motor abnormalities and rescues striatal pathology in transgenic murine models of HD. PMID:26195796

  12. Modulation of HIV-like particle assembly in vitro by inositol phosphates

    PubMed Central

    Campbell, Stephen; Fisher, Robert J.; Towler, Eric M.; Fox, Stephen; Issaq, Haleem J.; Wolfe, Tracy; Phillips, Lawrence R.; Rein, Alan

    2001-01-01

    HIV-1 Gag protein assembles into 100- to 120-nm diameter particles in mammalian cells. Recombinant HIV-1 Gag protein assembles in a fully defined system in vitro into particles that are only 25–30 nm in diameter and that differ significantly in other respects from authentic particles. However, particles with the size and other properties of authentic virions were obtained in vitro by addition of inositol phosphates or phosphatidylinsitol phosphates to the assembly system. Thus, the interactions between HIV-1 Gag protein molecules are altered by binding of inositol derivatives; this binding is apparently essential for normal HIV-1 particle assembly. This requirement is not seen in a deleted Gag protein lacking residues 16–99 within the matrix domain. PMID:11526217

  13. Purification, crystallization and preliminary X-ray analysis of inositol dehydrogenase (IDH) from Bacillus subtilis

    SciTech Connect

    Van Straaten, K. E.; Hoffort, A.; Palmer, D. R. J.; Sanders, D. A. R.

    2008-02-01

    Selenomethionine-substituted IDH was crystallized using the microbatch method. The crystals diffracted to beyond 2.0 Å resolution using synchrotron radiation. Inositol dehydrogenase (IDH) is an enzyme that catalyses the NAD{sup +}-dependent oxidation of myo-inositol to scyllo-inosose. The enzyme has been purified to homogeneity by means of Ni{sup 2+}-affinity chromatography and was crystallized in both native and selenomethionine (SeMet) labelled forms using the microbatch method. SAD X-ray diffraction data were collected to 2.0 Å resolution from a SeMet-labelled crystal at the Advanced Photon Source (APS) and a MAD data set was collected to 1.75 Å resolution at the Canadian Light Source (CLS); this is the first reported anomalous diffraction experiment from the CLS. The crystals belong to space group I222 and contain one molecule per asymmetric unit.

  14. Altered coupling between aortic adrenergic-receptors and inositol phosphate accumulation in aldosterone hypertension

    SciTech Connect

    Jones, A.W.; Bylund, D.B.; Shukla, S.D.; Magliola, L.; Smith, J.M.; Ray-Pranger, C.; Bailey, B.

    1986-03-05

    The authors previous studies of /sup 42/K efflux have shown a 6-10 fold increase in sensitivity to ..cap alpha..-receptor agonists in aortas from rats (AHR) made hypertensive with aldosterone infusion, uninephrectomy plus high salt intake. Analyses of ..cap alpha..-receptor properties, however, showed no significant difference in sub-type, affinity or maximum binding. They initiated this study to determine whether supersensitivity is associated with altered accumulation of inositol phosphates. Aortic strips were equilibrated in /sup 3/H-inositol PSS for 2 hrs, followed by 10 min exposures to inositol (10 mM) PSS then Li (10 mM) PSS. Strips were placed in Li-PSS +/- norepinephrine (NE) for 30 minutes followed by freeze clamping and analyses of inositol phosphates (IP, IP/sub 2/, IP/sub 3/) by standard methods. In controls (C) NE (3 ..mu..M) increased the CPM 2, 14, and 9 fold respectively (p < 0.001). Sufficient CPM were available to analyze the NE concentration effects on IP and IP/sub 2/. NE = 3 ..mu..M yielded similar CPM per cell water in C (n=6) and AHR (n=5). The EC/sub 50/ for AHR was 5-6 fold lower than C for IP (56 +/- 18 versus 300 +/- 130 nM, p < 0.03) and IP/sub 2/ (57 +/- 14 versus 410 +/- 60 nM, p < 0.001). The K/sub a/ for Ne was 480 nM in C and AHR as determined by fractional receptor inactivation with dibenamine. These preliminary findings indicate a close relation in C between ..cap alpha..-receptor occupancy and accumulation of IP and IP/sub 2/, while supersensitivity in AHR is associated with an increased efficacy in coupling between ..cap alpha..-receptors and polyphosphoinositide metabolism.

  15. Phorbol esters promote alpha 1-adrenergic receptor phosphorylation and receptor uncoupling from inositol phospholipid metabolism.

    PubMed Central

    Leeb-Lundberg, L M; Cotecchia, S; Lomasney, J W; DeBernardis, J F; Lefkowitz, R J; Caron, M G

    1985-01-01

    DDT1 MF-2 cells, which are derived from hamster vas deferens smooth muscle, contain alpha 1-adrenergic receptors (54,800 +/- 2700 sites per cell) that are coupled to stimulation of inositol phospholipid metabolism. Incubation of these cells with tumor-promoting phorbol esters, which stimulate calcium- and phospholipid-dependent protein kinase, leads to a marked attenuation of the ability of alpha 1-receptor agonists such as norepinephrine to stimulate the turnover of inositol phospholipids. This turnover was measured by determining the 32P content of phosphatidylinositol and phosphatidic acid after prelabeling of the cellular ATP pool with 32Pi. These phorbol ester-treated cells also displayed a decrease in binding affinity of cellular alpha 1 receptors for agonists with no change in antagonist affinity. By using affinity chromatography on the affinity resin Affi-Gel-A55414, the alpha 1 receptors were purified approximately equal to 300-fold from control and phorbol ester-treated 32Pi-prelabeled cells. As assessed by NaDodSO4/polyacrylamide gel electrophoresis, the Mr 80,000 alpha 1-receptor ligand-binding subunit is a phosphopeptide containing 1.2 mol of phosphate per mol of alpha 1 receptor. After phorbol ester treatment this increased to 3.6 mol of phosphate per mol of alpha 1 receptor. The effect of phorbol esters on norepinephrine-stimulated inositol phospholipid turnover and alpha 1-receptor phosphorylation showed the same rapid time course with a t1/2 less than 2 min. These results indicate that calcium- and phospholipid-dependent protein kinase may play an important role in regulating the function of receptors that are coupled to the inositol phospholipid cycle by phosphorylating and deactivating them. Images PMID:2994039

  16. Transient calcium release induced by successive increments of inositol 1,4,5-trisphosphate.

    PubMed Central

    Meyer, T; Stryer, L

    1990-01-01

    Many hormonal, neurotransmitter, and sensory stimuli trigger the formation of inositol 1,4,5-trisphosphate, which in turn releases calcium from intracellular stores. We report here that inositol 1,4,5-trisphosphate-induced calcium release from saponin-permeabilized rat basophilic leukemia cells at 37 degrees C is markedly biphasic, in contrast with nearly monophasic release kinetics at 11 degrees C. Hepatoma, PC-12 neuronal cells, and several other cell types exhibit similar biphasic release at 37 degrees C. The biphasic kinetics are not due to degradation of inositol 1,4,5-trisphosphate or to increased Ca2(+)-ATPase pump activity. Biphasic calcium release was also seen when ATP was quenched to less than 0.4 microM by adding hexokinase and glucose, suggesting that phosphorylation is not involved. External calcium (100 nM-600 nM) range had little influence on the biphasic kinetics. Rapid-mixing experiments revealed that rapid efflux of calcium is followed in approximately 0.5 s by a 30-fold slower efflux. Most striking, successive additions of the same amount of inositol 1,4,5-trisphosphate induced short bursts of calcium release of similar size. This retention of responsiveness, which we term increment detection, may be a distinct mode of signal transduction. Like inactivation and adaptation, increment detection gives rise to transient responses to sustained stimuli. Systems exhibiting inactivation, adaptation, and increment detection differ in their responsiveness (none, partial, and full, respectively) to stepwise increases in stimulus intensity. Increment detection could be advantageous in generating receptor-triggered calcium oscillations. Images PMID:2339124

  17. An uncharacterized member of the ribokinase family in Thermococcus kodakarensis exhibits myo-inositol kinase activity.

    PubMed

    Sato, Takaaki; Fujihashi, Masahiro; Miyamoto, Yukika; Kuwata, Keiko; Kusaka, Eriko; Fujita, Haruo; Miki, Kunio; Atomi, Haruyuki

    2013-07-19

    Here we performed structural and biochemical analyses on the TK2285 gene product, an uncharacterized protein annotated as a member of the ribokinase family, from the hyperthermophilic archaeon Thermococcus kodakarensis. The three-dimensional structure of the TK2285 protein resembled those of previously characterized members of the ribokinase family including ribokinase, adenosine kinase, and phosphofructokinase. Conserved residues characteristic of this protein family were located in a cleft of the TK2285 protein as in other members whose structures have been determined. We thus examined the kinase activity of the TK2285 protein toward various sugars recognized by well characterized ribokinase family members. Although activity with sugar phosphates and nucleosides was not detected, kinase activity was observed toward d-allose, d-lyxose, d-tagatose, d-talose, d-xylose, and d-xylulose. Kinetic analyses with the six sugar substrates revealed high Km values, suggesting that they were not the true physiological substrates. By examining activity toward amino sugars, sugar alcohols, and disaccharides, we found that the TK2285 protein exhibited prominent kinase activity toward myo-inositol. Kinetic analyses with myo-inositol revealed a greater kcat and much lower Km value than those obtained with the monosaccharides, resulting in over a 2,000-fold increase in kcat/Km values. TK2285 homologs are distributed among members of Thermococcales, and in most species, the gene is positioned close to a myo-inositol monophosphate synthase gene. Our results suggest the presence of a novel subfamily of the ribokinase family whose members are present in Archaea and recognize myo-inositol as a substrate.

  18. Inositol-related gene knockouts mimic lithium's effect on mitochondrial function.

    PubMed

    Toker, Lilach; Bersudsky, Yuly; Plaschkes, Inbar; Chalifa-Caspi, Vered; Berry, Gerard T; Buccafusca, Roberto; Moechars, Dieder; Belmaker, R H; Agam, Galila

    2014-01-01

    The inositol-depletion hypothesis proposes that lithium attenuates phosphatidylinositol signaling. Knockout (KO) mice of two genes (IMPA1 or Slc5a3), each encoding for a protein related to inositol metabolism, were studied in comparison with lithium-treated mice. Since we previously demonstrated that these KO mice exhibit a lithium-like neurochemical and behavioral phenotype, here we searched for pathways that may mediate lithium's/the KO effects. We performed a DNA-microarray study searching for pathways affected both by chronic lithium treatment and by the KO of each of the genes. The data were analyzed using three different bioinformatics approaches. We found upregulation of mitochondria-related genes in frontal cortex of lithium-treated, IMPA1 and Slc5a3 KO mice. Three out of seven genes differentially expressed in all three models, Cox5a, Ndufs7, and Ndufab, all members of the mitochondrial electron transfer chain, have previously been associated with bipolar disorder and/or lithium treatment. Upregulation of the expression of these genes was verified by real-time PCR. To further support the link between mitochondrial function and lithium's effect on behavior, we determined the capacity of chronic low-dose rotenone, a mitochondrial respiratory chain complex I inhibitor, to alter lithium-induced behavior as measured by the forced-swim and the amphetamine-induced hyperlocomotion paradigms. Rontenone treatment counteracted lithium's effect on behavior, supporting the proposition suggested by the bioinformatics analysis for a mitochondrial function involvement in behavioral effects of lithium mediated by inositol metabolism alterations.The results provide support for the notion that mitochondrial dysfunction is linked to bipolar disorder and can be ameliorated by lithium. The phenotypic similarities between lithium-treated wild-type mice and the two KO models suggest that lithium may affect behavior by altering inositol metabolism.

  19. Salinity-induced regulation of the myo-inositol biosynthesis pathway in tilapia gill epithelium.

    PubMed

    Sacchi, Romina; Li, Johnathon; Villarreal, Fernando; Gardell, Alison M; Kültz, Dietmar

    2013-12-15

    The myo-inositol biosynthesis (MIB) pathway converts glucose-6-phosphate to the compatible osmolyte myo-inositol that protects cells from osmotic stress. Using proteomics, the enzymes that constitute the MIB pathway, myo-inositol phosphate synthase (MIPS) and inositol monophosphatase 1 (IMPA1), are identified in tilapia (Oreochromis mossambicus) gill epithelium. Targeted, quantitative, label-free proteomics reveals that they are both upregulated during salinity stress. Upregulation is stronger when fish are exposed to severe (34 ppt acute and 90 ppt gradual) relative to moderate (70 ppt gradual) salinity stress. IMPA1 always responds more strongly than MIPS, suggesting that MIPS is more stable during salinity stress. MIPS is N-terminally acetylated and the corresponding peptide increases proportionally to MIPS protein, while non-acetylated N-terminal peptide is not detectable, indicating that MIPS acetylation is constitutive and may serve to stabilize the protein. Hyperosmotic induction of MIPS and IMPA1 is confirmed using western blot and real-time qPCR and is much higher at the mRNA than at the protein level. Two distinct MIPS mRNA variants are expressed in the gill, but one is more strongly regulated by salinity than the other. A single MIPS gene is encoded in the tilapia genome whereas the zebrafish genome lacks MIPS entirely. The genome of euryhaline tilapia contains four IMPA genes, two of which are expressed, but only one is salinity regulated in gill epithelium. The genome of stenohaline zebrafish contains a single IMPA gene. We conclude that the MIB pathway represents a major salinity stress coping mechanism that is regulated at multiple levels in euryhaline fish but absent in stenohaline zebrafish.

  20. Haplofungins, new inositol phosphorylceramide synthase inhibitors, from Lauriomyces bellulus SANK 26899 II. Structure elucidation.

    PubMed

    Ohnuki, Takashi; Yano, Tatsuya; Takatsu, Toshio

    2009-10-01

    Eight new inositol phosphorylceramide synthase inhibitors: haplofungin A, B, C, D, E, F, G and H, were discovered in a culture broth of the fungus Lauriomyces bellulus SANK 26899. The planar structures for these haplofungins were elucidated by various spectroscopic analyses and a GC/MS analysis of their degradation products. All eight compounds were found to comprise an arabinonic acid moiety linked through an ester bond to a modified long alkyl chain.

  1. An Uncharacterized Member of the Ribokinase Family in Thermococcus kodakarensis Exhibits myo-Inositol Kinase Activity*

    PubMed Central

    Sato, Takaaki; Fujihashi, Masahiro; Miyamoto, Yukika; Kuwata, Keiko; Kusaka, Eriko; Fujita, Haruo; Miki, Kunio; Atomi, Haruyuki

    2013-01-01

    Here we performed structural and biochemical analyses on the TK2285 gene product, an uncharacterized protein annotated as a member of the ribokinase family, from the hyperthermophilic archaeon Thermococcus kodakarensis. The three-dimensional structure of the TK2285 protein resembled those of previously characterized members of the ribokinase family including ribokinase, adenosine kinase, and phosphofructokinase. Conserved residues characteristic of this protein family were located in a cleft of the TK2285 protein as in other members whose structures have been determined. We thus examined the kinase activity of the TK2285 protein toward various sugars recognized by well characterized ribokinase family members. Although activity with sugar phosphates and nucleosides was not detected, kinase activity was observed toward d-allose, d-lyxose, d-tagatose, d-talose, d-xylose, and d-xylulose. Kinetic analyses with the six sugar substrates revealed high Km values, suggesting that they were not the true physiological substrates. By examining activity toward amino sugars, sugar alcohols, and disaccharides, we found that the TK2285 protein exhibited prominent kinase activity toward myo-inositol. Kinetic analyses with myo-inositol revealed a greater kcat and much lower Km value than those obtained with the monosaccharides, resulting in over a 2,000-fold increase in kcat/Km values. TK2285 homologs are distributed among members of Thermococcales, and in most species, the gene is positioned close to a myo-inositol monophosphate synthase gene. Our results suggest the presence of a novel subfamily of the ribokinase family whose members are present in Archaea and recognize myo-inositol as a substrate. PMID:23737529

  2. Salinity-induced regulation of the myo-inositol biosynthesis pathway in tilapia gill epithelium

    PubMed Central

    Sacchi, Romina; Li, Johnathon; Villarreal, Fernando; Gardell, Alison M.; Kültz, Dietmar

    2013-01-01

    SUMMARY The myo-inositol biosynthesis (MIB) pathway converts glucose-6-phosphate to the compatible osmolyte myo-inositol that protects cells from osmotic stress. Using proteomics, the enzymes that constitute the MIB pathway, myo-inositol phosphate synthase (MIPS) and inositol monophosphatase 1 (IMPA1), are identified in tilapia (Oreochromis mossambicus) gill epithelium. Targeted, quantitative, label-free proteomics reveals that they are both upregulated during salinity stress. Upregulation is stronger when fish are exposed to severe (34 ppt acute and 90 ppt gradual) relative to moderate (70 ppt gradual) salinity stress. IMPA1 always responds more strongly than MIPS, suggesting that MIPS is more stable during salinity stress. MIPS is N-terminally acetylated and the corresponding peptide increases proportionally to MIPS protein, while non-acetylated N-terminal peptide is not detectable, indicating that MIPS acetylation is constitutive and may serve to stabilize the protein. Hyperosmotic induction of MIPS and IMPA1 is confirmed using western blot and real-time qPCR and is much higher at the mRNA than at the protein level. Two distinct MIPS mRNA variants are expressed in the gill, but one is more strongly regulated by salinity than the other. A single MIPS gene is encoded in the tilapia genome whereas the zebrafish genome lacks MIPS entirely. The genome of euryhaline tilapia contains four IMPA genes, two of which are expressed, but only one is salinity regulated in gill epithelium. The genome of stenohaline zebrafish contains a single IMPA gene. We conclude that the MIB pathway represents a major salinity stress coping mechanism that is regulated at multiple levels in euryhaline fish but absent in stenohaline zebrafish. PMID:24072791

  3. Inositol hexakisphosphate inhibits mineralization of MC3T3-E1 osteoblast cultures.

    PubMed

    Addison, William N; McKee, Marc D

    2010-04-01

    Inositol hexakisphosphate (IP6, phytic acid) is an endogenous compound present in mammalian cells and tissues. Differentially phosphorylated forms of inositol are well-documented to have important roles in signal transduction, cell proliferation and differentiation, and IP6 in particular has been suggested to inhibit soft tissue calcification (specifically renal and vascular calcification) by binding extracellularly to calcium oxalate and calcium phosphate crystals. However, the effects of IP6 on bone mineralization are largely unknown. In this study, we used MC3T3-E1 osteoblast cultures to examine the effects of exogenous IP6 on osteoblast function and matrix mineralization. IP6 at physiologic concentrations caused a dose-dependent inhibition of mineralization without affecting cell viability, proliferation or collagen deposition. Osteoblast differentiation markers, including tissue-nonspecific alkaline phosphatase activity, bone sialoprotein and osteocalcin mRNA levels, were not adversely affected by IP6 treatment. On the other hand, IP6 markedly increased protein and mRNA levels of osteopontin, a potent inhibitor of crystal growth and matrix mineralization. Inositol alone (without phosphate), as well as inositol hexakis-sulphate, a compound with a high negative charge similar to IP6, had no effect on mineralization or osteopontin induction. Binding of IP6 to mineral crystals from the osteoblast cultures, as well as to synthetic hydroxyapatite crystals, was confirmed by a colorimetric assay for IP6. In summary, IP6 inhibits mineralization of osteoblast cultures by binding to growing crystals through negatively charged phosphate groups and by induction of inhibitory osteopontin expression. These data suggest that IP6 may regulate physiologic bone mineralization by directly acting extracellularly, and by serving as a specific signal at the cellular level for the regulation of osteopontin gene expression.

  4. A Combined Therapy with Myo-Inositol and D-Chiro-Inositol Improves Endocrine Parameters and Insulin Resistance in PCOS Young Overweight Women.

    PubMed

    Benelli, Elena; Del Ghianda, Scilla; Di Cosmo, Caterina; Tonacchera, Massimo

    2016-01-01

    Introduction. We evaluated the effects of a therapy that combines myo-inositol (MI) and D-chiro-inositol (DCI) in young overweight women affected by polycystic ovary syndrome (PCOS), characterized by oligo- or anovulation and hyperandrogenism, correlated to insulin resistance. Methods. We enrolled 46 patients affected by PCOS and, randomly, we assigned them to two groups, A and B, treated, respectively, with the association of MI plus DCI, in a 40 : 1 ratio, or with placebo (folic acid) for six months. Thus, we analyzed pretreatment and posttreatment FSH, LH, 17-beta-Estradiol, Sex Hormone Binding Globulin, androstenedione, free testosterone, dehydroepiandrosterone sulphate, HOMA index, and fasting glucose and insulin. Results. We recorded a statistically significant reduction of LH, free testosterone, fasting insulin, and HOMA index only in the group treated with the combined therapy of MI plus DCI; in the same patients, we observed a statistically significant increase of 17-beta-Estradiol levels. Conclusions. The combined therapy of MI plus DCI is effective in improving endocrine and metabolic parameters in young obese PCOS affected women. PMID:27493664

  5. A Combined Therapy with Myo-Inositol and D-Chiro-Inositol Improves Endocrine Parameters and Insulin Resistance in PCOS Young Overweight Women

    PubMed Central

    Benelli, Elena; Del Ghianda, Scilla

    2016-01-01

    Introduction. We evaluated the effects of a therapy that combines myo-inositol (MI) and D-chiro-inositol (DCI) in young overweight women affected by polycystic ovary syndrome (PCOS), characterized by oligo- or anovulation and hyperandrogenism, correlated to insulin resistance. Methods. We enrolled 46 patients affected by PCOS and, randomly, we assigned them to two groups, A and B, treated, respectively, with the association of MI plus DCI, in a 40 : 1 ratio, or with placebo (folic acid) for six months. Thus, we analyzed pretreatment and posttreatment FSH, LH, 17-beta-Estradiol, Sex Hormone Binding Globulin, androstenedione, free testosterone, dehydroepiandrosterone sulphate, HOMA index, and fasting glucose and insulin. Results. We recorded a statistically significant reduction of LH, free testosterone, fasting insulin, and HOMA index only in the group treated with the combined therapy of MI plus DCI; in the same patients, we observed a statistically significant increase of 17-beta-Estradiol levels. Conclusions. The combined therapy of MI plus DCI is effective in improving endocrine and metabolic parameters in young obese PCOS affected women. PMID:27493664

  6. Studies of inositol 1-phosphate analogues as inhibitors of the phosphatidylinositol phosphate synthase in mycobacteria.

    PubMed

    Morii, Hiroyuki; Okauchi, Tatsuo; Nomiya, Hiroki; Ogawa, Midori; Fukuda, Kazumasa; Taniguchi, Hatsumi

    2013-03-01

    We previously reported a novel pathway for the biosynthesis of phosphatidylinositol in mycobacteria via phosphatidylinositol phosphate (PIP) [Morii H., Ogawa, M., Fukuda, K., Taniguchi, H., and Koga, Y (2010) J. Biochem. 148, 593-602]. PIP synthase in the pathway is a promising target for the development of new anti-mycobacterium drugs. In the present study, we evaluated the characteristics of the PIP synthase of Mycobacterium tuberculosis. Four types of compounds were chemically synthesized based on the assumption that structural homologues of inositol 1-phosphate, a PIP synthase substrate, would act as PIP synthase inhibitors, and the results confirmed that all synthesized compounds inhibited PIP synthase activity. The phosphonate analogue of inositol 1-phosphate (Ino-C-P) had the greatest inhibitory effect among the synthesized compounds examined. Kinetic analysis indicated that Ino-C-P acted as a competitive inhibitor of inositol 1-phosphate. The IC(50) value for Ino-C-P inhibition of the PIP synthase activity was estimated to be 2.0 mM. Interestingly, Ino-C-P was utilized in the same manner as the normal PIP synthase substrate, leading to the synthesis of a phosphonate analogue of PIP (PI-C-P), which had a structure similar to that of the natural product, PIP. In addition, PI-C-P had high inhibitory activity against PIP synthase.

  7. Molecular Basis of Phosphatidyl-myo-inositol Mannoside Biosynthesis and Regulation in Mycobacteria*

    PubMed Central

    Guerin, Marcelo E.; Korduláková, Jana; Alzari, Pedro M.; Brennan, Patrick J.; Jackson, Mary

    2010-01-01

    Phosphatidyl-myo-inositol mannosides (PIMs) are unique glycolipids found in abundant quantities in the inner and outer membranes of the cell envelope of all Mycobacterium species. They are based on a phosphatidyl-myo-inositol lipid anchor carrying one to six mannose residues and up to four acyl chains. PIMs are considered not only essential structural components of the cell envelope but also the structural basis of the lipoglycans (lipomannan and lipoarabinomannan), all important molecules implicated in host-pathogen interactions in the course of tuberculosis and leprosy. Although the chemical structure of PIMs is now well established, knowledge of the enzymes and sequential events leading to their biosynthesis and regulation is still incomplete. Recent advances in the identification of key proteins involved in PIM biogenesis and the determination of the three-dimensional structures of the essential phosphatidyl-myo-inositol mannosyltransferase PimA and the lipoprotein LpqW have led to important insights into the molecular basis of this pathway. PMID:20801880

  8. Differential insulin response to myo-inositol administration in obese polycystic ovary syndrome patients.

    PubMed

    Genazzani, Alessandro D; Prati, Alessia; Santagni, Susanna; Ricchieri, Federica; Chierchia, Elisa; Rattighieri, Erica; Campedelli, Annalisa; Simoncini, Tommaso; Artini, Paolo G

    2012-12-01

    Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism, chronic anovulation, polycystic ovaries at ultrasound evaluation, and quite frequently by insulin resistance or compensatory hyperinsulinemia. Attention has been given to the role of inositol-phosphoglycan (IPG) mediators of insulin action and growing evidences suggest that a deficiency of D-chiro-inositol (DCI) containing IPG might be at the basis of insulin resistance, frequent in PCOS patients. On such basis, we investigated the efficacy on insulin sensitivity and hormonal parameters of 8 weeks treatment with myo-inositol (MYO) (Inofert, ItalPharmaco, Milano, Italy) at the dosage of 2 g day in a group (n = 42) of obese PCOS patients,. After the treatment interval body mass index (BMI) and insulin resistance decreased together with luteinizing hormone (LH), LH/FSH and insulin. When subdividing the patients according to their fasting insulin levels, Group A (n = 15) insulin below 12 µU/ml and Group B (n = 27) insulin above 12 µU/ml, MYO treatment induced similar changes in both groups but only patients of Group B showed the significant decrease of both fasting insulin plasma levels (from 20.3 ± 1.8 to 12.9 ± 1.8 µU/ml, p < 0.00001) and of area under the curve (AUC) of insulin under oral glucose tolerance test (OGTT). In conclusion, our study supports the hypothesis that MYO administration is more effective in obese patients with high fasting insulin plasma levels.

  9. Desymmetrization of myo-inositol derivatives by lanthanide catalyzed phosphitylation with C2-symmetric phosphites.

    PubMed

    Duss, Michael; Capolicchio, Samanta; Linden, Anthony; Ahmed, Nisar; Jessen, Henning J

    2015-06-15

    Desymmetrization by phosphorylation represents a promising method with potential impact in many different areas of research. C2-Symmetric phosphoramidites have been used to desymmetrize myo-inositol derivatives by functionalization at different positions. With this method, 1:1 mixtures of diastereomers are obtained that can be separated subsequently. In this work, activation of a C2-symmetric phosphoramidite is achieved by addition of pentafluorophenol (PFP) and leads to a reactive PFP phosphite, which can then be coupled to protected myo-inositol derivatives with reactive OH groups at the 1, 3, 4 and 6 positions. This strategy enhances the diastereoselectivity of the coupling reaction with a preference towards phosphitylation at position 6 (up to 3:1) or position 3 (up to 2:1). The concept of attenuative activation of phosphoramidites via in situ generated pentafluorophenol phosphite triesters is thus proven in these studies. It is further shown that Lewis-Acid catalysis enhances the rate of phosphite triester coupling without affecting the diastereoselectivity. This novel strategy improves access to different phosphorylated myo-inositol derivatives and will thus enable further studies into the function of these important intracellular second messengers.

  10. Optimization of pressurized liquid extraction of inositols from pine nuts (Pinus pinea L.).

    PubMed

    Ruiz-Aceituno, L; Rodríguez-Sánchez, S; Sanz, J; Sanz, M L; Ramos, L

    2014-06-15

    Pressurized liquid extraction (PLE) has been used for the first time to extract bioactive inositols from pine nuts. The influence of extraction time, temperature and cycles of extraction in the yield and composition of the extract was studied. A quadratic lineal model using multiple linear regression in the stepwise mode was used to evaluate possible trends in the process. Under optimised PLE conditions (50°C, 18 min, 3 cycles of 1.5 mL water each one) at 10 MPa, a noticeable reduction in extraction time and solvent volume, compared with solid-liquid extraction (SLE; room temperature, 2h, 2 cycles of 5 mL water each one) was achieved; 5.7 mg/g inositols were extracted by PLE, whereas yields of only 3.7 mg/g were obtained by SLE. Subsequent incubation of PLE extracts with Saccharomyces cerevisiae (37°C, 5h) allowed the removal of other co-extracted low molecular weight carbohydrates which may interfere in the bioactivity of inositols.

  11. Inositol Trisphosphate and Diacylglycerol Can Differentially Modulate Gene Expression in Dictyostelium

    NASA Astrophysics Data System (ADS)

    Ginsburg, Gail; Kimmel, Alan R.

    1989-12-01

    We have previously shown that several genes expressed during Dictyostelium development could be induced in shaking culture by exogenous cAMP, even though the accumulation of intracellular cAMP was inhibited. The use of selected cAMP analogs indicated that the exogenous cAMP functioned by activating the cell surface cAMP receptor and not by interacting with the regulatory subunit of the intracellular cAMP-dependent protein kinase. Although some genes in Dictyostelium appear to be regulated by intracellular cAMP, these data suggest that this is not the case for all genes regulated by cAMP. Intracellular second messengers other than cAMP may, therefore, promote the expression of these other genes. Here, we have examined inositol trisphosphate and diacylglycerol as candidates for such mediators of signal transduction. We have studied three genes that exhibit disparate modes of temporal and spatial expression during development of Dictyostelium. In shaking cultures, maximal levels of expression of each are dependent on the accumulation of or exposure to extracellular cAMP. We show that the addition of inositol trisphosphate and/or diacylglycerol to cells in shaking culture has distinct effects on the expression of each gene and, under specific conditions, can bypass the requirement for extracellular cAMP. These data suggest that extracellular cAMP interacting with its cell surface receptor may promote synthesis of inositol trisphosphate and diacylglycerol to regulate gene expression and aspects of differentiation in Dictyostelium.

  12. Isolation and identification of myo-inositol crystals from dragon fruit (Hylocereus polyrhizus).

    PubMed

    Rebecca, Ow Phui San; Boyce, Amru Nasrulhaq; Somasundram, Chandran

    2012-04-17

    Crystals isolated from Hylocereus polyrhizus were analyzed using four different approaches--X-ray Crystallography, High Performance Liquid Chromatography (HPLC), Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) and Nuclear Magnetic Resonance (NMR) and identified as myo-inositol. The X-ray crystallography analysis showed that the unit-cell parameters were: a = 6.6226 (3) Å, b = 12.0462 (5) Å, c = 18.8942 (8) Å, α = 90.00, β = 93.98, δ = 90.00. The purity of the crystals were checked using HPLC, whereupon a clean single peak was obtained at 4.8 min with a peak area of 41232 μV*s. The LC-MS/MS technique, which is highly sensitive and selective, was used to provide a comparison of the isolated crystals with a myo-inositol standard where the results gave an identical match for both precursor and product ions. NMR was employed to confirm the molecular structure and conformation of the crystals, and the results were in agreement with the earlier results in this study. The discovery of myo-inositol crystals in substantial amount in H. polyrhizus has thus far not been reported and this is an important finding which will increase the marketability and importance of H. polyrhizus as a crop with a wide array of health properties.

  13. Anion-exchange high-performance liquid chromatography with post-column detection for the analysis of phytic acid and other inositol phosphates

    NASA Technical Reports Server (NTRS)

    Rounds, M. A.; Nielsen, S. S.; Mitchell, C. A. (Principal Investigator)

    1993-01-01

    The use of gradient anion-exchange HPLC, with a simple post-column detection system, is described for the separation of myo-inositol phosphates, including "phytic acid" (myo-inositol hexaphosphate). Hexa-, penta-, tetra-, tri- and diphosphate members of this homologous series are clearly resolved within 30 min. This method should facilitate analysis and quantitation of "phytic acid" and other inositol phosphates in plant, food, and soil samples.

  14. Sugar-metal ion interactions: the complicated coordination structures of cesium ion with D-ribose and myo-inositol.

    PubMed

    Hu, Haijian; Xue, Junhui; Wen, Xiaodong; Li, Weihong; Zhang, Chao; Yang, Limin; Xu, Yizhuang; Zhao, Guozhong; Bu, Xiaoxia; Liu, Kexin; Chen, Jia'er; Wu, Jinguang

    2013-11-18

    The novel cesium chloride-D-ribose complex (CsCl·C5H10O5; Cs-R) and cesium chloride-myo-inositol complex (CsCl·C6H12O6; Cs-I) have been synthesized and characterized using X-ray diffraction and FTIR, FIR, THz, and Raman spectroscopy. Cs(+) is eight-coordinated to three chloride ions, O1 and O2 from one D-ribose molecule, O1 from another D-ribose molecule, and O4 and O5 from the third D-ribose molecule in Cs-R. For one D-ribose molecule, the oxygen atom O1 in the ring is coordinated to two cesium ions as an oxygen bridge, O2 is cocoordinated with O1 to one of the two cesium ions, and O4 and O5 are coordinated to the third cesium ion, respectively. O3 does not coordinate to metal ions and only takes part in forming hydrogen bonds. One chloride ion is connected to three cesium ions. Thus, a complicated structure of Cs-D-ribose forms. For Cs-I, Cs(+) is 10-coordinated to three chloride ions, O1 and O2 from one myo-inositol molecule, O3 and O4 from another myo-inositol molecule, O5 and O6 from the third myo-inositol molecule, and O6 from the fourth myo-inositol molecule. One metal ion is connected to four ligands, and one myo-inositol is coordinated to four Cs(+) ions, which is also a complicated coordination structure. Crystal structure results, FTIR, FIR, THz, and Raman spectra provide detailed information on the structure and coordination of hydroxyl groups to metal ions in the cesium chloride-D-ribose and cesium chloride-myo-inositol complexes.

  15. Variations in myo-inositol in fronto-limbic regions and clinical response to electroconvulsive therapy in major depression.

    PubMed

    Njau, Stephanie; Joshi, Shantanu H; Leaver, Amber M; Vasavada, Megha; Van Fleet, Jessica; Espinoza, Randall; Narr, Katherine L

    2016-09-01

    Though electroconvulsive therapy (ECT) is an established treatment for severe depression, the neurobiological factors accounting for the clinical effects of ECT are largely unknown. Myo-inositol, a neurometabolite linked with glial activity, is reported as reduced in fronto-limbic regions in patients with depression. Whether changes in myo-inositol relate to the antidepressant effects of ECT is unknown. Using magnetic resonance spectroscopy ((1)H-MRS), we measured dorsomedial anterior cingulate cortex (dmACC) and left and right hippocampal myo-inositol in 50 ECT patients (mean age: 43.78, 14 SD) and 33 controls (mean age: 39.33, 12 SD) to determine cross sectional effects of diagnosis and longitudinal effects of ECT. Patients were scanned prior to treatment, after the second ECT and at completion of the ECT index series. Controls were scanned twice at intervals corresponding to patients' baseline and end of treatment scans. Myo-inositol increased over the course of ECT in the dmACC (p = 0.042). A significant hemisphere by clinical response effect was observed for the hippocampus (p = 0.003) where decreased myo-inositol related to symptom improvement in the left hippocampus. Cross-sectional differences between patients and controls at baseline were not detected. Changes in myo-inositol observed in the dmACC in association with ECT and in the hippocampus in association with ECT-related clinical response suggest the mechanisms of ECT could include gliogenesis or a reversal of gliosis that differentially affect dorsal and ventral limbic regions. Change in dmACC myo-inositol diverged from control values with ECT suggesting compensation, while hippocampal change suggested normalization.

  16. Variations in myo-inositol in fronto-limbic regions and clinical response to electroconvulsive therapy in major depression.

    PubMed

    Njau, Stephanie; Joshi, Shantanu H; Leaver, Amber M; Vasavada, Megha; Van Fleet, Jessica; Espinoza, Randall; Narr, Katherine L

    2016-09-01

    Though electroconvulsive therapy (ECT) is an established treatment for severe depression, the neurobiological factors accounting for the clinical effects of ECT are largely unknown. Myo-inositol, a neurometabolite linked with glial activity, is reported as reduced in fronto-limbic regions in patients with depression. Whether changes in myo-inositol relate to the antidepressant effects of ECT is unknown. Using magnetic resonance spectroscopy ((1)H-MRS), we measured dorsomedial anterior cingulate cortex (dmACC) and left and right hippocampal myo-inositol in 50 ECT patients (mean age: 43.78, 14 SD) and 33 controls (mean age: 39.33, 12 SD) to determine cross sectional effects of diagnosis and longitudinal effects of ECT. Patients were scanned prior to treatment, after the second ECT and at completion of the ECT index series. Controls were scanned twice at intervals corresponding to patients' baseline and end of treatment scans. Myo-inositol increased over the course of ECT in the dmACC (p = 0.042). A significant hemisphere by clinical response effect was observed for the hippocampus (p = 0.003) where decreased myo-inositol related to symptom improvement in the left hippocampus. Cross-sectional differences between patients and controls at baseline were not detected. Changes in myo-inositol observed in the dmACC in association with ECT and in the hippocampus in association with ECT-related clinical response suggest the mechanisms of ECT could include gliogenesis or a reversal of gliosis that differentially affect dorsal and ventral limbic regions. Change in dmACC myo-inositol diverged from control values with ECT suggesting compensation, while hippocampal change suggested normalization. PMID:27285661

  17. Metabolism of inositol(1,4,5)trisphosphate by a soluble enzyme fraction from pea (Pisum sativum) roots

    SciTech Connect

    Drobak, B.K.; Watkins, P.A.C.; Roberts, K. ); Chattaway, J.A. Univ. of East Anglia, Norwich ); Dawson, A.P. )

    1991-02-01

    Metabolism of the putative messenger molecule D-myo-inositol(1,4,5)trisphosphate (Ins(1,4,5)P{sub 3}) in plant cells has been studied using a soluble fraction from pea (pisum sativum) roots as enzyme source and (5-{sup 32}P)Ins(1,4,5)P{sub 3} and (2-{sup 3}H)Ins(1,4,5)P{sub 3} as tracers. Ins(1,4,5)P{sub 3} was rapidly converted into both lower and higher inositol phosphates. The major dephosphorylation product was inositol (4,5) bisphosphate (Ins(4,5)P{sub 2}) whereas inositol(1,4)bisphosphate (Ins(1,4)P{sub 2}) was only present in very small quantities throughout a 15 minute incubation period. In addition to these compounds, small amounts of nine other metabolites were produced including inositol and inositol(1,4,5,X)P{sub 4}. Dephosphorylation of Ins(1,4,5)P{sub 3} to Ins(4,5)P{sub 2} was dependent on Ins(1,4,5)P{sub 3} concentration and was partially inhibited by the phosphohydrolase inhibitors 2,3-diphosphoglycerate, glucose 6-phosphate, and p-nitrophenylphosphate. Conversion of Ins(1,4,5)P{sub 3} to Ins(4,5)P{sub 2} and Ins(1,4,5,X)P{sub 4} was inhibited by 55 micromolar Ca{sup 2+}. This study demonstrates that enzymes are present in plant tissues which are capable of rapidly converting Ins(1,4,5)P{sub 3} and that pathways of inositol phosphate metabolism exist which may prove to be unique to the plant kingdom.

  18. [3H]Indole-3-acetyl-myo-inositol hydrolysis by extracts of Zea mays L. vegetative tissue

    NASA Technical Reports Server (NTRS)

    Hall, P. J.; Bandurski, R. S.

    1986-01-01

    [3H]Indole-3-acetyl-myo-inositol was hydrolyzed by buffered extracts of acetone powders prepared from 4 day shoots of dark grown Zea mays L. seedlings. The hydrolytic activity was proportional to the amount of extract added and was linear for up to 6 hours at 37 degrees C. Boiled or alcohol denatured extracts were inactive. Analysis of reaction mixtures by high performance liquid chromatography demonstrated that not all isomers of indole-3-acetyl-myo-inositol were hydrolyzed at the same rate. Buffered extracts of acetone powders were prepared from coleoptiles and mesocotyls. The rates of hydrolysis observed with coleoptile extracts were greater than those observed with mesocotyl extracts. Active extracts also catalyzed the hydrolysis of esterase substrates such as alpha-naphthyl acetate and the methyl esters of indoleacetic acid and naphthyleneacetic acid. Attempts to purify the indole-3-acetyl-myo-inositol hydrolyzing activity by chromatographic procedures resulted in only slight purification with large losses of activity. Chromatography over hydroxylapatite allowed separation of two enzymically active fractions, one of which catalyzed the hydrolysis of both indole-3-acetyl-myo-inositol and esterase substrates. With the other enzymic hydrolysis of esterase substrates was readily demonstrated, but no hydrolysis of indole-3-acetyl-myo-inositol was ever detected.

  19. Asymmetric Distribution of Glucose and Indole-3-Acetyl-myo-Inositol in Geostimulated Zea mays Seedlings 1

    PubMed Central

    Momonoki, Yoshie S.

    1988-01-01

    Indole-3-acetyl-myo-inositol occurs in both the kernel and vegetative shoot of germinating Zea mays seedlings. The effect of a gravitational stimulus on the transport of [3H]-5-indole-3-acetyl-myo-inositol and [U-14C]-d-glucose from the kernel to the seedling shoot was studied. Both labeled glucose and labeled indole-3-acetyl-myo-inositol become asymmetrically distributed in the mesocotyl cortex of the shoot with more radioactivity occurring in the bottom half of a horizontally placed seedling. Asymmetric distribution of [3H]indole-3-acetic acid, derived from the applied [3H]indole-3-acetyl-myo-inositol, occurred more rapidly than distribution of total 3H-radioactivity. These findings demonstrate that the gravitational stimulus can induce an asymmetric distribution of substances being transported from kernel to shoot. They also indicate that, in addition to the transport asymmetry, gravity affects the steady state amount of indole-3-acetic acid derived from indole-3-acetyl-myo-inositol. PMID:11537873

  20. Certain Malvaceae Plants Have a Unique Accumulation of myo-Inositol 1,2,4,5,6-Pentakisphosphate

    PubMed Central

    Phillippy, Brian Q.; Perera, Imara Y.; Donahue, Janet L.; Gillaspy, Glenda E.

    2015-01-01

    Methods used to quantify inositol phosphates in seeds lack the sensitivity and specificity necessary to accurately detect the lower concentrations of these compounds contained in the leaves of many plants. In order to measure inositol hexakisphosphate (InsP6) and inositol pentakisphosphate (InsP5) levels in leaves of different plants, a method was developed to concentrate and pre-purify these compounds prior to analysis. Inositol phosphates were extracted from leaves with diluted HCl and concentrated on small anion exchange columns. Reversed-phase solid phase extraction cartridges were used to remove compounds that give peaks that sometimes interfere during HPLC. The method permitted the determination of InsP6 and InsP5 concentrations in leaves as low as 10 µM and 2 µM, respectively. Most plants analyzed contained a high ratio of InsP6 to InsP5. In contrast, certain members of the Malvaceae family, such as cotton (Gossypium) and some hibiscus (Hibiscus) species, had a preponderance of InsP5. Radiolabeling of cotton seedlings also showed increased amounts of InsP5 relative to InsP6. Why some Malvaceae species exhibit a reversal of the typical ratios of these inositol phosphates is an intriguing question for future research. PMID:27135328

  1. Formation of inositol 1,3,4,6-tetrakisphosphate during angiotensin II action in bovine adrenal glomerulosa cells

    SciTech Connect

    Balla, T.; Guillemette, G.; Baukal, A.J.; Catt, K.J.

    1987-10-14

    Angiotensin II stimulates the formation of several inositol polyphosphates in cultured bovine adrenal glomerulosa cells prelabelled with (/sup 3/H) inositol. Analysis by high performance anion exchange chromatography of the inositol-phosphate compounds revealed the existence of two additional inositol tetrakisphosphate (InsP4) isomers in proximity to Ins-1,3,4,5-P4, the known phosphorylation product of Ins-1,4,5-trisphosphate and precursor of Ins-1,3,4-trisphosphate. Both of these new compounds showed a slow increase after stimulation with angiotensin II. The structure of one of these new InsP4 isomers, which is a phosphorylation product of Ins-1,3,4-P3, was deduced by its resistance to periodate oxidation to be Ins-1,3,4,6-P4. The existence of multiple cycles of phosphorylation-dephosphorylation reactions for the processing of Ins-1,4,5-P4 may represent a new aspect of the inositol-lipid related signalling mechanism in agonist-activated target cells.

  2. Effect of atropine and gammahydroxybutyrate on ischemically induced changes in the level of radioactivity in (/sup 3/H)inositol phosphates in gerbil brain in vivo

    SciTech Connect

    Wikiel, H.; Halat, G.; Strosznajder, J.

    1988-05-01

    Brain ischemia in gerbils was induced by ligation of both common carotid arteries for 1 min or 10 min. Sham-operated animals served as controls. Intracerebral injection of (3H)inositol into gerbil brain 16 hr before ischemic insult resulted in equilibration of the label between inositol lipids and water-soluble inositol phosphate. A short ischemic period (1 min) resulted in a statistically significant increase in the radioactivity of inositol triphosphate (IP3) and inositol monophosphate (IP), by about 48% and 79%, respectively, with little change in that of the intermediate inositol biphosphate (IP2), which increased by about 16%. When the ischemic period was prolonged (10 min), an increase in the radioactivity of inositol monophosphate exclusively, by about 84%, was observed. The level of radioactivity in inositol phosphates IP2 and IP3 decreased by about 50%, probably as a consequence of phosphatase activation by the ischemic insult. The agonist of the cholinergic receptor, carbachol, injected intracerebrally (40 micrograms per animal) increased accumulation of radioactivity in all inositol phosphates. The level of radioactivity in IP3, IP2, and IP was elevated by about 40, 23, and 147%, respectively. The muscarinic cholinergic antagonist, atropine, injected intraperitoneally in doses of 100 mg/kg body wt. depressed phosphoinositide metabolism in control animals. The level of radioactivity in water-soluble inositol metabolites in the brain of animals pretreated with atropine was evidently about 32% lower than in untreated animals. Pretreatment with atropine decreased the radioactivity of all inositol phosphates in the brain of animals subjected to 1-min ischemia and the radioactivity of IP in the case of 10-min brain ischemia.

  3. myo-Inositol is an osmolyte in rat liver macrophages (Kupffer cells) but not in RAW 264.7 mouse macrophages.

    PubMed Central

    Warskulat, U; Weik, C; Häussinger, D

    1997-01-01

    The role of myo-inositol as an osmolyte was studied in cultured rat liver macrophages (Kupffer cells). Hyperosmotic exposure of Kupffer cells stimulated myo-inositol uptake and led to an increase in the mRNA levels for the sodium/myo-inositol cotransporter (SMIT). Conversely, hypo-osmotic (205 m-osM) exposure diminished myo-inositol uptake when compared with normo-osmotic (305 m-osM) control incubations. The hyperosmolarity-induced SMIT mRNA increase was counteracted by added myo-inositol or betaine. In contrast with Kupffer cells, there was only a slight hyperosmotic stimulation of myo-inositol uptake in RAW 264.7 mouse macrophages, and the myo-inositol transporter (SMIT) mRNA was not detectable. Further, a slight stimulation of taurine uptake and an increase in taurine transporter (TAUT) mRNA level by hyperosmolarity was observed in RAW 264.7 cells, whereas hypo-osmolarity led to a decrease in taurine uptake and TAUT mRNA level. When Kupffer cells were preloaded with myo-inositol, hypo-osmotic exposure led to a rapid efflux of myo-inositol from the cells. Myo-inositol efflux was also stimulated by phagocytosis of latex particles; however, latex was without effect on the hyperosmolarity-induced increase of SMIT mRNA levels. The results suggest a role of myo-inositol as an osmolyte in rat Kupffer cells but not in RAW 264.7 mouse macrophages. The functional relevance of this osmolyte strategy might lie in the maintenance of cell volume homeostasis during phagocytosis in Kupffer cells; however, the interplay with the other osmolytes betaine and taurine remains to be established. PMID:9337881

  4. Prefrontal inositol levels and implicit decision-making in healthy individuals and depressed patients.

    PubMed

    Jollant, Fabrice; Richard-Devantoy, Stéphane; Ding, Yang; Turecki, Gustavo; Bechara, Antoine; Near, Jamie

    2016-08-01

    Risky decision-making is found in several mental disorders and is associated with deleterious consequences. Current research aims at understanding the biological underpinnings of this complex cognitive function and the basis of individual variability. We used 3T proton Magnetic Resonance Spectroscopy to measure in vivo glutamate, GABA, N-acetyl-aspartate (NAA), and myo-inositol levels at rest in the right dorsal prefrontal cortex of 54 participants, comprising 24 unmedicated depressed patients and 30 healthy individuals. Participants were also tested with the Iowa Gambling Task (IGT), a classical measure of value-based decision-making. No group differences were found in terms of compound levels or decision-making performance. However, high inositol levels were associated with lower decision-making scores independently from group, notably during the initial stage of the task when explicit rules are still unknown and decisions are largely based on implicit processes (whole sample: F=4.0; p=0.02), with a large effect size (Cohen׳s d=0.8, 95% [0.2-1.5]). This effect was stronger when explicit knowledge was taken into account, with explicit knowledge showing an independent effect on performance. There was no association with other compounds. This study suggests, for the first time, a role for the inositol pathway on the implicit learning component of decision-making, without any direct effect on the explicit component. Hypothesized mechanisms implicate intracellular calcium modulation and subsequent synaptic plasticity. These findings represent a first step in the understanding of the biochemical mechanisms underlying decision-making and the identification of therapeutic targets. They also emphasize a dimensional approach in the study of the neurobiological determinants of mental disorders.

  5. Fungal Inositol Pyrophosphate IP7 Is Crucial for Metabolic Adaptation to the Host Environment and Pathogenicity

    PubMed Central

    Lev, Sophie; Li, Cecilia; Desmarini, Desmarini; Saiardi, Adolfo; Fewings, Nicole L.; Schibeci, Stephen D.; Sharma, Raghwa; Sorrell, Tania C.

    2015-01-01

    ABSTRACT Inositol pyrophosphates (PP-IPs) comprising inositol, phosphate, and pyrophosphate (PP) are essential for multiple functions in eukaryotes. Their role in fungal pathogens has never been addressed. Cryptococcus neoformans is a model pathogenic fungus causing life-threatening meningoencephalitis. We investigate the cryptococcal kinases responsible for the production of PP-IPs (IP7/IP8) and the hierarchy of PP-IP importance in pathogenicity. Using gene deletion and inositol polyphosphate profiling, we identified Kcs1 as the major IP6 kinase (producing IP7) and Asp1 as an IP7 kinase (producing IP8). We show that Kcs1-derived IP7 is the most crucial PP-IP for cryptococcal drug susceptibility and the production of virulence determinants. In particular, Kcs1 kinase activity is essential for cryptococcal infection of mouse lungs, as reduced fungal burdens were observed in the absence of Kcs1 or when Kcs1 was catalytically inactive. Transcriptome and carbon source utilization analysis suggested that compromised growth of the KCS1 deletion strain (Δkcs1 mutant) in the low-glucose environment of the host lung is due to its inability to utilize alternative carbon sources. Despite this metabolic defect, the Δkcs1 mutant established persistent, low-level asymptomatic pulmonary infection but failed to elicit a strong immune response in vivo and in vitro and was not readily phagocytosed by primary or immortalized monocytes. Reduced recognition of the Δkcs1 cells by monocytes correlated with reduced exposure of mannoproteins on the Δkcs1 mutant cell surface. We conclude that IP7 is essential for fungal metabolic adaptation to the host environment, immune recognition, and pathogenicity. PMID:26037119

  6. Is inositol (1,3,4,5)-tetrakisphosphate a new second messenger

    SciTech Connect

    Hansen, C.A.; Williamson, J.R.

    1986-05-01

    Hormone-stimulated hydrolysis of inositol (Ins) lipids results in the rapid formation of Ins(1,4,5)P/sub 3/, the second messenger for intracellular Ca/sup 2 +/ mobilization. Recently, a more polar inositol phosphate, Ins(1,3,4,5)P/sub 4/ as well as its probable hydrolysis product Ins(1,3,4)P/sub 3/ have been reported to accumulate in carbachol-stimulated brain slices. Vasopressin addition to hepatocytes prelabeled with (/sup 3/H)-Ins also showed a rapid increase of Ins(1,3,4,5)P/sub 4/, which was similar to that of Ins(1,4,5)P/sub 3/, while the accumulation of Ins(1,3,4)P/sub 3/ was slower. In order to examine whether Ins(1,3,4,5)P/sub 4/ has any functional effects on Ca/sup 2 +/ homeostasis, it was synthesized enzymatically from (/sup 3/H)-Ins(1,4,5)P/sub 3/ using a partially purified phosphoinositol kinase activity from rat brain cortex. (/sup 3/H)-labeled inositol phosphates were separated by anion exchange chromatography and analyzed by HPLC using ammonium formate/phosphoric acid gradient elution. Preliminary experiments indicate that Ins(1,3,4,5)P/sub 4/ up to 10 ..mu..M does not release Ca/sup 2 +/ from vesicular pools in saponin-permeabilized hepatocytes. It has a slight inhibitory effect on Ins(1,4,5)P/sub 3/-induced Ca/sup 2 +/ release. The effect of Ins(1,3,4,5)P/sub 4/ on plasma membrane Ca/sup 2 +/ fluxes are presently being investigated.

  7. Production of inositol trisphosphates upon. cap alpha. -adrenergic stimulation in BC3H-1 muscle cells

    SciTech Connect

    Ambler, S.K.; Thompson, B.; Brown, J.H.; Taylor, P.

    1986-05-01

    Activation of ..cap alpha../sub 1/-adrenergic receptors in BC3H-1 muscle cells rapidly mobilizes intracellular and results in a paradoxically slower accumulation of inositol trisphosphate. A possible explanation for this discrepancy may be provided by the recent findings of Irvine et al. of additional Ins P3 isomers besides the Ca/sup + +/-mobilizing isomer, Ins 1,4,5-P3. They have eluted and separated the inositol phosphates of BC3H-1 cells with an NH/sub 4//sup +/ x HCO/sub 2//sup -//H/sub 3/PO/sub 4/ gradient on a Whatman Partisil 10SAX column using Hewlett-Packard HPLC. Commercial (/sup 3/H)Ins 1,4,5-P3 and (/sup 3/H)inositol phosphates from carbachol-stimulated parotid glands were used as standards. Little or no Ins 1,3,4-P3 could be detected in control or phenylephrine-treated BC3H-1 cells. Ins 1,4,5-P3 followed the pattern of agonist stimulation observed previously. As a positive control, Ins P3 isomers were also measured in 1321N1 astrocytoma cells. Muscarinic stimulation of 1321N1 cells results in both the rapid accumulation of Ins P3 and Ca/sup + +/ mobilization. There is no detectable basal Ins 1,3,4-P3, but carbachol stimulates a rapid production of this compound in 1321N1 cells. Agonist activation also results in a rapid increase in Ins 1,4,5-P3 above basal values. These studies indicate that Ins 1,3,4-P3 does not contribute to the InsP3 signal in BC3H-1 cells and multiple mechanisms may exist for the coupling of receptors to PI turnover.

  8. Sustained diacylglycerol formation from inositol phospholipids in angiotensin II-stimulated vascular smooth muscle cells

    SciTech Connect

    Griendling, K.K.; Rittenhouse, S.E.; Brock, T.A.; Ekstein, L.S.; Gimbrone, M.A. Jr.; Alexander, R.W.

    1986-05-05

    Angiotensin II acts on cultured rat aortic vascular smooth muscle cells to stimulate phospholipase C-mediated hydrolysis of membrane phosphoinositides and subsequent formation of diacylglycerol and inositol phosphates. In intact cells, angiotensin II induces a dose-dependent increase in diglyceride which is detectable after 5 s and sustained for at least 20 min. Angiotensin II (100 nM)-stimulated diglyceride formation is biphasic, peaking at 15 s (227 +/- 19% control) and at 5 min (303 +/- 23% control). Simultaneous analysis of labeled inositol phospholipids shows that at 15 s phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-phosphate (PIP) decline to 52 +/- 6% control and 63 +/- 5% control, respectively, while phosphatidylinositol (PI) remains unchanged. In contrast, at 5 min, PIP2 and PIP have returned toward control levels (92 +/- 2 and 82 +/- 4% control, respectively), while PI has decreased substantially (81 +/- 2% control). The calcium ionophore ionomycin (15 microM) stimulates diglyceride accumulation but does not cause PI hydrolysis. 4 beta-Phorbol 12-myristate 13-acetate, an activator of protein kinase C, inhibits early PIP and PIP2 breakdown and diglyceride formation, without inhibiting late-phase diglyceride accumulation. Thus, angiotensin II induces rapid transient breakdown of PIP and PIP2 and delayed hydrolysis of PI. The rapid attenuation of polyphosphoinositide breakdown is likely caused by a protein kinase C-mediated inhibition of PIP and PIP2 hydrolysis. While in vascular smooth muscle stimulated with angiotensin II inositol 1,4,5-trisphosphate formation is transient, diglyceride production is biphasic, suggesting that initial and sustained diglyceride formation from the phosphoinositides results from different biochemical and/or cellular processes.

  9. Inositol Metabolism in Plants. IV. Biosynthesis of Apiose in Lemna and Petroselinum

    PubMed Central

    Roberts, R. M.; Shah, R. H.; Loewus, F.

    1967-01-01

    The biosynthesis of apiose was investigated in cell wall polysaccharide of Lemna gibba G3 (duckweed) and in detached leaves of Petroselinum crispum (parsley). Lemna grown either in short days or in continuous light incorporated 14C from a medium containing myo-inositol-2-14C into d-apiosyl and d-xylosyl units of cell wall polysaccharides. Labeled d-apiose was characterized by paper chromatography, by formation of labeled crystalline di-O-isopropylidene d-apiose, and by gas chromatography of trimethylsilyl derivatives of apiose and of its sodium borohydride reduction product, apiitol. Periodate oxidation of labeled apiose revealed 86 to 94% of the 14C was located in formaldehyde fragments corresponding to C3′ and C4. Comparison of this result with work reported by Grisebach and Doebereiner and by Beck and Kandler supports the conclusion that myo-inositol-2-14C was converted to d-apiose labeled specifically at C4. When l-arabinose-l-14C was supplied to Lemna, both l-arabinosyl and d-xylosyl units of cell wall polysaccharides became labeled, but no 14C was found in d-apiose. Analysis of the medium external to the plants revealed the presence of a polysaccharide-like polymer that also contained labeled xylose and arabinose. Petroselinum leaves utilized myo-inositol-2-3H for the synthesis of apiose in apiin. These results provide direct evidence for a pathway of apiose biosynthesis involving d-glucuronic acid metabolism. PMID:16656551

  10. Effects of epinephrine on ADP-induced changes in platelet inositol phosphates

    SciTech Connect

    Vickers, J.D.; Keraly, C.L.; Kinlough-Rathbone, R.L.; Mustard, J.F.

    1986-03-01

    Epinephrine (EPI) does not aggregate rabbit platelets, but it does increase the labelling of inositol phosphate (IP) at 60s (21%, p < 0.05) in the presence of 20 mM Li/sup +/, in platelets prelabelled with (/sup 3/H) inositol. In contrast, 0.5 ..mu..M ADP which causes aggregation, increases the labelling of inositol bisphosphate (IP/sub 2/) by 30% (p < 0.01) at 10s and by 46% (p < 0.05) at 60s and IP by 26% (p < 0.05) at 60s. The combination of 0.5 ..mu..M ADP and 50 ..mu..M EPI causes more extensive aggregation and increases IP/sub 2/ by 154% (p < 0.01) and IP by 65% (p < 0.01) at 60s. The increase in IP/sub 2/ stimulated by ADP + EPI was greater than the increase caused by ADP (p < 0.05). The authors examined the effects of ..cap alpha..- and ..beta..-adrenergic receptor blockers on EPI + ADP-induced changes in the inositol phosphates. The ..beta..-adrenergic blocker Sotalol (50 ..mu..M), which had no effect by itself, enhanced the accumulation of IP/sub 2/ due to 0.2 ..mu..M ADP + 0.6 ..mu..M EPI by 70% (p < 0.01) at 60s, as well as aggregation. This is consistent with EPI inhibition mediated through stimulation of adenylate cyclase via the ..beta..-adrenergic receptor. The ..cap alpha..-adrenergic blocker phentolamine (50 ..mu..M), reduced aggregation stimulated by 0.5 ..mu..M ADP + 50 ..mu..M EPI, and reduced the accumulation of IP by 53% (p < 0.05) and IP/sub 2/ by 108% (0 < 0.05). These data are compatible with the hypothesis that the effect of EPI on ADP-induced aggregation involves IP/sub 2/ metabolism stimulated via the ..cap alpha..-adrenergic receptor.

  11. Labeling Cell Surface GPIs and GPI-Anchored Proteins through Metabolic Engineering with Artificial Inositol Derivatives.

    PubMed

    Lu, Lili; Gao, Jian; Guo, Zhongwu

    2015-08-10

    Glycosylphosphatidylinositol (GPI) anchoring of proteins to the cell surface is important for various biological processes, but GPI-anchored proteins are difficult to study. An effective strategy was developed for the metabolic engineering of cell-surface GPIs and GPI-anchored proteins by using inositol derivatives carrying an azido group. The azide-labeled GPIs and GPI-anchored proteins were then tagged with biotin on live cells through a click reaction, which allows further elaboration with streptavidin-conjugated dyes or other molecules. The strategy can be used to label GPI-anchored proteins with various tags for biological studies.

  12. A molecular dynamics simulation of the melting points and glass transition temperatures of myo- and neo-inositol.

    PubMed

    Watt, Stephen W; Chisholm, James A; Jones, William; Motherwell, Sam

    2004-11-15

    The heat of sublimation, density, melting point, and glass transition temperature are calculated for myo- and neo-inositol, using the condensed-phase optimized molecular potentials for atomistic simulation studies (COMPASS) force field and molecular dynamics techniques. Our results show that the calculated heats of sublimation and density are very close to the experimental values for both compounds. Furthermore, our simulated melting temperatures for myo- and neo-inositol also compare very well to the experimentally obtained data. The glass transition temperatures for myo- and neo-inositol have been calculated to be ca. 494 K and ca. 518 K, respectively, and the shape of the volume versus temperature plots produced are typical for a glass transition. As a result, it is our view that the COMPASS force field suitably describes these two compounds in molecular simulations and that molecular dynamics techniques, combined with this force field, can be used to simulate the melt and glass transitions for such molecules.

  13. Myo-inositol esters of indole-3-acetic acid are endogenous components of Zea mays L. shoot tissue

    NASA Technical Reports Server (NTRS)

    Chisnell, J. R.

    1984-01-01

    Indole-3-acetyl-myo-inositol esters have been demonstrated to be endogenous components of etiolated Zea mays shoots tissue. This was accomplished by comparison of the putative compounds with authentic, synthetic esters. The properties compared were liquid and gas-liquid chromatographic retention times and the 70-ev mass spectral fragmentation pattern of the pentaacetyl derivative. The amount of indole-3-acetyl-myo-inositol esters in the shoots was determined to be 74 nanomoles per kilogram fresh weight as measured by isotope dilution, accounting for 19% of the ester indole-3-acetic acid of the shoot. This work is the first characterization of an ester conjugate of indole-3-acetate acid from vegetative shoot tissue using multiple chromatographic properties and mass spectral identification. The kernel and the seedling shoot both contain indole-3-acetyl-myo-inositol esters, and these esters comprise approximately the same percentage of the total ester content of the kernel and of the shoot.

  14. Cloning, expression, purification, crystallization and X-ray analysis of inositol monophosphatase from Mus musculus and Homo sapiens

    PubMed Central

    Singh, Nisha; Halliday, Amy C.; Knight, Matthew; Lack, Nathan A.; Lowe, Edward; Churchill, Grant C.

    2012-01-01

    Inositol monophosphatase (IMPase) catalyses the hydrolysis of inositol monophosphate to inositol and is crucial in the phosphatidylinositol (PI) signalling pathway. Lithium, which is the drug of choice for bipolar disorder, inhibits IMPase at therapeutically relevant plasma concentrations. Both mouse IMPase 1 (MmIMPase 1) and human IMPase 1 (HsIMPase 1) were cloned into pRSET5a, expressed in Escherichia coli, purified and crystallized using the sitting-drop method. The structures were solved at resolutions of 2.4 and 1.7 Å, respectively. Comparison of MmIMPase 1 and HsIMPase 1 revealed a core r.m.s. deviation of 0.516 Å. PMID:23027737

  15. Genome-wide screen for inositol auxotrophy in Saccharomyces cerevisiae implicates lipid metabolism in stress response signaling

    PubMed Central

    Villa-García, Manuel J.; Choi, Myung Sun; Hinz, Flora I.; Gaspar, María L.; Jesch, Stephen A.

    2011-01-01

    Inositol auxotrophy (Ino− phenotype) in budding yeast has classically been associated with misregulation of INO1 and other genes involved in lipid metabolism. To identify all non-essential yeast genes that are necessary for growth in the absence of inositol, we carried out a genome-wide phenotypic screening for deletion mutants exhibiting Ino− phenotypes under one or more growth conditions. We report the identification of 419 genes, including 385 genes not previously reported, which exhibit this phenotype when deleted. The identified genes are involved in a wide range of cellular processes, but are particularly enriched in those affecting transcription, protein modification, membrane trafficking, diverse stress responses, and lipid metabolism. Among the Ino− mutants involved in stress response, many exhibited phenotypes that are strengthened at elevated temperature and/or when choline is present in the medium. The role of inositol in regulation of lipid metabolism and stress response signaling is discussed. PMID:21136082

  16. Synthesis of Differentially Protected myo- and chiro-Inositols from D-Xylose; Stereoselectivity in Intramolecular SmI(2)-Promoted Pinacol Reactions.

    PubMed

    Luchetti, Giovanni; Ding, Kejia; Kornienko, Alexander; d'Alarcao, Marc

    2008-10-01

    Methods for the enantioselective conversion of D-xylose to differentially protected myo-inositol and L-chiro-inositol have been developed. The key transformation is a highly diastereoselective intramolecular SmI(2)-promoted pinacol coupling. The stereoselectivity was extremely dependent on the conditions, suggesting a change in mechanism. Preliminary mechanistic experiments and possible explanations for this behavior are discussed.

  17. A cold-induced myo-inositol transporter-like gene confers tolerance to multiple abiotic stresses in transgenic tobacco plants.

    PubMed

    Sambe, Mame Abdou Nahr; He, Xueying; Tu, Qinghua; Guo, Zhenfei

    2015-03-01

    A full length cDNA encoding a myo-inositol transporter-like protein, named as MfINT-like, was cloned from Medicago sativa subsp. falcata (herein falcata), a species with greater cold tolerance than alfalfa (M. sativa subsp. sativa). MfINT-like is located on plasma membranes. MfINT-like transcript was induced 2-4 h after exogenous myo-inositol treatment, 24-96 h with cold, and 96 h by salinity. Given that myo-inositol accumulates higher in falcata after 24 h of cold treatment, myo-inositol is proposed to be involved in cold-induced expression of MfINT-like. Higher levels of myo-inositol was observed in leaves of transgenic tobacco plants overexpressing MfINT-like than the wild-type but not in the roots of plants grown on myo-inositol containing medium, suggesting that transgenic plants had higher myo-inositol transport activity than the wild-type. Transgenic plants survived better to freezing temperature, and had lower ion leakage and higher maximal photochemical efficiency of photosystem II (Fv /Fm ) after chilling treatment. In addition, greater plant fresh weight was observed in transgenic plants as compared with the wild-type when plants were grown under drought or salinity stress. The results suggest that MfINT-like mediated transport of myo-inositol is associated with plant tolerance to abiotic stresses.

  18. Protection against the synaptic targeting and toxicity of Alzheimer's-associated Aβ oligomers by insulin mimetic chiro-inositols

    PubMed Central

    Pitt, Jason; Thorner, Michael; Brautigan, David; Larner, Joseph; Klein, William L.

    2013-01-01

    Alzheimer's disease (AD) is a progressive dementia that correlates highly with synapse loss. This loss appears due to the synaptic accumulation of toxic Aβ oligomers (ADDLs), which damages synapse structure and function. Although it has been reported that oligomer binding and toxicity can be prevented by stimulation of neuronal insulin signaling with PPARγ agonists, these agonists have problematic side effects. We therefore investigated the therapeutic potential of chiro-inositols, insulin-sensitizing compounds safe for human consumption. Chiro-inositols have been studied extensively for treatment of diseases associated with peripheral insulin resistance, but their insulin mimetic function in memory-relevant central nervous system (CNS) cells is unknown. Here we demonstrate that mature cultures of hippocampal neurons respond to d-chiro-inositol (DCI), pinitol (3-O-methyl DCI), and the inositol glycan INS-2 (pinitol β-1-4 galactosamine) with increased phosphorylation in key upstream components in the insulin-signaling pathway (insulin receptor, insulin receptor substrate-1, and Akt). Consistent with insulin stimulation, DCI treatment promotes rapid withdrawal of dendritic insulin receptors. With respect to neuroprotection, DCI greatly enhances the ability of insulin to prevent ADDL-induced synapse damage (EC50 of 90 nM). The mechanism comprises inhibition of oligomer binding at synapses and requires insulin/IGF signaling. DCI showed no effects on Aβ oligomerization. We propose that inositol glycans and DCI, a compound already established as safe for human consumption, have potential as AD therapeutics by protecting CNS synapses against Aβ oligomers through their insulin mimetic activity.—Pitt, J., Thorner, M., Brautigan, D., Larner, J., Klein, W. L. Protection against the synaptic targeting and toxicity of Alzheimer's-associated Aβ oligomers by insulin mimetic chiro-inositols. PMID:23073831

  19. Potentiometric and ³¹P NMR studies on inositol phosphates and their interaction with iron(III) ions.

    PubMed

    Sala, Martin; Makuc, Damjan; Kolar, Jana; Plavec, Janez; Pihlar, Boris

    2011-03-01

    Potentiometric, conductometric and ³¹P NMR titrations have been applied to study interactions between myo-inositol hexakisphosphate (phytic acid), (±)-myo-inositol 1,2,3,5-tetrakisphosphate and (±)-myo-inositol 1,2,3-trisphosphate with iron(III) ions. Potentiometric and conductometric titrations of myo-inositol phosphates show that addition of iron increases acidity and consumption of hydroxide titrant. By increasing the Fe(III)/InsP(6) ratio (from 0.5 to 4) 3 mol of protons are released per 2 mol of iron(III). At first, phytates coordinate iron octahedrally between P2 and P1,3. The second coordination site represents P5 and neighbouring P4,6 phosphate groups. Complexation is accompanied with the deprotonation of P1,3 and P4,6 phosphate oxygens. At higher concentration of iron(III) intermolecular P-O-Fe-O-P bonds trigger formation of a polymeric network and precipitation of the amorphous Fe(III)-InsP(6) aggregates. (31)P NMR titration data complement the above results and display the largest chemical shift changes at pD values between 5 and 10 in agreement with strong interactions between iron and myo-inositol phosphates. The differences in T(1) relaxation times of phosphorous atoms have shown that phosphate groups at positions 1, 2 and 3 are complexated with iron(III). The interactions between iron(III) ions and inositol phosphates depend significantly on the metal to ligand ratio and an attempt to coordinate more than two irons per InsP(6) molecule results in an unstable heterogeneous system.

  20. Investigation of myo-inositol catabolism in Rhizobium leguminosarum bv. viciae and its effect on nodulation competitiveness.

    PubMed

    Fry, J; Wood, M; Poole, P S

    2001-08-01

    Three discrete loci required for growth on myo-inositol in Rhizobium leguminosarum bv. viciae have been characterized. Two of these are catabolic loci that code for malonate semialdehyde dehydrogenase (iolA) and malonate semialdehyde dehydrogenase (iolD). IolD is part of a possible operon, iolDEB, although the functions of IolE and IolB are unknown. The third locus, int, codes for an ABC transport system that is highly specific for myo-inositol. LacZ analysis showed that mutation of iolD, which codes for one of the last steps in the catabolic pathway, prevents increased transcription of the entire pathway. It is likely that the pathway is induced by an end product of catabolism rather than myo-inositol itself. Mutants in any of the loci nodulated peas (Pisum sativum) and vetch (Vicia sativa) at the same rate as the wild type. Acetylene reduction rates and plant dry weights also were the same in the mutants and wild type, indicating no defects in nitrogen fixation. When wild-type 3841 was coinoculated onto vetch plants with either catabolic mutant iolD (RU360) or iolA (RU361), however, >95% of the nodules were solely infected with the wild type. The competitive advantage of the wild type was unaffected, even when the mutants were at 100-fold excess. The myo-inositol transport mutant (RU1487), which grows slowly on myo-inositol, was only slightly less competitive than the wild type. The nodulation advantage of the wild type was not the result of superior growth in the rhizosphere. Instead, it appears that the wild type may displace the mutants early on in the infection and nodulation process, suggesting an important role for myo-inositol catabolism. PMID:11497462

  1. Rv2131c gene product: An unconventional enzyme that is both inositol monophosphatase and fructose-1,6-bisphosphatase

    SciTech Connect

    Gu Xiaoling; Chen Mao; Shen Hongbo; Jiang Xin; Huang Yishu; Wang Honghai . E-mail: hhwang@fudan.edu.cn

    2006-01-20

    Inositol monophosphatase is an enzyme in the biosynthesis of myo-inostiol, a crucial substrate for the synthesis of phosphatidylinositol, which has been demonstrated to be an essential component of mycobacteria. In this study, the Rv2131c gene from Mycobacterium tuberculosis H37Rv was cloned into the pET28a vector and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) strain, allowing the expression of the enzyme in fusion with a histidine-rich peptide on the N-terminal. The fusion protein was purified from the soluble fraction of the lysed cells under native conditions by immobilized metal affinity chromatography (IMAC). The purified Rv2131c gene product showed inositol monophosphatase activity but with substrate specificity that was broader than those of several bacterial and eukaryotic inositol monophosphatases, and it also acted as fructose-1,6-bisphosphatase. The dimeric enzyme exhibited dual activities of IMPase and FBPase, with K {sub m} of 0.22 {+-} 0.03 mM for inositol-1-phosphate and K {sub m} of 0.45 {+-} 0.05 mM for fructose-1,6-bisphosphatase. To better understand the relationship between the function and structure of the Rv2131c enzyme, we constructed D40N, L71A, and D94N mutants and purified these corresponding proteins. Mutations of D40N and D94N caused the proteins to almost completely lose both the inositol monophosphatase and fructose-1,6-bisphosphatase activities. However, L71A mutant did not cause loss either of the activities, but the activity toward the inositol was 12-fold more resistant to inhibition by lithium (IC{sub 5} {approx} 60 mM). Based on the substrate specificity and presence of conserved sequence motifs of the M. tuberculosis Rv2131c, we proposed that the enzyme belonged to class IV fructose-1,6-bisphosphatase (FBPase IV)

  2. Metabolic Studies on Intermediates in the myo-Inositol Oxidation Pathway in Lilium longiflorum Pollen: I. Conversion to Hexoses.

    PubMed

    Rosenfield, C L; Fann, C; Loewus, F A

    1978-01-01

    The myo-inositol oxidation pathway was investigated in regard to its role as a source of carbon for products of hexose monophosphate metabolism in germinated pollen of Lilium longiflorum Thunb., cv. Ace. myo-[2-(14)]Inositol and d-[1-(14)C]glucuronate had similar distributions of radioactivity, contributing about three times more label to polysaccharide-bound glucose than myo-[2-(3)H]inositol. In the course of glucogenesis label from the latter appeared as tritiated water in the medium. This exchange could be enhanced by supplying d-[5R,5S-(3)H]xylose instead of myo-[2-(3)H]inositol. When the former was administered, [(3)H]glucose was the only labeled sugar residue found in polysaccharide products. The soluble constituents of d-[5R,5S-(3)H]xylose-labeled pollen contained no traces of labeled xylose despite massive uptake and utilization.l-[1-(14)C]- and l-[5-(14)C]Arabinose produced similar labeling patterns in germinated pollen including incorporation of arabinosyl units into pollen tube polysaccharides and substantial glucogenesis which led to utilization of arabinose for respiration and further incorporation of labeled glucosyl units into pollen tube polysaccharides.d-[5-(3)H]Galacturonate was rapidly taken up by germinated pollen but slowly utilized, without conversion to other sugars, for incorporation into pollen tube polysaccharides. l-[6-(14)C]Gulonate was not taken up by pollen.Results strongly support a scheme of conversion from myo-inositol to hexose monophosphate and subsequent products of glucose metabolism that involves the myo-inositol oxidation pathway.

  3. Diastereoselective synthesis of new O-alkylated and C-branched inositols and their corresponding fluoro analogues

    PubMed Central

    Chrétien, Françoise; Chapleur, Yves

    2016-01-01

    Summary Efficient routes were developed for the diastereoselective synthesis of new O-alkylated and C-branched inositols and their corresponding fluoro analogues. The key steps of the synthesis were the easy accessibility of different types of arms in term of configuration (myo and scyllo), the linking method and length, which could modulate the biological properties. These inositol derivatives, bearing an arm terminated either with a hydroxy group or a fluorine atom, could be interesting candidates for diastereoisomeric intermediates and biological evaluations, especially for PET imaging experiments. PMID:26977196

  4. Asymmetric phosphorylation through catalytic P(III) phosphoramidite transfer: Enantioselective synthesis of d-myo-inositol-6-phosphate

    PubMed Central

    Jordan, Peter A.; Kayser-Bricker, Katherine J.; Miller, Scott J.

    2010-01-01

    Despite the ubiquitous use of phosphoramidite chemistry in the synthesis of biophosphates, catalytic asymmetric phosphoramidite transfer remains largely unexplored for phosphate ester synthesis. We have discovered that a tetrazole-functionalized peptide, in the presence of 10-Å molecular sieves, functions as an enantioselective catalyst for phosphite transfer. This chemistry in turn has been used as the key step in a streamlined synthesis of myo-inositol-6-phosphate. Mechanistic insights implicate phosphate as a directing group for a highly selective kinetic resolution of a protected inositol monophosphate. This work represents a distinct and efficient method for the selective catalytic phosphorylation of natural products. PMID:20439750

  5. Synthesis of bradyrhizose, a unique inositol-fused monosaccharide relevant to a Nod-factor independent nitrogen fixation.

    PubMed

    Li, Wei; Silipo, Alba; Molinaro, Antonio; Yu, Biao

    2015-04-25

    The symbiosis of Bradyrhizobium sp. BTAi1 with its host plant Aeschynomene indica relies on a Nod-factor independent mechanism, wherein the Bradyrhizobium O-antigen is regarded as a key factor. This O-antigen polysaccharide is composed of a unique C10 monosaccharide, namely bradyrhizose, which has a galactose-inositol trans-fused scaffold, via a homogeneous α-(1 → 7)-linkage. Herein, we report the first synthesis of bradyrhizose. The scalable synthesis requires 26 steps in a high overall yield of 9%, with the inositol scaffold being constructed effectively via a Ferrier II rearrangement from a fully functionalized C2 and C4 branched pyranose derivative.

  6. Myo-Inositol in the Treatment of Teenagers Affected by PCOS

    PubMed Central

    Barbakadze, Ludmila; Kvashilava, Nana

    2016-01-01

    Objective. To compare the effectiveness of myo-inositol (MI) and oral contraceptive pills (OCPs) in monotherapy and MI in combination with OCPs in the treatment of teenagers affected by polycystic ovary syndrome (PCOS). Methods. 61 adolescent girls aged 13–19 years, with PCOS, were involved in the prospective, open-label study. Patients were randomized into three groups: I group, 20 patients receiving drospirenone 3 mg/ethinyl estradiol 30 μg; II group, 20 patients receiving 4 g myo-inositol plus 400 mg folic acid; III group, 21 patients receiving both medications. Results. After receiving MI significant reduction in weight, BMI, glucose, C-peptide, insulin, HOMA-IR, FT, and LH was detected. The levels of SHBG, TT, FAI, DHEA-S, and AMH did not change statistically significantly. After receiving OCPs weight and BMI slightly increased, but metabolic parameters did not change. Combination of MI and OCPs did not change weight and BMI, but reduction in C-peptide, insulin, and HOMA-IR was detected. TT, FT, FAI, DHEA-S, LH, and AMH levels decreased and SHBG increased. Conclusions. Administration of MI is a safe and effective method to prevent and correct metabolic disorders in teenagers affected by PCOS. With combination of MI and OCPs antiandrogenic effects are enhanced, negative impact of OCPs on weight gain is balanced, and metabolic profile is improved.

  7. Chemogenetic Characterization of Inositol Phosphate Metabolic Pathway Reveals Druggable Enzymes for Targeting Kinetoplastid Parasites.

    PubMed

    Cestari, Igor; Haas, Paige; Moretti, Nilmar Silvio; Schenkman, Sergio; Stuart, Ken

    2016-05-19

    Kinetoplastids cause Chagas disease, human African trypanosomiasis, and leishmaniases. Current treatments for these diseases are toxic and inefficient, and our limited knowledge of drug targets and inhibitors has dramatically hindered the development of new drugs. Here we used a chemogenetic approach to identify new kinetoplastid drug targets and inhibitors. We conditionally knocked down Trypanosoma brucei inositol phosphate (IP) pathway genes and showed that almost every pathway step is essential for parasite growth and infection. Using a genetic and chemical screen, we identified inhibitors that target IP pathway enzymes and are selective against T. brucei. Two series of these inhibitors acted on T. brucei inositol polyphosphate multikinase (IPMK) preventing Ins(1,4,5)P3 and Ins(1,3,4,5)P4 phosphorylation. We show that IPMK is functionally conserved among kinetoplastids and that its inhibition is also lethal for Trypanosoma cruzi. Hence, IP enzymes are viable drug targets in kinetoplastids, and IPMK inhibitors may aid the development of new drugs. PMID:27133314

  8. Inositol Hexakisphosphate Mediates Apoptosis in Human Breast Adenocarcinoma MCF-7 Cell Line via Intrinsic Pathway

    NASA Astrophysics Data System (ADS)

    Agarwal, Rakhee; Ali, Nawab

    2010-04-01

    Inositol polyphosphates (InsPs) are naturally occurring compounds ubiquitously present in plants and animals. Inositol hexakisphosphate (InsP6) is the most abundant among all InsPs and constitutes the major portion of dietary fiber in most cereals, legumes and nuts. Certain derivatives of InsPs also regulate cellular signaling mechanisms. InsPs have also been shown to reduce tumor formation and induce apoptosis in cancerous cells. Therefore, in this study, the effects of InsPs on apoptosis were studied in an attempt to investigate their potential anti-cancer therapeutic application and understand their mechanism of action. Acridine orange and ethidium bromide staining suggested that InsP6 dose dependently induced apoptosis in human breast adenocarcinoma MCF-7 cells. Among InsPs tested (InsP3, InsP4, InsP5, and InsP6), InsP6 was found to be the most effective in inducing apoptosis. Furthermore, effects of InsP6 were found most potent inducing apoptosis. Etoposide, the drug known to induce apoptosis in both in vivo and in vitro, was used as a positive control. Western blotting experiments using specific antibodies against known apoptotic markers suggested that InsP6 induced apoptotic changes were mediated via an intrinsic apoptotic pathway.

  9. Potentiometric and spectroscopic study of the interaction of 3d transition metal ions with inositol hexakisphosphate

    NASA Astrophysics Data System (ADS)

    Veiga, Nicolás; Macho, Israel; Gómez, Kerman; González, Gabriel; Kremer, Carlos; Torres, Julia

    2015-10-01

    Among myo-inositol phosphates, the most abundant in nature is the myo-inositol hexakisphosphate, InsP6. Although it is known to be vital to cell functioning, the biochemical research into its metabolism needs chemical and structural analysis of all the protonation, complexation and precipitation processes that it undergoes in the biological media. In view of its high negative charge at physiological level, our group has been leading a thorough research into the InsP6 chemical and structural behavior in the presence of the alkali and alkaline earth metal ions essential for life. The aim of this article is to extend these studies, dealing with the chemical and structural features of the InsP6 interaction with biologically relevant 3d transition metal ions (Fe(II), Fe(III), Mn(II), Co(II), Ni(II), Cu(II) and Zn(II)), in a non-interacting medium and under simulated physiological conditions. The metal-complex stability constants were determined by potentiometry, showing under ligand-excess conditions the formation of mononuclear species in different protonation states. Under metal ion excess, polymetallic species were detected for Fe(II), Fe(III), Zn(II) and Cu(II). Additionally, the 31P NMR and UV-vis spectroscopic studies provided interesting structural aspects of the strong metal ion-InsP6 interaction.

  10. Inositol phosphate kinase 2 is required for imaginal disc development in Drosophila

    PubMed Central

    Seeds, Andrew M.; Tsui, Marco M.; Sunu, Christine; Spana, Eric P.; York, John D.

    2015-01-01

    Inositol phosphate kinase 2 (Ipk2), also known as IP multikinase IPMK, is an evolutionarily conserved protein that initiates production of inositol phosphate intracellular messengers (IPs), which are critical for regulating nuclear and cytoplasmic processes. Here we report that Ipk2 kinase activity is required for the development of the adult fruit fly epidermis. Ipk2 mutants show impaired development of their imaginal discs, the primordial tissues that form the adult epidermis. Although disk tissue seems to specify normally during early embryogenesis, loss of Ipk2 activity results in increased apoptosis and impairment of proliferation during larval and pupal development. The proliferation defect is in part attributed to a reduction in JAK/STAT signaling, possibly by controlling production or secretion of the pathway’s activating ligand, Unpaired. Constitutive activation of the JAK/STAT pathway downstream of Unpaired partially rescues the disk growth defects in Ipk2 mutants. Thus, IP production is essential for proliferation of the imaginal discs, in part, by regulating JAK/STAT signaling. Our work demonstrates an essential role for Ipk2 in producing inositide messengers required for imaginal disk tissue maturation and subsequent formation of adult body structures and provides molecular insights to signaling pathways involved in tissue growth and stability during development. PMID:26647185

  11. Brevis plant1, a putative inositol polyphosphate 5-phosphatase, is required for internode elongation in maize

    PubMed Central

    Avila, Luis M.; Cerrudo, Diego; Swanton, Clarence

    2016-01-01

    In maize (Zea mays L.), as in other grass species, stem elongation occurs during growth and most noticeably upon the transition to flowering. Genes that reduce stem elongation have been important to reduce stem breakage, or lodging. Stem elongation has been mediated by dwarf and brachytic/brevis plant mutants that affect giberellic acid and auxin pathways, respectively. Maize brevis plant1 (bv1) mutants, first identified over 80 years ago, strongly resemble brachytic2 mutants that have shortened internodes, short internode cells, and are deficient in auxin transport. Here, we characterized two novel bv1 maize mutants. We found that an inositol polyphosphate 5-phosphatase orthologue of the rice gene dwarf50 was the molecular basis for the bv1 phenotype, implicating auxin-mediated inositol polyphosphate and/or phosphoinositide signalling in stem elongation. We suggest that auxin-mediated internode elongation involves processes that also contribute to stem gravitropism. Genes misregulated in bv1 mutants included genes important for cell wall synthesis, transmembrane transport, and cytoskeletal function. Mutant and wild-type plants were indistinguishable early in development, responded similarly to changes in light quality, had unaltered flowering times, and had normal flower development. These attributes suggest that breeding could utilize bv1 alleles to increase crop grain yields. PMID:26767748

  12. Microtubule-Mediated Inositol Lipid Signaling Plays Critical Roles in Regulation of Blebbing

    PubMed Central

    Sugiyama, Tatsuroh; Pramanik, Md. Kamruzzaman; Yumura, Shigehiko

    2015-01-01

    Cells migrate by extending pseudopods such as lamellipodia and blebs. Although the signals leading to lamellipodia extension have been extensively investigated, those for bleb extension remain unclear. Here, we investigated signals for blebbing in Dictyostelium cells using a newly developed assay to induce blebbing. When cells were cut into two pieces with a microneedle, the anucleate fragments vigorously extended blebs. This assay enabled us to induce blebbing reproducibly, and analyses of knockout mutants and specific inhibitors identified candidate molecules that regulate blebbing. Blebs were also induced in anucleate fragments of leukocytes, indicating that this assay is generally applicable to animal cells. After cutting, microtubules in the anucleate fragments promptly depolymerized, followed by the extension of blebs. Furthermore, when intact cells were treated with a microtubule inhibitor, they frequently extended blebs. The depolymerization of microtubules induced the delocalization of inositol lipid phosphatidylinositol 3,4,5-trisphosphate from the cell membrane. PI3 kinase-null cells frequently extended blebs, whereas PTEN-null cells extended fewer blebs. From these observations, we propose a model in which microtubules play a critical role in bleb regulation via inositol lipid metabolism. PMID:26317626

  13. Inositol phosphate kinase 2 is required for imaginal disc development in Drosophila.

    PubMed

    Seeds, Andrew M; Tsui, Marco M; Sunu, Christine; Spana, Eric P; York, John D

    2015-12-22

    Inositol phosphate kinase 2 (Ipk2), also known as IP multikinase IPMK, is an evolutionarily conserved protein that initiates production of inositol phosphate intracellular messengers (IPs), which are critical for regulating nuclear and cytoplasmic processes. Here we report that Ipk2 kinase activity is required for the development of the adult fruit fly epidermis. Ipk2 mutants show impaired development of their imaginal discs, the primordial tissues that form the adult epidermis. Although disk tissue seems to specify normally during early embryogenesis, loss of Ipk2 activity results in increased apoptosis and impairment of proliferation during larval and pupal development. The proliferation defect is in part attributed to a reduction in JAK/STAT signaling, possibly by controlling production or secretion of the pathway's activating ligand, Unpaired. Constitutive activation of the JAK/STAT pathway downstream of Unpaired partially rescues the disk growth defects in Ipk2 mutants. Thus, IP production is essential for proliferation of the imaginal discs, in part, by regulating JAK/STAT signaling. Our work demonstrates an essential role for Ipk2 in producing inositide messengers required for imaginal disk tissue maturation and subsequent formation of adult body structures and provides molecular insights to signaling pathways involved in tissue growth and stability during development. PMID:26647185

  14. The role of the inositol phosphate cascade in visual excitation of invertebrate microvillar photoreceptors

    PubMed Central

    1991-01-01

    The identity of the transmitter(s) involved in visual transduction in invertebrate microvillar photoreceptors remains unresolved. In this study, the role of inositol 1,4,5-trisphosphate (IP3) was examined in Limulus ventral photoreceptors by studying the effects on the light response of heparin and neomycin, agents that inhibit the production or action of IP3. Both heparin and neomycin reduce responses to brief flashes of light and the transient component of responses to steps of light, and also inhibit IP3-induced calcium release, indicating that IP3 plays a direct role in invertebrate visual excitation. The effects of BAPTA, a calcium buffer, were also examined and shown to be consistent with a role for IP3-mediated calcium release in visual excitation. However, all three agents fail to block the plateau component of the response to a step of light, indicating that a single pathway involving IP3 and calcium cannot solely be responsible for visual excitation in invertebrates. We suggest that the inositol phosphate cascade and a second parallel process that is not dependent on IP3 are involved in the production of the light response. PMID:1905344

  15. α-Synuclein aggregation, seeding and inhibition by scyllo-inositol.

    PubMed

    Ibrahim, Tarek; McLaurin, JoAnne

    2016-01-15

    Recent literature demonstrates the accelerated aggregation of α-synuclein, a protein implicated in the pathogenesis of Parkinson's disease (PD), by the presence of preformed fibrillar conformers in vitro. Furthermore, these preformed fibrillar seeds are suggested to accelerate pathological induction in vivo when injected into the brains of mice. Variation in the results of in vivo studies is proposed to be caused by α-synuclein conformational variants. To investigate the impact of amino acid sequence on seeding efficiency, human and mouse α-synuclein seeds, which vary at 7 amino acid residues, were generated and cross-seeding kinetics studied. Using transmission electron microscopy (TEM), we confirmed that mouse α-synuclein aggregated more rapidly than human α-synuclein. Subsequently, we determined that seeding of human and mouse α-synuclein was more rapid in the presence of seeds generated from the same species. In addition, an established amyloid inhibitor, scyllo-inositol, was examined for potential inhibitory effects on α-synuclein aggregation. TEM analysis of protein:inhibitor assays demonstrated that scyllo-inositol inhibits the aggregation of α-synuclein, suggesting the therapeutic potential of the small molecule in PD.

  16. Activation of in vitro matured pig oocytes using activators of inositol triphosphate or ryanodine receptors.

    PubMed

    Petr, J; Urbánková, D; Tománek, M; Rozinek, J; Jílek, F

    2002-04-15

    In our study, we observed the activation of in vitro matured pig oocytes and their subsequent parthenogenetic cleavage after stimulation of ryanodine receptors (RyR) using ryanodine (Ry), caffeine or cyclic adenosine diphosphate ribose (cADPri) or after stimulation of inositol triphosphate receptors (IP(3)R) using D-myo-inositol 1,4,5-triphosphate (IP(3)). Heparin, a potent blocker of IP(3)R, prevented the activation of porcine oocytes using IP(3), but blockers of RyR (ruthenium red or procaine) prevented activation after stimulation by RyR and stimulation by IP(3)R using IP(3). The drugs were injected into oocytes matured to the stage of metaphase II and activation was determined by assessment of pronuclear formation. The activity of H1 kinase was determined and our results demonstrated a significant drop in H1 activity in the activated oocytes. The cleavage of parthenogenetic embryos progresses to more advanced stages after stimulation by IP(3)R than after stimulation by RyR. Our results could indicate that, in pig oocytes, the calcium released from IP(3)-sensitive stores triggers the calcium release from ryanodine-sensitive intracellular stores, which is necessary for oocyte activation. The calmodulin inhibitors ophiobolin A and W7 reduce the activation of oocytes induced by stimulation of RyR or IP(3)R.

  17. The Inositol 1,4,5-triphosphate kinase1 gene affects olfactory reception in Drosophila melanogaster.

    PubMed

    Gomez-Diaz, Carolina; Martin, Fernando; Alcorta, Esther

    2006-03-01

    The Inositol 1,4,5-triphosphate (IP3) route is one of the two main transduction cascades that mediate olfactory reception in Drosophila melanogaster. The activity of IP3 kinase1 reduces the levels of this substrate by phosphorylation into inositol 1,3,4,5-tetrakiphosphate (IP4). We show here that the gene is expressed in olfactory sensory organs as well as in the rest of the head. To evaluate in vivo the olfactory functional effects of up-regulating IP3K1, individuals with directed genetic changes at the reception level only were generated using the UAS/Gal4 method. In this report, we described the consequences in olfactory perception of overexpressing the IP3Kinase1 gene at eight different olfactory receptor-neuron subsets. Six out of the eight studied Gal-4/UAS-IP3K1 hybrids displayed abnormal behavioral responses to ethyl acetate, acetone, ethanol or propionaldehyde. Specific behavioral defects corresponded to the particular neuronal olfactory profile. These data confirm the role of the IP3kinase1 gene, and consequently the IP3 transduction cascade, in mediating olfactory information at the reception level.

  18. Myo-Inositol in the Treatment of Teenagers Affected by PCOS.

    PubMed

    Pkhaladze, Lali; Barbakadze, Ludmila; Kvashilava, Nana

    2016-01-01

    Objective. To compare the effectiveness of myo-inositol (MI) and oral contraceptive pills (OCPs) in monotherapy and MI in combination with OCPs in the treatment of teenagers affected by polycystic ovary syndrome (PCOS). Methods. 61 adolescent girls aged 13-19 years, with PCOS, were involved in the prospective, open-label study. Patients were randomized into three groups: I group, 20 patients receiving drospirenone 3 mg/ethinyl estradiol 30 μg; II group, 20 patients receiving 4 g myo-inositol plus 400 mg folic acid; III group, 21 patients receiving both medications. Results. After receiving MI significant reduction in weight, BMI, glucose, C-peptide, insulin, HOMA-IR, FT, and LH was detected. The levels of SHBG, TT, FAI, DHEA-S, and AMH did not change statistically significantly. After receiving OCPs weight and BMI slightly increased, but metabolic parameters did not change. Combination of MI and OCPs did not change weight and BMI, but reduction in C-peptide, insulin, and HOMA-IR was detected. TT, FT, FAI, DHEA-S, LH, and AMH levels decreased and SHBG increased. Conclusions. Administration of MI is a safe and effective method to prevent and correct metabolic disorders in teenagers affected by PCOS. With combination of MI and OCPs antiandrogenic effects are enhanced, negative impact of OCPs on weight gain is balanced, and metabolic profile is improved. PMID:27635134

  19. Inositol hexakisphosphate kinase-1 interacts with perilipin1 to modulate lipolysis.

    PubMed

    Ghoshal, Sarbani; Tyagi, Richa; Zhu, Qingzhang; Chakraborty, Anutosh

    2016-09-01

    Lipolysis leads to the breakdown of stored triglycerides (TAG) to release free fatty acids (FFA) and glycerol which is utilized by energy expenditure pathways to generate energy. Therefore, a decrease in lipolysis augments fat accumulation in adipocytes which promotes weight gain. Conversely, if lipolysis is not complemented by energy expenditure, it leads to FFA induced insulin resistance and type-2 diabetes. Thus, lipolysis is under stringent physiological regulation, although the precise mechanism of the regulation is not known. Deletion of inositol hexakisphosphate kinase-1 (IP6K1), the major inositol pyrophosphate biosynthetic enzyme, protects mice from high fat diet (HFD) induced obesity and insulin resistance. IP6K1-KO mice are lean due to enhanced energy expenditure. Therefore, IP6K1 is a target in obesity and type-2 diabetes. However, the mechanism/s by which IP6K1 regulates adipose tissue lipid metabolism is yet to be understood. Here, we demonstrate that IP6K1-KO mice display enhanced basal lipolysis. IP6K1 modulates lipolysis via its interaction with the lipolytic regulator protein perilipin1 (PLIN1). Furthermore, phosphorylation of IP6K1 at a PKC/PKA motif modulates its interaction with PLIN1 and lipolysis. Thus, IP6K1 is a novel regulator of PLIN1 mediated lipolysis. PMID:27373682

  20. Myo-Inositol in the Treatment of Teenagers Affected by PCOS

    PubMed Central

    Barbakadze, Ludmila; Kvashilava, Nana

    2016-01-01

    Objective. To compare the effectiveness of myo-inositol (MI) and oral contraceptive pills (OCPs) in monotherapy and MI in combination with OCPs in the treatment of teenagers affected by polycystic ovary syndrome (PCOS). Methods. 61 adolescent girls aged 13–19 years, with PCOS, were involved in the prospective, open-label study. Patients were randomized into three groups: I group, 20 patients receiving drospirenone 3 mg/ethinyl estradiol 30 μg; II group, 20 patients receiving 4 g myo-inositol plus 400 mg folic acid; III group, 21 patients receiving both medications. Results. After receiving MI significant reduction in weight, BMI, glucose, C-peptide, insulin, HOMA-IR, FT, and LH was detected. The levels of SHBG, TT, FAI, DHEA-S, and AMH did not change statistically significantly. After receiving OCPs weight and BMI slightly increased, but metabolic parameters did not change. Combination of MI and OCPs did not change weight and BMI, but reduction in C-peptide, insulin, and HOMA-IR was detected. TT, FT, FAI, DHEA-S, LH, and AMH levels decreased and SHBG increased. Conclusions. Administration of MI is a safe and effective method to prevent and correct metabolic disorders in teenagers affected by PCOS. With combination of MI and OCPs antiandrogenic effects are enhanced, negative impact of OCPs on weight gain is balanced, and metabolic profile is improved. PMID:27635134

  1. Quantification of myo-inositol, 1,5-anhydro- D-sorbitol, and D-chiro-inositol using high-performance liquid chromatography with electrochemical detection in very small volume clinical samples.

    PubMed

    Schimpf, Karen J; Meek, Claudia C; Leff, Richard D; Phelps, Dale L; Schmitz, Daniel J; Cordle, Christopher T

    2015-11-01

    Inositol is a six-carbon sugar alcohol and is one of nine biologically significant isomers of hexahydroxycyclohexane. Myo-inositol is the primary biologically active form and is present in higher concentrations in the fetus and newborn than in adults. It is currently being examined for the prevention of retinopathy of prematurity in newborn preterm infants. A robust method for quantifying myo-inositol (MI), D-chiro-inositol (DCI) and 1,5-anhydro- D-sorbitol (ADS) in very small-volume (25 μL) urine, blood serum and/or plasma samples was developed. Using a multiple-column, multiple mobile phase liquid chromatographic system with electrochemical detection, the method was validated with respect to (a) selectivity, (b) accuracy/recovery, (c) precision/reproducibility, (d) sensitivity, (e) stability and (f) ruggedness. The standard curve was linear and ranged from 0.5 to 30 mg/L for each of the three analytes. Above-mentioned performance measures were within acceptable limits described in the Food and Drug Administration's Guidance for Industry: Bioanalytical Method Validation. The method was validated using blood serum and plasma collected using four common anticoagulants, and also by quantifying the accuracy and sensitivity of MI measured in simulated urine samples recovered from preterm infant diaper systems. The method performs satisfactorily measuring the three most common inositol isomers on 25 μL clinical samples of serum, plasma, milk, and/or urine. Similar performance is seen testing larger volume samples of infant formulas and infant formula ingredients. MI, ADS and DCI may be accurately tested in urine samples collected from five different preterm infant diapers if the urine volume is greater than 2-5 mL.

  2. A comparison of d-inositol 1:2-cyclic phosphate 2-phosphohydrolase with other phosphodiesterases of kidney

    PubMed Central

    Dawson, R. M. C.; Clarke, N. G.

    1973-01-01

    1. The ability to hydrolyse various phosphodiesterase substrates was examined in subcellular fractions of rat kidney and in serial slices of the kidneys of mouse, rat, guinea pig and ox cut from the cortex perimeter inwards. 2. d-Inositol 1:2-cyclic phosphate 2-phosphohydrolase could be clearly distinguished from phosphodiesterases which hydrolyse 2′:3′- and 3′:5′-cyclic AMP and p-nitrophenyl thymidine 5′-phosphate (phosphodiesterase I). The hydrolysis of sn-glycero-3-phosphorylcholine showed a distribution identical with that of particle-bound d-inositol 1:2-cyclic phosphate 2-phosphodiesterase, but there was a 30-fold difference in the ratio of enzyme activities between the rat and guinea pig. 3. In rat and mouse kidney, d-inositol 1:2-cyclic phosphate 2-phosphohydrolase is virtually all membrane bound and in the outer cortex, whereas in guinea-pig kidney the enzyme is almost entirely soluble and located throughout the kidney tissue. Some properties of the soluble enzyme are described. 4. Distribution and histochemical studies indicated that in the rat and mouse, phosphodiesterase I is associated with the brush borders of the straight portion (pars recta) of the proximal tubule, whereas inositol 1:2-cyclic phosphate 2-phosphohydrolase and probably glycerylphosphorylcholine diesterase are associated with the brush borders of the convoluted part of the tubule (pars convoluta). ImagesPLATE 1 PMID:4353088

  3. Raman spectra of organic (myo-inositol hexakis phosphate) and inorganic P sepctra show pH dependence

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Understanding phosphorous fate and transport is in part limited by technical difficulties and/or access to expensive equipment associated with differentiating ortho-phosphate (P) from organic phosphate in complex environmental samples. Myo-inositol hexakis phosphate (IHP) is the most prevalent form...

  4. An improved Bacillus subtilis cell factory for producing scyllo-inositol, a promising therapeutic agent for Alzheimer’s disease

    PubMed Central

    2013-01-01

    Background Bacillus subtilis 168 possesses an efficient pathway to metabolize some of the stereoisomers of inositol, including myo-inositol (MI) and scyllo-inositol (SI). Previously we reported a prototype of a B. subtilis cell factory with modified inositol metabolism that converts MI into SI in the culture medium. However, it wasted half of initial 1.0% (w/v) MI, and the conversion was limited to produce only 0.4% (w/v) SI. To achieve a more efficient SI production, we attempted additional modifications. Results All “useless” genes involved in MI and SI metabolism were deleted. Although no elevation in SI production was observed in the deletion strain, it did result in no wastage of MI anymore. Thus additionally, overexpression of the key enzymes, IolG and IolW, was appended to demonstrate that simultaneous overexpression of them enabled complete conversion of all MI into SI. Conclusions The B. subtilis cell factory was improved to yield an SI production rate of 10 g/L/48 h at least. The improved conversion was achieved only in the presence of enriched nutrition in the form of 2% (w/v) Bacto soytone in the medium, which may be due to the increasing demand for regeneration of cofactors. PMID:24325193

  5. Accumulation of Mannosylglycerate and Di-myo-Inositol-Phosphate by Pyrococcus furiosus in Response to Salinity and Temperature

    PubMed Central

    Martins, L. O.; Santos, H.

    1995-01-01

    (sup13)C and (sup1)H nuclear magnetic resonance spectroscopy was used to identify and quantify organic solutes accumulated by the hyperthermophilic archaeon Pyrococcus furiosus in response to temperature and salinity. Di-myo-inositol-phosphate and 2-O-(beta)-mannosylglycerate were the major organic solutes accumulated in these cells. The total intracellular organic solutes increased significantly in response either to an increase in temperature or to an increase in salinity, but (beta)-mannosylglycerate accumulated mainly at high salinities, whereas the concentration of di-myo-inositol-phosphate increased dramatically at supraoptimal growth temperatures. Glutamate was present at concentrations detectable by nuclear magnetic resonance only in cells grown in low-salinity media. The intracellular levels of K(sup+) are clearly dependent on the salinity of the medium, and the concentrations of this cation are high enough to counterbalance the negative charges of (beta)-mannosylglycerate and di-myo-inositol-phosphate in the cell. The results presented here together with those previously reported for Pyrococcus woesei (S. Scholz, J. Sonnenbichler, W. Schafer, and R. Hensel, FEBS Lett. 306:239-242, 1992) strongly support a role for di-myo-inositol-phosphate in thermoprotection. PMID:16535119

  6. Role of Inositol Phosphosphingolipid Phospholipase C1, the Yeast Homolog of Neutral Sphingomyelinases in DNA Damage Response and Diseases

    PubMed Central

    Tripathi, Kaushlendra

    2015-01-01

    Sphingolipids play a very crucial role in many diseases and are well-known as signaling mediators in many pathways. Sphingolipids are produced during the de novo process in the ER (endoplasmic reticulum) from the nonsphingolipid precursor and comprise both structural and bioactive lipids. Ceramide is the central core of the sphingolipid pathway, and its production has been observed following various treatments that can induce several different cellular effects including growth arrest, DNA damage, apoptosis, differentiation, and senescence. Ceramides are generally produced through the sphingomyelin hydrolysis and catalyzed by the enzyme sphingomyelinase (SMase) in mammals. Presently, there are many known SMases and they are categorized into three groups acid SMases (aSMases), alkaline SMases (alk-SMASES), and neutral SMases (nSMases). The yeast homolog of mammalians neutral SMases is inositol phosphosphingolipid phospholipase C. Yeasts generally have inositol phosphosphingolipids instead of sphingomyelin, which may act as a homolog of mammalian sphingomyelin. In this review, we shall explain the structure and function of inositol phosphosphingolipid phospholipase C1, its localization inside the cells, mechanisms, and its roles in various cell responses during replication stresses and diseases. This review will also give a new basis for our understanding for the mechanisms and nature of the inositol phosphosphingolipid phospholipase C1/nSMase. PMID:26346287

  7. Comparison of muscarine- and vasopressin-stimulated inositol phospholipid metabolism in the superior cervical ganglion of the rat

    SciTech Connect

    Horwitz, J.; Anderson, C.; Perlman, R.L.

    1986-03-05

    Both muscarine and vasopressin have previously been shown to increase the accumulation of /sup 3/H-inositol phosphates (/sup 3/H-IP) in superior cervical ganglia in which the phospholipids were labeled with /sup 3/H-inositol. They have compared the effects of muscarine and vasopressin on phospholipid metabolism in the ganglion. The effects of these agents on /sup 3/H-IP accumulation are additive. The response to muscarine plateaus after approximately 10 min whereas the response to vasopressin increases for at least 30 min. Decentralization and maintenance in organ culture appear to potentiate the effect of muscarine on /sup 3/H-IP accumulation but do not effect the response of the ganglia to vasopressin. Muscarine and vasopressin also increase the incorporation of /sup 3/H-inositol into phospholipids in the ganglion. Autoradiographic techniques were used to localize the inositol-containing phospholipids in the ganglion. Muscarine increases phospholipid labeling primarily in the cell bodies of the principal ganglionic neurons, whereas vasopressin increases phospholipid labeling primarily in the neuropil. These data are consistent with the hypothesis that muscarine and vasopressin stimulate the metabolism of different pools of phospholipids.

  8. Pharmacokinetics and tissue distribution of inositol hexaphosphate in C.B17 SCID mice bearing human breast cancer xenografts.

    PubMed

    Eiseman, Julie; Lan, Jing; Guo, Jianxia; Joseph, Erin; Vucenik, Ivana

    2011-10-01

    Inositol hexaphosphate (IP(6)) is effective in preclinical cancer prevention and chemotherapy. In addition to cancer, IP(6) has many other beneficial effects for human health, such as reduction in risk of developing cardiovascular disease and diabetes and inhibition of kidney stone formation. Studies presented here describe the pharmacokinetics, tissue distribution, and metabolism of IP(6) following intravenous (IV) or per os (PO) administration to mice. SCID mice bearing MDA-MB-231 xenografts were treated with 20 mg/kg IP(6) (3 μCi per mouse [(14)C]-uniformly ring-labeled IP(6)) and euthanized at various times after IP(6) treatment. Plasma and tissues were analyzed for [(14)C]-IP(6) and metabolites by high-performance liquid chromatography with radioactivity detection. Following IV administration of IP(6), plasma IP(6) concentrations peaked at 5 minutes and were detectable until 45 minutes. Liver IP(6) concentrations were more than 10-fold higher than plasma concentrations, whereas other normal tissue concentrations were similar to plasma. Only inositol was detected in xenografts. After PO administration, IP(6) was detected in liver; but only inositol was detectable in other tissues. After both IV and PO administration, exogenous IP(6) was rapidly dephosphorylated to inositol; however, alterations in endogenous IPs were not examined.

  9. Enhancement of inositol phospholipid metabolism and activation of protein kinase C in ras-transformed rat fibroblasts

    SciTech Connect

    Huang, M.; Chida, K.; Kamata, N.; Nose, K.; Kato, M.; Homma, Y.; Takenawa, T.; Kuroki, T.

    1988-12-05

    The inositol phospholipid metabolism is one of the main pathways of signal transduction in cells. We measured the activities of its key enzymes in v-Ha-ras-transformed 208F rat fibroblasts. In the ras-transformed clones, incorporation of (TSP)Pi into intermediates of the inositol phospholipid metabolism was stimulated. The activities of phosphatidylinositol and phosphatidylinositol-4-phosphate kinases in the transformed clones were about 35-50% more than in untransformed cells, indicating increased inositol phospholipid metabolism. However, the activity of diacylglycerol kinase in their membrane fraction was 25-35% less than that of untransformed cells, although the total diacylglycerol kinase activity did not change. The imbalance of these kinases could constitute one of the main reasons leading to the increased level of inositol phosphates and the accumulation of diacylglycerol to 2-2.2 times that in control 208F cells. Phosphatidylinositol-4,5-bisphosphate-phospholipase C activity did not change on the transformation when assayed under various conditions. The increased level of diacylglycerol caused intracellular translocation, activation, and down-regulation of protein kinase C changes which may be one of the essential events in transformation by the v-Ha-ras gene.

  10. Effects of omega-3 fatty acids on vascular smooth muscle cells: reduction in arachidonic acid incorporation into inositol phospholipids.

    PubMed

    Yerram, N R; Spector, A A

    1989-07-01

    A rapid increase in arachidonic acid incorporation into phosphatidylinositol (PI) occurred following exposure of cultured porcine pulmonary artery smooth muscle cells to calcium ionophore A23187. This response was specific for PI and phosphatidic acid; none of the other phosphoglycerides showed any increase in arachidonic acid incorporation. The incorporation of [3H]inositol also was increased, indicating that complete synthesis of PI rather than only fatty acylation occurred in response to the ionophore. The presence of omega-3 fatty acids, especially eicosapentaenoic acid (EPA), reduced arachidonic acid but not inositol incorporation into PI. Stimulated incorporation of EPA also occurred under these conditions, suggesting that EPA replaces arachidonic acid in the newly synthesized pool of PI. Although much less arachidonic acid was incorporated into the polyphosphoinositides following exposure to the ionophore, arachidonic acid incorporation into these phosphorylated derivatives also decreased when EPA was present. These findings suggest that when omega-3 fatty acids are available, less arachidonic acid is channeled into the inositol phospholipids of activated smooth muscle cells because of replacement by EPA. This may represent a mechanism whereby omega-3 fatty acids, especially EPA, can accumulate in the metabolically active pools of inositol phospholipids and thereby possibly influence the properties or responsiveness of vascular smooth muscle.

  11. Involvement of Inositol Biosynthesis and Nitric Oxide in the Mediation of UV-B Induced Oxidative Stress

    PubMed Central

    Lytvyn, Dmytro I.; Raynaud, Cécile; Yemets, Alla I.; Bergounioux, Catherine; Blume, Yaroslav B.

    2016-01-01

    The involvement of NO-signaling in ultraviolet B (UV-B) induced oxidative stress (OS) in plants is an open question. Inositol biosynthesis contributes to numerous cellular functions, including the regulation of plants tolerance to stress. This work reveals the involvement of inositol-3-phosphate synthase 1 (IPS1), a key enzyme for biosynthesis of myo-inositol and its derivatives, in the response to NO-dependent OS in Arabidopsis. Homozygous mutants deficient for IPS1 (atips1) and wild-type plants were transformed with a reduction- grx1-rogfp2 and used for the dynamic measurement of UV-B-induced and SNP (sodium nitroprusside)-mediated oxidative stresses by confocal microscopy. atips1 mutants displayed greater tissue-specific resistance to the action of UV-B than the wild type. SNP can act both as an oxidant or repairer depending on the applied concentration, but mutant plants were more tolerant than the wild type to nitrosative effects of high concentration of SNP. Additionally, pretreatment with low concentrations of SNP (10, 100 μM) before UV-B irradiation resulted in a tissue-specific protective effect that was enhanced in atips1. We conclude that the interplay between nitric oxide and inositol signaling can be involved in the mediation of UV-B-initiated oxidative stress in the plant cell. PMID:27148278

  12. Metabolomics Reveals Relationship between Plasma Inositols and Birth Weight: Possible Markers for Fetal Programming of Type 2 Diabetes

    PubMed Central

    Nissen, Pia Marlene; Nebel, Caroline; Oksbjerg, Niels; Bertram, Hanne Christine

    2011-01-01

    Epidemiological studies in man and with experimental animal models have shown that intrauterine growth restriction (IUGR) resulting in low birth weight is associated with higher risk of programming welfare diseases in later life. In the pig, severe IUGR occurs naturally and contribute substantially to a large intralitter variation in birth weight and may therefore be a good model for man. In the present paper the natural form of IUGR in pigs was studied close to term by nuclear magnetic resonance (NMR-)based metabolomics. The NMR-based investigations revealed different metabolic profiles of plasma samples from low-birth weight (LW) and high-birth weight (HW) piglets, respectively, and differences were assigned to levels of glucose and myo-inositol. Further studies by GC-MS revealed that LW piglets had a significant higher concentration of myoinositol and D-chiro-inositol in plasma compared to larger littermates. Myo-inositol and D-chiro-inositol have been coupled with glucose intolerance and insulin resistance in adults, and the present paper therefore suggests that IUGR is related to impaired glucose metabolism during fetal development, which may cause type 2 diabetes in adulthood. PMID:20814537

  13. A limitation of the continuous spectrophotometric assay for the measurement of myo-inositol-1-phosphate synthase activity.

    PubMed

    Huang, Xinyi; Hernick, Marcy

    2011-10-15

    Myo-inositol-1-phosphate synthase (MIPS) catalyzes the conversion of glucose-6-phosphate to myo-inositol-1-phosphate. The reaction catalyzed by MIPS is the first step in the biosynthesis of inositol and inositol-containing molecules that serve important roles in both eukaryotes and prokaryotes. Consequently, MIPS is a target for the development of therapeutic agents for the treatment of infectious diseases and bipolar disorder. We recently reported a continuous spectrophotometric method for measuring MIPS activity using a coupled assay that allows the rapid characterization of MIPS in a multiwell plate format. Here we validate the continuous assay as a high-throughput alternative for measuring MIPS activity and report on one limitation of this assay-the inability to examine the effect of divalent metal ions (at high concentrations) on MIPS activity. In addition, we demonstrate that the activity of MIPS from Arabidopsis thaliana is moderately enhanced by the addition Mg(2+) and is not enhanced by other divalent metal ions (Zn(2+) and Mn(2+)), consistent with what has been observed for other eukaryotic MIPS enzymes. Our findings suggest that the continuous assay is better suited for characterizing eukaryotic MIPS enzymes that require monovalent cations as cofactors than for characterizing bacterial or archeal MIPS enzymes that require divalent metal ions as cofactors. PMID:21729692

  14. Inositol Metabolism in Plants. III. Conversion of Myo-inositol-2-H to Cell Wall Polysaccharides in Sycamore (Acer pseudoplatanus L.) Cell Culture.

    PubMed

    Roberts, R M; Loewus, F

    1966-11-01

    Prolonged growth of cell cultures of sycamore (Acer pseudoplatanus L.) on agar medium containing myo-inositol-2-(3)H resulted in incorporation of label predominately into uronosyl and pentosyl units of cell wall polysaccharides. Procedures normally used to distinguish between pectic substance and hemicellulose yielded carbohydrate-rich fractions with solubility characteristics ranging from pectic substance to hemicellulose yet the uronic acid and pentose composition of these fractions was decidedly pectic. Galacturonic acid was the only uronic acid present in each fraction. Subfractionation of alkali-soluble (hemicellulosic) polysaccharide by neutralization followed by ethanol precipitation gave 3 fractions, a water-insoluble, an ethanol-insoluble, and an ethanol-soluble fraction, each progressively poorer in galacturonic acid units and progressively richer in arabinose units; all relatively poor in xylose units.Apparently, processes involved in biosynthesis of primary cell wall continued to produce pectic substance during cell enlargement while processes leading to biosynthesis of typically secondary cell wall polysaccharide such as 4-0-methyl glucuronoxylan were not activated.

  15. A Novel Inositol Pyrophosphate Phosphatase in Saccharomyces cerevisiae: Siw14 PROTEIN SELECTIVELY CLEAVES THE β-PHOSPHATE FROM 5-DIPHOSPHOINOSITOL PENTAKISPHOSPHATE (5PP-IP5).

    PubMed

    Steidle, Elizabeth A; Chong, Lucy S; Wu, Mingxuan; Crooke, Elliott; Fiedler, Dorothea; Resnick, Adam C; Rolfes, Ronda J

    2016-03-25

    Inositol pyrophosphates are high energy signaling molecules involved in cellular processes, such as energetic metabolism, telomere maintenance, stress responses, and vesicle trafficking, and can mediate protein phosphorylation. Although the inositol kinases underlying inositol pyrophosphate biosynthesis are well characterized, the phosphatases that selectively regulate their cellular pools are not fully described. The diphosphoinositol phosphate phosphohydrolase enzymes of the Nudix protein family have been demonstrated to dephosphorylate inositol pyrophosphates; however, theSaccharomyces cerevisiaehomolog Ddp1 prefers inorganic polyphosphate over inositol pyrophosphates. We identified a novel phosphatase of the recently discovered atypical dual specificity phosphatase family as a physiological inositol pyrophosphate phosphatase. Purified recombinant Siw14 hydrolyzes the β-phosphate from 5-diphosphoinositol pentakisphosphate (5PP-IP5or IP7)in vitro. In vivo,siw14Δ yeast mutants possess increased IP7levels, whereas heterologousSIW14overexpression eliminates IP7from cells. IP7levels increased proportionately whensiw14Δ was combined withddp1Δ orvip1Δ, indicating independent activity by the enzymes encoded by these genes. We conclude that Siw14 is a physiological phosphatase that modulates inositol pyrophosphate metabolism by dephosphorylating the IP7isoform 5PP-IP5to IP6. PMID:26828065

  16. Effect of co-solutes and process variables on crystallinity and the crystal form of freeze-dried myo-inositol.

    PubMed

    Izutsu, Ken-Ichi; Kusano, Riho; Arai, Ryoko; Yoshida, Hiroyuki; Ito, Masataka; Shibata, Hiroko; Sugano, Kiyohiko; Goda, Yukihiro; Terada, Katsuhide

    2016-07-25

    The purpose of this study was to elucidate how co-solutes affect the crystallization of small solute molecules during freeze-drying and subsequent storage. Crystallization profiles of myo-inositol and its mixture with dextran 40k in frozen solutions and dried solids were assessed by thermal analysis (DSC), powder-X-ray diffraction, and simultaneous DSC and PXRD analysis. Higher mass ratios of dextran maintained myo-inositol in the non-crystalline mixture state, in frozen solutions, during freeze-drying process, and exposure of dried solids to higher temperatures. Co-lyophilization with a lower mass ratio of dextran resulted in solids containing a variety of myo-inositol crystal forms and crystallinity depending on the composition and thermal history of the process. Heating of some inositol-rich amorphous solids showed crystallization of myo-inositol in the metastable form and its transition to stable form before melting. Heat-treatment of inositol-rich frozen solutions resulted in high crystallinity stable-form inositol solids, leaving dextran in the amorphous state. Sufficient direct molecular interactions (e.g., hydrogen bonding) should explain the stability of dextran-rich amorphous solids. Optimizing solute composition and processes should be a potent way to control crystal form and crystallinity of components in freeze-dried formulations. PMID:27282535

  17. Activation of protein kinase C-mitogen-activated protein kinase signaling in response to inositol starvation triggers Sir2p-dependent telomeric silencing in yeast.

    PubMed

    Lee, Sojin; Gaspar, Maria L; Aregullin, Manuel A; Jesch, Stephen A; Henry, Susan A

    2013-09-27

    Depriving wild type yeast of inositol, a soluble precursor for phospholipid, phosphoinositide, and complex sphingolipid synthesis, activates the protein kinase C (PKC)-MAPK signaling pathway, which plays a key role in the activation of NAD(+)-dependent telomeric silencing. We now report that triggering PKC-MAPK signaling by inositol deprivation or by blocking inositol-containing sphingolipid synthesis with aureobasidin A results in increased telomeric silencing regulated by the MAPK, Slt2p, and the NAD(+)-dependent deacetylase, Sir2p. Consistent with the dependence on NAD(+) in Sir2p-regulated silencing, we found that inositol depletion induces the expression of BNA2, which is required for the de novo synthesis of NAD(+). Moreover, telomeric silencing is greatly reduced in bna2Δ and npt1Δ mutants, which are defective in de novo and salvage pathways for NAD(+) synthesis, respectively. Surprisingly, however, omitting nicotinic acid from the growth medium, which reduces cellular NAD(+) levels, leads to increased telomeric silencing in the absence of inositol and/or at high temperature. This increase in telomeric silencing in response to inositol starvation is correlated to chronological life span extension but is Sir2p-independent. We conclude that activation of the PKC-MAPK signaling by interruption of inositol sphingolipid synthesis leads to increased Sir2p-dependent silencing and is dependent upon the de novo and salvage pathways for NAD(+) synthesis but is not correlated with cellular NAD(+) levels.

  18. Detection of orally administered inositol stereoisomers in mouse blood plasma and their effects on translocation of glucose transporter 4 in skeletal muscle cells.

    PubMed

    Yamashita, Yoko; Yamaoka, Masaru; Hasunuma, Tomohisa; Ashida, Hitoshi; Yoshida, Ken-ichi

    2013-05-22

    Simple pharmacological studies on inositol stereoisomers are presented in this study. Male ICR mice were orally administered 1 g/kg BW of three inositol stereoisomers, myo-inositol (MI), d-chiro-inositol (DCI), and scyllo-inositol (SI), and blood plasma samples and skeletal muscle fractions were prepared after an hour. The plasma samples were subjected to gas chromatography-coupled time-of-flight mass spectrometry (GC-TOF-MS) analysis. None of the three stereoisomers was seen in untreated samples, but substantial amounts ranging from 2.5 to 6.5 mM were detected only after administration, indicating that orally administered inositol stereoisomers were readily absorbed and their levels elevated in the bloodstream. In addition, plasma of SI-administered animals contained substantial MI, suggesting a possible metabolic conversion of SI to MI. In the skeletal muscle fractions, glucose transporter type 4 (GLUT4) content in the plasma membrane increased, indicating that inositol stereoisomers stimulated GLUT4 translocation.

  19. 4-hydroxyphenylacetic acid derivatives of inositol from dandelion (Taraxacum officinale) root characterised using LC-SPE-NMR and LC-MS techniques.

    PubMed

    Kenny, O; Smyth, T J; Hewage, C M; Brunton, N P; McLoughlin, P

    2014-02-01

    The combination of hyphenated techniques, LC-SPE-NMR and LC-MS, to isolate and identify minor isomeric compounds from an ethyl acetate fraction of Taraxacum officinale root was employed in this study. Two distinct fractions of 4-hydroxyphenylacetic acid derivatives of inositol were isolated and characterised by spectroscopic methods. The (1)H NMR spectra and MS data revealed two groups of compounds, one of which were derivatives of the di-4-hydroxyphenylacetic acid derivative of the inositol compound tetrahydroxy-5-[2-(4-hydroxyphenyl)acetyl] oxycyclohexyl-2-(4-hydroxyphenyl) acetate, while the other group consisted of similar tri-substituted inositol derivatives. For both fractions the derivatives of inositols vary in the number of 4-hydroxyphenylacetic acid groups present and their position and geometry on the inositol ring. In total, three di-substituted and three tri-substituted 4-hydroxyphenylacetic acid inositol derivates were identified for the first time along with a further two previously reported di-substituted inositol derivatives.

  20. The separation of ( sup 32 P)inositol phosphates by ion-pair chromatography: Optimization of the method and biological applications

    SciTech Connect

    Sulpice, J.C.; Gascard, P.; Journet, E.; Rendu, F.; Renard, D.; Poggioli, J.; Giraud, F. )

    1989-05-15

    We have developed an ion-pair reverse-phase HPLC method to measure inositol phosphates in {sup 32}P-labeled cells. The different chromatographic parameters were analyzed to optimize the resolution of the {sup 32}P-labeled metabolites. Analysis of inositol phosphates in biological samples was improved by a single charcoal pretreatment which eliminated interfering nucleotides without removing inositol phosphates. The kinetics of production of inositol phosphates in calcium-activated erythrocytes, vasopressin-stimulated hepatocytes, and thrombin-activated platelets were analyzed. Original data on the activation of phosphoinositide phospholipase C were obtained in intact erythrocytes by direct measurement of inositol (1,4,5)P3. Data from agonist-stimulated hepatocytes and platelets were consistent with those from previous studies. In conclusion, this technique offers many advantages over the methodologies currently employed involving anion-exchange chromatography and ({sup 3}H)inositol labeling: (i) {sup 32}P labeling is less expensive and more efficient than {sup 3}H labeling and can be used with all types of cells without permeabilization treatments and (ii) ion-pair HPLC gives good resolution of inositol phosphates from nucleotides with shorter retention times, and long reequilibration periods are not required.

  1. A Novel Inositol Pyrophosphate Phosphatase in Saccharomyces cerevisiae: Siw14 PROTEIN SELECTIVELY CLEAVES THE β-PHOSPHATE FROM 5-DIPHOSPHOINOSITOL PENTAKISPHOSPHATE (5PP-IP5).

    PubMed

    Steidle, Elizabeth A; Chong, Lucy S; Wu, Mingxuan; Crooke, Elliott; Fiedler, Dorothea; Resnick, Adam C; Rolfes, Ronda J

    2016-03-25

    Inositol pyrophosphates are high energy signaling molecules involved in cellular processes, such as energetic metabolism, telomere maintenance, stress responses, and vesicle trafficking, and can mediate protein phosphorylation. Although the inositol kinases underlying inositol pyrophosphate biosynthesis are well characterized, the phosphatases that selectively regulate their cellular pools are not fully described. The diphosphoinositol phosphate phosphohydrolase enzymes of the Nudix protein family have been demonstrated to dephosphorylate inositol pyrophosphates; however, theSaccharomyces cerevisiaehomolog Ddp1 prefers inorganic polyphosphate over inositol pyrophosphates. We identified a novel phosphatase of the recently discovered atypical dual specificity phosphatase family as a physiological inositol pyrophosphate phosphatase. Purified recombinant Siw14 hydrolyzes the β-phosphate from 5-diphosphoinositol pentakisphosphate (5PP-IP5or IP7)in vitro. In vivo,siw14Δ yeast mutants possess increased IP7levels, whereas heterologousSIW14overexpression eliminates IP7from cells. IP7levels increased proportionately whensiw14Δ was combined withddp1Δ orvip1Δ, indicating independent activity by the enzymes encoded by these genes. We conclude that Siw14 is a physiological phosphatase that modulates inositol pyrophosphate metabolism by dephosphorylating the IP7isoform 5PP-IP5to IP6.

  2. A High-Throughput Screening-Compatible Strategy for the Identification of Inositol Pyrophosphate Kinase Inhibitors

    PubMed Central

    Wang, Huanchen; An, Yi; Kireev, Dmitri; Stashko, Michael A.; Jessen, Henning J.; Pearce, Kenneth H.; Frye, Stephen V.; Shears, Stephen B.

    2016-01-01

    Pharmacological tools—‘chemical probes’—that intervene in cell signaling cascades are important for complementing genetically-based experimental approaches. Probe development frequently begins with a high-throughput screen (HTS) of a chemical library. Herein, we describe the design, validation, and implementation of the first HTS-compatible strategy against any inositol phosphate kinase. Our target enzyme, PPIP5K, synthesizes ‘high-energy’ inositol pyrophosphates (PP-InsPs), which regulate cell function at the interface between cellular energy metabolism and signal transduction. We optimized a time-resolved, fluorescence resonance energy transfer ADP-assay to record PPIP5K-catalyzed, ATP-driven phosphorylation of 5-InsP7 to 1,5-InsP8 in 384-well format (Z’ = 0.82 ± 0.06). We screened a library of 4745 compounds, all anticipated to be membrane-permeant, which are known—or conjectured based on their structures—to target the nucleotide binding site of protein kinases. At a screening concentration of 13 μM, fifteen compounds inhibited PPIP5K >50%. The potency of nine of these hits was confirmed by dose-response analyses. Three of these molecules were selected from different structural clusters for analysis of binding to PPIP5K, using isothermal calorimetry. Acceptable thermograms were obtained for two compounds, UNC10112646 (Kd = 7.30 ± 0.03 μM) and UNC10225498 (Kd = 1.37 ± 0.03 μM). These Kd values lie within the 1–10 μM range generally recognized as suitable for further probe development. In silico docking data rationalizes the difference in affinities. HPLC analysis confirmed that UNC10225498 and UNC10112646 directly inhibit PPIP5K-catalyzed phosphorylation of 5-InsP7 to 1,5-InsP8; kinetic experiments showed inhibition to be competitive with ATP. No other biological activity has previously been ascribed to either UNC10225498 or UNC10112646; moreover, at 10 μM, neither compound inhibits IP6K2, a structurally-unrelated PP-InsP kinase. Our

  3. Uncoupling of attenuated myo-(3H)inositol uptake and dysfunction in Na(+)-K(+)-ATPase pumping activity in hypergalactosemic cultured bovine lens epithelial cells

    SciTech Connect

    Cammarata, P.R.; Tse, D.; Yorio, T. )

    1991-06-01

    Attenuation of both the active transport of myo-inositol and Na(+)-K(+)-ATPase pumping activity has been implicated in the onset of sugar cataract and other diabetic complications in cell culture and animal models of the disease. Cultured bovine lens epithelial cells (BLECs) maintained in galactose-free Eagle's minimal essential medium (MEM) or 40 mM galactose with and without sorbinil for up to 5 days were examined to determine the temporal effects of hypergalactosemia on Na(+)-K(+)-ATPase and myo-inositol uptake. The Na(+)-K(+)-ATPase pumping activity after 5 days of continuous exposure to galactose did not change, as demonstrated by 86Rb uptake. The uptake of myo-(3H)inositol was lowered after 20 h of incubation in galactose and remained below that of the control throughout the 5-day exposure period. The coadministration of sorbinil to the galactose medium normalized the myo-(3H)inositol uptake. No significant difference in the rates of passive efflux of myo-(3H)inositol or 86Rb from preloaded galactose-treated and control cultures was observed. Culture-media reversal studies were also carried out to determine whether the galactose-induced dysfunction in myo-inositol uptake could be corrected. BLECs were incubated in galactose for 5 days, then changed to galactose-free physiological medium with and without sorbinil for a 1-day recovery period. myo-Inositol uptake was reduced to 34% of control after 6 days of continuous exposure to galactose. Within 24 h of media reversal, myo-inositol uptake returned to or exceeded control values in BLECs switched to either MEM or MEM with sorbinil.2+ reversible and occurred independently of changes in Na(+)-K(+)-ATPase pumping activity in cultured lens epithelium, indicating that the two parameters are not strictly associated and that the deficit in myo-inositol uptake occurs rapidly during hypergalactosemia.

  4. Effect of myo-inositol and alpha-lipoic acid on oocyte quality in polycystic ovary syndrome non-obese women undergoing in vitro fertilization: a pilot study.

    PubMed

    Rago, R; Marcucci, I; Leto, G; Caponecchia, L; Salacone, P; Bonanni, P; Fiori, C; Sorrenti, G; Sebastianelli, A

    2015-01-01

    The aim of the present study was to evaluate the effectiveness of the combined administration of myo-inositol and α-lipoic acid in polycystic ovary syndrome (PCOS) patients with normal body mass index (BMI), who had previously undergone intracytoplasmic sperm injection (ICSI) and received myo-inositol alone. Thirty-six of 65 normal-weight patients affected by PCOS who did not achieve pregnancy and one patient who had a spontaneous abortion were re-enrolled and given a cycle of treatment with myo-inositol and α-lipoic acid. For all female partners of the treated couples, the endocrine-metabolic and ultrasound parameters, ovarian volume, oocyte and embryo quality, and pregnancy rates were assessed before and after three months of treatment and compared with those of previous in vitro fertilization (IVF) cycle(s). After supplementation of myo-inositol with α-lipoic acid, insulin levels, BMI and ovarian volume were significantly reduced compared with myo-inositol alone. No differences were found in the fertilization and cleavage rate or in the mean number of transferred embryos between the two different treatments, whereas the number of grade 1 embryos was significantly increased, with a significant reduction in the number of grade 2 embryos treated with myo-inositol plus α-lipoic acid. Clinical pregnancy was not significantly different with a trend for a higher percentage for of myo-inositol and α-lipoic acid compared to the myo-inositol alone group. Our preliminary data suggest that the supplementation of myo-inositol and α-lipoic acid in PCOS patients undergoing an IVF cycle can help to improve their reproductive outcome and also their metabolic profiles, opening potential for their use in long-term prevention of PCOS.

  5. The regulation of runt-related transcription factor 2 by fibroblast growth factor-2 and connexin43 requires the inositol polyphosphate/protein kinase Cδ cascade.

    PubMed

    Niger, Corinne; Luciotti, Maria A; Buo, Atum M; Hebert, Carla; Ma, Vy; Stains, Joseph P

    2013-06-01

    Connexin43 (Cx43) plays a critical role in osteoblast function and bone mass accrual, yet the identity of the second messengers communicated by Cx43 gap junctions, the targets of these second messengers and how they regulate osteoblast function remain largely unknown. We have shown that alterations of Cx43 expression in osteoblasts can impact the responsiveness to fibroblast growth factor-2 (FGF2), by modulating the transcriptional activity of runt-related transcription factor 2 (Runx2). In this study, we examined the contribution of the phospholipase Cγ1/inositol polyphosphate/protein kinase C delta (PKCδ) cascade to the Cx43-dependent transcriptional response of MC3T3 osteoblasts to FGF2. Knockdown of expression and/or inhibition of function of phospholipase Cγ1, inositol polyphosphate multikinase, which generates inositol 1,3,4,5-tetrakisphosphate (InsP₄) and InsP₅, and inositol hexakisphosphate kinase 1/2, which generates inositol pyrophosphates, prevented the ability of Cx43 to potentiate FGF2-induced signaling through Runx2. Conversely, overexpression of phospholipase Cγ1 and inositol hexakisphosphate kinase 1/2 enhanced FGF2 activation of Runx2 and the effect of Cx43 overexpression on this response. Disruption of these pathways blocked the nuclear accumulation of PKCδ and the FGF2-dependent interaction of PKCδ and Runx2, reducing Runx2 transcriptional activity. These data reveal that FGF2-signaling involves the inositol polyphosphate cascade, including inositol hexakisphosphate kinase (IP6K), and demonstrate that IP6K regulates Runx2 and osteoblast gene expression. Additionally, these data implicate the water-soluble inositol polyphosphates as mediators of the Cx43-dependent amplification of the osteoblast response to FGF2, and suggest that these low molecular weight second messengers may be biologically relevant mediators of osteoblast function that are communicated by Cx43-gap junctions.

  6. Effect of myo-inositol and alpha-lipoic acid on oocyte quality in polycystic ovary syndrome non-obese women undergoing in vitro fertilization: a pilot study.

    PubMed

    Rago, R; Marcucci, I; Leto, G; Caponecchia, L; Salacone, P; Bonanni, P; Fiori, C; Sorrenti, G; Sebastianelli, A

    2015-01-01

    The aim of the present study was to evaluate the effectiveness of the combined administration of myo-inositol and α-lipoic acid in polycystic ovary syndrome (PCOS) patients with normal body mass index (BMI), who had previously undergone intracytoplasmic sperm injection (ICSI) and received myo-inositol alone. Thirty-six of 65 normal-weight patients affected by PCOS who did not achieve pregnancy and one patient who had a spontaneous abortion were re-enrolled and given a cycle of treatment with myo-inositol and α-lipoic acid. For all female partners of the treated couples, the endocrine-metabolic and ultrasound parameters, ovarian volume, oocyte and embryo quality, and pregnancy rates were assessed before and after three months of treatment and compared with those of previous in vitro fertilization (IVF) cycle(s). After supplementation of myo-inositol with α-lipoic acid, insulin levels, BMI and ovarian volume were significantly reduced compared with myo-inositol alone. No differences were found in the fertilization and cleavage rate or in the mean number of transferred embryos between the two different treatments, whereas the number of grade 1 embryos was significantly increased, with a significant reduction in the number of grade 2 embryos treated with myo-inositol plus α-lipoic acid. Clinical pregnancy was not significantly different with a trend for a higher percentage for of myo-inositol and α-lipoic acid compared to the myo-inositol alone group. Our preliminary data suggest that the supplementation of myo-inositol and α-lipoic acid in PCOS patients undergoing an IVF cycle can help to improve their reproductive outcome and also their metabolic profiles, opening potential for their use in long-term prevention of PCOS. PMID:26753656

  7. The inositol high-polyphosphate series blocks synaptic transmission by preventing vesicular fusion: a squid giant synapse study.

    PubMed Central

    Llinás, R; Sugimori, M; Lang, E J; Morita, M; Fukuda, M; Niinobe, M; Mikoshiba, K

    1994-01-01

    Presynaptic injection of inositol 1,3,4,5-tetraphosphate, inositol 1,3,4,5,6-pentakisphosphate, or inositol 1,2,3,4,5,6-hexakisphosphate--which we denote here the inositol high-polyphosphate series (IHPS)--is shown to block synaptic transmission when injected into the preterminal of the squid giant synapse. This effect is not produced by injection of inositol 1,4,5-trisphosphate. The synaptic block is characterized by a time course in the order of 15-45 min, depending on the injection site in the preterminal fiber; the fastest block occurs when the injection is made at the terminal release site. Presynaptic voltage clamp during transmitter release demonstrates that IHPS block did not modify the presynaptic inward, calcium current. Analysis of synaptic noise at the postsynaptic axon shows that both the evoked and spontaneous transmitter release are blocked by the IHPS. Tetanic stimulation of the presynaptic fiber at frequencies of 100 Hz indicates that block is accompanied by gradual reduction of the postsynaptic response, demonstrating that the block interferes with vesicular fusion rather than with vesicular docking. These results, in combination with the recently demonstrated observation that the IHPS bind the C2B domain in synaptotagmin [Fukada, M., Aruga, J., Niinobe, M., Aimoto, S. & Mikoshiba, K. (1994) J. Biol. Chem. 269, 29206-29211], suggest that IHPS elements are involved in vesicle fusion and exocytosis. In addition, a scheme is proposed in which synaptotagmin triggers transmitter release directly by promoting the fusion of synaptic vesicles with the presynaptic plasmalemma, in agreement with the very rapid nature of transmitter release in chemical synapses. Images Fig. 4 PMID:7809161

  8. Mechanism of myo-inositol hexakisphosphate sorption on amorphous aluminum hydroxide: spectroscopic evidence for rapid surface precipitation.

    PubMed

    Yan, Yupeng; Li, Wei; Yang, Jun; Zheng, Anmin; Liu, Fan; Feng, Xionghan; Sparks, Donald L

    2014-06-17

    Inositol hexakisphosphates are the most abundant organic phosphates (OPs) in most soils and sediments. Adsorption, desorption, and precipitation reactions at environmental interfaces govern the reactivity, speciation, mobility, and bioavailability of inositol hexakisphosphates in terrestrial and aquatic environments. However, surface complexation and precipitation reactions of inositol hexakisphosphates on soil minerals have not been well understood. Here we investigate the surface complexation-precipitation process and mechanism of myo-inositol hexakisphosphate (IHP, phytate) on amorphous aluminum hydroxide (AAH) using macroscopic sorption experiments and multiple spectroscopic tools. The AAH (16.01 μmol m(-2)) exhibits much higher sorption density than boehmite (0.73 μmol m(-2)) and α-Al2O3 (1.13 μmol m(-2)). Kinetics of IHP sorption and accompanying OH(-) release, as well as zeta potential measurements, indicate that IHP is initially adsorbed on AAH through inner-sphere complexation via ligand exchange, followed by AAH dissolution and ternary complex formation; last, the ternary complexes rapidly transform to surface precipitates and bulk phase analogous to aluminum phytate (Al-IHP). The pH level, reaction time, and initial IHP loading evidently affect the interaction of IHP on AAH. In situ ATR-FTIR and solid-state NMR spectra further demonstrate that IHP sorbs on AAH and transforms to surface precipitates analogous to Al-IHP, consistent with the results of XRD analysis. This study indicates that active metal oxides such as AAH strongly mediate the speciation and behavior of IHP via rapid surface complexation-precipitation reactions, thus controlling the mobility and bioavailability of inositol phosphates in the environment.

  9. Osmotic regulation and tissue localization of the myo-inositol biosynthesis pathway in tilapia (Oreochromis mossambicus) larvae.

    PubMed

    Sacchi, Romina; Gardell, Alison M; Chang, Nicole; Kültz, Dietmar

    2014-10-01

    The myo-inositol biosynthesis (MIB) pathway converts glucose-6-phosphate to the compatible osmolyte myo-inositol, which protects cells from salinity stress. We exposed tilapia larvae just after yolk sac resorption to various hypersaline environments and recorded robust induction of the enzymes that constitute the MIB pathway, myo-inositol-phosphate synthase (MIPS), and inositol monophosphatase 1 (IMPA1). Strong up-regulation of these enzymes is evident at both mRNA (quantitative real-time PCR) and protein (densitometric analysis of Western blots) levels. The highest level of induction of these enzymes occurs at the highest salinity that larvae were exposed to (90 ppt). Less severe salinity stress causes a proportionately reduced induction of the MIB pathway. Two distinct MIPS mRNA variants are present in tilapia larvae and both are induced at comparable levels for all the salinity challenges tested (34, 70, and 90 ppt). Immunohistochemical localization of IMPA1 protein in sagittal sections of salinity stressed and control larvae identified tissues that are particularly potent in inducing the MIB pathway. These tissues include the skin (epidermis), gills, eye (ciliary epithelium) and heart. In particular, the epidermis directly facing the external milieu showed a very strong induction of IMPA1 immunoreactivity. IMPA1 induction in response to salinity stress was not observed in other tissues suggesting that tilapia larvae may also utilize compatible organic osmolytes other than solely myo-inositol for osmoprotection. We conclude that the MIB pathway plays an important role in protecting multiple (but not all) tissues of tilapia larvae from hyperosmotic salinity stress.

  10. Mechanism of myo-inositol hexakisphosphate sorption on amorphous aluminum hydroxide: spectroscopic evidence for rapid surface precipitation.

    PubMed

    Yan, Yupeng; Li, Wei; Yang, Jun; Zheng, Anmin; Liu, Fan; Feng, Xionghan; Sparks, Donald L

    2014-06-17

    Inositol hexakisphosphates are the most abundant organic phosphates (OPs) in most soils and sediments. Adsorption, desorption, and precipitation reactions at environmental interfaces govern the reactivity, speciation, mobility, and bioavailability of inositol hexakisphosphates in terrestrial and aquatic environments. However, surface complexation and precipitation reactions of inositol hexakisphosphates on soil minerals have not been well understood. Here we investigate the surface complexation-precipitation process and mechanism of myo-inositol hexakisphosphate (IHP, phytate) on amorphous aluminum hydroxide (AAH) using macroscopic sorption experiments and multiple spectroscopic tools. The AAH (16.01 μmol m(-2)) exhibits much higher sorption density than boehmite (0.73 μmol m(-2)) and α-Al2O3 (1.13 μmol m(-2)). Kinetics of IHP sorption and accompanying OH(-) release, as well as zeta potential measurements, indicate that IHP is initially adsorbed on AAH through inner-sphere complexation via ligand exchange, followed by AAH dissolution and ternary complex formation; last, the ternary complexes rapidly transform to surface precipitates and bulk phase analogous to aluminum phytate (Al-IHP). The pH level, reaction time, and initial IHP loading evidently affect the interaction of IHP on AAH. In situ ATR-FTIR and solid-state NMR spectra further demonstrate that IHP sorbs on AAH and transforms to surface precipitates analogous to Al-IHP, consistent with the results of XRD analysis. This study indicates that active metal oxides such as AAH strongly mediate the speciation and behavior of IHP via rapid surface complexation-precipitation reactions, thus controlling the mobility and bioavailability of inositol phosphates in the environment. PMID:24871399

  11. Analysis of Membrane Topology and Identification of Essential Residues for the Yeast Endoplasmic Reticulum Inositol Acyltransferase Gwt1p

    PubMed Central

    Sagane, Koji; Umemura, Mariko; Ogawa-Mitsuhashi, Kaoru; Tsukahara, Kappei; Yoko-o, Takehiko; Jigami, Yoshifumi

    2011-01-01

    Glycosylphosphatidylinositol (GPI) is a post-translational modification that anchors cell surface proteins to the plasma membrane, and GPI modifications occur in all eukaryotes. Biosynthesis of GPI starts on the cytoplasmic face of the endoplasmic reticulum (ER) membrane, and GPI precursors flip from the cytoplasmic side to the luminal side of the ER, where biosynthesis of GPI precursors is completed. Gwt1p and PIG-W are inositol acyltransferases that transfer fatty acyl chains to the inositol moiety of GPI precursors in yeast and mammalian cells, respectively. To ascertain whether flipping across the ER membrane occurs before or after inositol acylation of GPI precursors, we identified essential residues of PIG-W and Gwt1p and determined the membrane topology of Gwt1p. Guided by algorithm-based predictions of membrane topology, we experimentally identified 13 transmembrane domains in Gwt1p. We found that Gwt1p, PIG-W, and their orthologs shared four conserved regions and that these four regions in Gwt1p faced the luminal side of the ER membrane. Moreover, essential residues of Gwt1p and PIG-W faced the ER lumen or were near the luminal edge of transmembrane domains. The membrane topology of Gwt1p suggested that inositol acylation occurred on the luminal side of the ER membrane. Rather than stimulate flipping of the GPI precursor across the ER membrane, inositol acylation of GPI precursors may anchor the precursors to the luminal side of the ER membrane, preventing flip-flops. PMID:21367863

  12. Purification, crystallization and room-temperature X-ray diffraction of inositol dehydrogenase LcIDH2 from Lactobacillus casei BL23

    PubMed Central

    Bertwistle, Drew; Vogt, Linda; Aamudalapalli, Hari Babu; Palmer, David R. J.; Sanders, David A. R.

    2014-01-01

    Lactobacillus casei BL23 contains two genes, iolG1 and iolG2, homologous with inositol dehydrogenase encoding genes from many bacteria. Inositol dehydrogenase catalyzes the oxidation of inositol with concomitant reduction of NAD+. The protein encoded by iolG2, LcIDH2, has been purified to homogeneity, crystallized and cryoprotected for diffraction at 77 K. The crystals had a high mosaicity and poor processing statistics. Subsequent diffraction measurements were performed without cryoprotectant at room temperature. These crystals were radiation-resistant and a full diffraction data set was collected at room temperature to 1.6 Å resolution. PMID:25005103

  13. Purification, crystallization and room-temperature X-ray diffraction of inositol dehydrogenase LcIDH2 from Lactobacillus casei BL23.

    PubMed

    Bertwistle, Drew; Vogt, Linda; Aamudalapalli, Hari Babu; Palmer, David R J; Sanders, David A R

    2014-07-01

    Lactobacillus casei BL23 contains two genes, iolG1 and iolG2, homologous with inositol dehydrogenase encoding genes from many bacteria. Inositol dehydrogenase catalyzes the oxidation of inositol with concomitant reduction of NAD+. The protein encoded by iolG2, LcIDH2, has been purified to homogeneity, crystallized and cryoprotected for diffraction at 77 K. The crystals had a high mosaicity and poor processing statistics. Subsequent diffraction measurements were performed without cryoprotectant at room temperature. These crystals were radiation-resistant and a full diffraction data set was collected at room temperature to 1.6 Å resolution. PMID:25005103

  14. Myo-Inositol trisphosphate mobilizes calcium from fusogenic carrot (Daucus carota L. ) protoplasts

    SciTech Connect

    Rincon, M.; Boss, W.F.

    1987-02-01

    To determine whether or not inositol trisphosphate (IP/sub 3/) mobilizes calcium in higher plant cells; they investigated the effect of IP/sub 3/ on Ca/sup 2 +/ fluxes in fusogenic carrot (Daucus carota L.) protoplasts. The protoplasts were incubated in /sup 45/Ca/sup 2 +/-containing medium and the /sup 45/Ca/sup 2 +/ associated with the protoplasts was monitored with time. Addition of IP/sub 3/ (20 micromolar) caused a 17% net loss of the accumulated /sup 45/Ca/sup 2 +/ within 4 minutes. There was a reuptake of /sup 45/Ca/sup 2 +/ and the protoplasts recovered to their initial value by 10 minutes. Phytic acid (IP/sub 6/), also stimulated /sup 45/Ca/sup 2 +/ efflux from the protoplasts. Both the IP/sub 3/- and the IP/sub 6/-induced /sup 45/Ca/sup 2 +/ efflux were inhibited by the calmodulin antagonist, trifluoperazine.

  15. Isolation of the Inositol Phosphoceramide Synthase Gene (AUR1) from Stress-Tolerant Yeast Pichia kudriavzevii.

    PubMed

    Yoo, Boung-Hyuk; Kim, Myoung-Dong

    2015-11-01

    This study is the first report of the entire nucleotide sequence of an inositol phosphoceramide synthase gene from the stress-tolerant yeast Pichia kudriavzevii (PkAUR1). Sequence analysis revealed an open reading frame that spans 1,443 bp and encodes a 480-amino-acid-residue protein with the highest sequence similarity (41.7%) to Aur1 from Spathaspora passalidarum. A phenotypic assay with transformed S. cerevisiae and P. kudriavzevii indicated that two amino acid residues, Phe166 and Gly249, play crucial roles in the resistance to aureobasidin A, which is consistent with previous reports for other fungal Aur1s. The GenBank Accession No. for PkAUR1 is KP729614. PMID:26323269

  16. Novel structural features of the immunocompetent ceramide phospho-inositol glycan core from Trichomonas vaginalis.

    PubMed

    Heiss, Christian; Wang, Zhirui; Black, Ian; Azadi, Parastoo; Fichorova, Raina N; Singh, Bibhuti N

    2016-01-01

    The ceramide phosphoinositol glycan core (CPI-GC) of the lipophosphoglycan of Trichomonas vaginalis is a major virulent factor of this common genitourinary parasite. While its carbohydrate composition has been reported before, its structure has remained largely unknown. We isolated the glycan portions of CPI-GC by nitrous acid deamination and hydrofluoric acid treatment and investigated their structures by methylation analysis and 1- and 2-D NMR. We found that the α-anomer of galactose is a major constituent of CPI-GC. The β-anomer was found exclusively at the non-reducing end of CPI-GC side chains. Furthermore the data showed that the rhamnan backbone is more complex than previously thought and that the inositol residue at the reducing end is linked to a 4-linked α-glucuronic acid (GlcA) residue. This appears to be the most striking and novel feature of this GPI-anchor type molecule.

  17. Linking structure to function: Recent lessons from inositol 1,4,5-trisphosphate receptor mutagenesis.

    PubMed

    Yule, David I; Betzenhauser, Matthew J; Joseph, Suresh K

    2010-06-01

    Great insight has been gained into the structure and function of the inositol 1,4,5 trisphosphate receptor (InsP(3)R) by studies employing mutagenesis of the cDNA encoding the receptor. Notably, early studies using this approach defined the key constituents required for InsP(3) binding in the N-terminus and the membrane spanning regions in the C-terminal domain responsible for channel formation, targeting and function. In this article we evaluate recent studies which have used a similar approach to investigate key residues underlying the in vivo modulation by select regulatory factors. In addition, we review studies defining the structural requirements in the channel domain which comprise the conduction pathway and are suggested to be involved in the gating of the channel.

  18. Inositol hexakisphosphate is bound in the ADAR2 core and required for RNA editing.

    PubMed

    Macbeth, Mark R; Schubert, Heidi L; Vandemark, Andrew P; Lingam, Arunth T; Hill, Christopher P; Bass, Brenda L

    2005-09-01

    We report the crystal structure of the catalytic domain of human ADAR2, an RNA editing enzyme, at 1.7 angstrom resolution. The structure reveals a zinc ion in the active site and suggests how the substrate adenosine is recognized. Unexpectedly, inositol hexakisphosphate (IP6) is buried within the enzyme core, contributing to the protein fold. Although there are no reports that adenosine deaminases that act on RNA (ADARs) require a cofactor, we show that IP6 is required for activity. Amino acids that coordinate IP6 in the crystal structure are conserved in some adenosine deaminases that act on transfer RNA (tRNA) (ADATs), related enzymes that edit tRNA. Indeed, IP6 is also essential for in vivo and in vitro deamination of adenosine 37 of tRNAala by ADAT1.

  19. Inositol 1,4,5‐trisphosphate receptors and their protein partners as signalling hubs

    PubMed Central

    Taylor, Colin W.

    2016-01-01

    Abstract Inositol 1,4,5‐trisphosphate receptors (IP3Rs) are expressed in nearly all animal cells, where they mediate the release of Ca2+ from intracellular stores. The complex spatial and temporal organization of the ensuing intracellular Ca2+ signals allows selective regulation of diverse physiological responses. Interactions of IP3Rs with other proteins contribute to the specificity and speed of Ca2+ signalling pathways, and to their capacity to integrate information from other signalling pathways. In this review, we provide a comprehensive survey of the proteins proposed to interact with IP3Rs and the functional effects that these interactions produce. Interacting proteins can determine the activity of IP3Rs, facilitate their regulation by multiple signalling pathways and direct the Ca2+ that they release to specific targets. We suggest that IP3Rs function as signalling hubs through which diverse inputs are processed and then emerge as cytosolic Ca2+ signals. PMID:26830355

  20. Inositol trisphosphate receptor and ion channel models based on single-channel data

    NASA Astrophysics Data System (ADS)

    Gin, Elan; Wagner, Larry E.; Yule, David I.; Sneyd, James

    2009-09-01

    The inositol trisphosphate receptor (IPR) plays an important role in controlling the dynamics of intracellular Ca2+. Single-channel patch-clamp recordings are a typical way to study these receptors as well as other ion channels. Methods for analyzing and using this type of data have been developed to fit Markov models of the receptor. The usual method of parameter fitting is based on maximum-likelihood techniques. However, Bayesian inference and Markov chain Monte Carlo techniques are becoming more popular. We describe the application of the Bayesian methods to real experimental single-channel data in three ion channels: the ryanodine receptor, the K+ channel, and the IPR. One of the main aims of all three studies was that of model selection with different approaches taken. We also discuss the modeling implications for single-channel data that display different levels of channel activity within one recording.

  1. Inositol Phosphate Recycling Regulates Glycolytic and Lipid Metabolism That Drives Cancer Aggressiveness

    PubMed Central

    2015-01-01

    Cancer cells possess fundamentally altered metabolism that supports their pathogenic features, which includes a heightened reliance on aerobic glycolysis to provide precursors for synthesis of biomass. We show here that inositol polyphosphate phosphatase 1 (INPP1) is highly expressed in aggressive human cancer cells and primary high-grade human tumors. Inactivation of INPP1 leads to a reduction in glycolytic intermediates that feed into the synthesis of the oncogenic signaling lipid lysophosphatidic acid (LPA), which in turn impairs LPA signaling and further attenuates glycolytic metabolism in a feed-forward mechanism to impair cancer cell motility, invasiveness, and tumorigenicity. Taken together these findings reveal a novel mode of glycolytic control in cancer cells that can serve to promote key oncogenic lipid signaling pathways that drive cancer pathogenicity. PMID:24738946

  2. Inositol polyphosphate multikinase is a transcriptional coactivator required for immediate early gene induction.

    PubMed

    Xu, Risheng; Paul, Bindu D; Smith, Dani R; Tyagi, Richa; Rao, Feng; Khan, A Basit; Blech, Daniel J; Vandiver, M Scott; Harraz, Maged M; Guha, Prasun; Ahmed, Ishrat; Sen, Nilkantha; Gallagher, Michela; Snyder, Solomon H

    2013-10-01

    Profound induction of immediate early genes (IEGs) by neural activation is a critical determinant for plasticity in the brain, but intervening molecular signals are not well characterized. We demonstrate that inositol polyphosphate multikinase (IPMK) acts noncatalytically as a transcriptional coactivator to mediate induction of numerous IEGs. IEG induction by electroconvulsive stimulation is virtually abolished in the brains of IPMK-deleted mice, which also display deficits in spatial memory. Neural activity stimulates binding of IPMK to the histone acetyltransferase CBP and enhances its recruitment to IEG promoters. Interestingly, IPMK regulation of CBP recruitment and IEG induction does not require its catalytic activities. Dominant-negative constructs, which prevent IPMK-CBP binding, substantially decrease IEG induction. As IPMK is ubiquitously expressed, its epigenetic regulation of IEGs may influence diverse nonneural and neural biologic processes.

  3. Ionophore A23187 induces a refractory state in thrombin-activated release of inositol phosphates.

    PubMed Central

    Moscat, G; Moreno, F; Iglesias, S; Garcia-Barreno, P; Municio, A M

    1986-01-01

    The phosphatidylinositol cycle has been proposed to be involved in the regulation of platelet functionality through the control of cytoplasmic Ca2+ levels. However, the requirements of phospholipase C for Ca2+ has not yet been elucidated in intact platelets. The primary purpose of the present study was to investigate the Ca2+ requirements of this enzyme in platelets from miniature swine by taking advantage of the permeabilizing properties of the ionophore A23187. Our results strongly suggest that the treatment of platelets with A23187 induces a refractory state in thrombin-stimulated release of inositol phosphates while 5-hydroxytryptamine (serotonin)-secretory capacity in response to thrombin remained constant. This refractory state seems to be dependent on some cytochalasin-inhibitable cytoskeletal phenomena. PMID:3099773

  4. Direct Ionic Regulation of the Activity of Myo-Inositol Biosynthesis Enzymes in Mozambique Tilapia.

    PubMed

    Villarreal, Fernando D; Kültz, Dietmar

    2015-01-01

    Myo-inositol (Ins) is a major compatible osmolyte in many cells, including those of Mozambique tilapia (Oreochromis mossambicus). Ins biosynthesis is highly up-regulated in tilapia and other euryhaline fish exposed to hyperosmotic stress. In this study, enzymatic regulation of two enzymes of Ins biosynthesis, Ins phosphate synthase (MIPS) and inositol monophosphatase (IMPase), by direct ionic effects is analyzed. Specific MIPS and IMPase isoforms from Mozambique tilapia (MIPS-160 and IMPase 1) were selected based on experimental, phylogenetic, and structural evidence supporting their role for Ins biosynthesis during hyperosmotic stress. Recombinant tilapia IMPase 1 and MIPS-160 activity was assayed in vitro at ionic conditions that mimic changes in the intracellular milieu during hyperosmotic stress. The in vitro activities of MIPS-160 and IMPase 1 are highest at alkaline pH of 8.8. IMPase 1 catalytic efficiency is strongly increased during hyperosmolality (particularly for the substrate D-Ins-3-phosphate, Ins-3P), mainly as a result of [Na+] elevation. Furthermore, the substrate-specificity of IMPase 1 towards D-Ins-1-phosphate (Ins-1P) is lower than towards Ins-3P. Because MIPS catalysis results in Ins-3P this results represents additional evidence for IMPase 1 being the isoform that mediates Ins biosynthesis in tilapia. Our data collectively demonstrate that the Ins biosynthesis enzymes are activated under ionic conditions that cells are exposed to during hypertonicity, resulting in Ins accumulation, which, in turn, results in restoration of intracellular ion homeostasis. We propose that the unique and direct ionic regulation of the activities of Ins biosynthesis enzymes represents an efficient biochemical feedback loop for regulation of intracellular physiological ion homeostasis during hyperosmotic stress.

  5. Dietary arginine silicate inositol complex increased bone healing: histologic and histomorphometric study

    PubMed Central

    Yaman, Ferhan; Acikan, Izzet; Dundar, Serkan; Simsek, Sercan; Gul, Mehmet; Ozercan, Ibrahim Hanifi; Komorowski, James; Sahin, Kazim

    2016-01-01

    Background Arginine silicate inositol complex (ASI; arginine 49.5%, silicon 8.2%, and inositol 25%) is a novel material that is a bioavailable source of silicon and arginine. ASI offers potential benefits for vascular and bone health. Objective The aim of this study was to evaluate the potential effects of ASI complex on bone healing of critical-sized defects in rats. Methods The rats were randomly assigned to two groups of 21 rats each. The control group was fed a standard diet for 12 weeks; after the first 8 weeks, a calvarial critical-sized defect was created, and the rats were sacrificed 7, 14, and 28 days later. The ASI group was fed a diet containing 1.81 g/kg of ASI for 12 weeks; after the first 8 weeks, a calvarial critical-sized defect was created, and the rats were sacrificed 7, 14, and 28 days later. The calvarial bones of all the rats were then harvested for evaluation. Results Osteoblasts and osteoclasts were detected at higher levels in the ASI group compared with the control group at days 7, 14, and 28 of the calvarial defect (P<0.05). New bone formation was detected at higher levels in the ASI group compared with the controls at day 28 (P<0.05). However, new bone formation was not detected at days 7 and 14 in both the groups (P>0.05). Conclusion ASI supplementation significantly improved bone tissue healing in rats with critical-sized defects. This study demonstrated that ASI can enhance bone repair and has potential as a therapeutic regimen in humans. PMID:27390517

  6. Direct Ionic Regulation of the Activity of Myo-Inositol Biosynthesis Enzymes in Mozambique Tilapia

    PubMed Central

    Villarreal, Fernando D.; Kültz, Dietmar

    2015-01-01

    Myo-inositol (Ins) is a major compatible osmolyte in many cells, including those of Mozambique tilapia (Oreochromis mossambicus). Ins biosynthesis is highly up-regulated in tilapia and other euryhaline fish exposed to hyperosmotic stress. In this study, enzymatic regulation of two enzymes of Ins biosynthesis, Ins phosphate synthase (MIPS) and inositol monophosphatase (IMPase), by direct ionic effects is analyzed. Specific MIPS and IMPase isoforms from Mozambique tilapia (MIPS-160 and IMPase 1) were selected based on experimental, phylogenetic, and structural evidence supporting their role for Ins biosynthesis during hyperosmotic stress. Recombinant tilapia IMPase 1 and MIPS-160 activity was assayed in vitro at ionic conditions that mimic changes in the intracellular milieu during hyperosmotic stress. The in vitro activities of MIPS-160 and IMPase 1 are highest at alkaline pH of 8.8. IMPase 1 catalytic efficiency is strongly increased during hyperosmolality (particularly for the substrate D-Ins-3-phosphate, Ins-3P), mainly as a result of [Na+] elevation. Furthermore, the substrate-specificity of IMPase 1 towards D-Ins-1-phosphate (Ins-1P) is lower than towards Ins-3P. Because MIPS catalysis results in Ins-3P this results represents additional evidence for IMPase 1 being the isoform that mediates Ins biosynthesis in tilapia. Our data collectively demonstrate that the Ins biosynthesis enzymes are activated under ionic conditions that cells are exposed to during hypertonicity, resulting in Ins accumulation, which, in turn, results in restoration of intracellular ion homeostasis. We propose that the unique and direct ionic regulation of the activities of Ins biosynthesis enzymes represents an efficient biochemical feedback loop for regulation of intracellular physiological ion homeostasis during hyperosmotic stress. PMID:26066044

  7. Specific receptor for inositol-1,4,5-trisphosphate in permeabilized rabbit neutrophils

    SciTech Connect

    Bradford, P.G.; Spat, A.; Rubin, R.P.

    1986-03-05

    Neutrophil chemotaxis and degranulation are resultant, in part, from the mobilization of intracellular calcium by inositol-1,4,5-trisphosphate ((1,4,5)IP/sub 3/), one of the products of chemoattractant-stimulated phospholipase C activity. High specific activity (ca. 40 Ci/mmol) (/sup 32/P)(1,4,5)IP/sub 3/ was prepared from (..gamma..-/sup 32/P)ATP-labeled human erythrocyte ghosts and was used in binding assays with saponin-permeabilized rabbit peritoneal neutrophils. At 4/sup 0/C and in the presence of inhibitors of the IP/sub 3/ 5-phosphomonoesterase, (/sup 32/P)(1,4,5)IP/sub 3/ rapidly associated with a specific binding component which saturated within 60s. Nonspecific binding, taken as the residual binding in the presence of 10 ..mu..M (1,4,5)IP/sub 3/, was 15% of the total. No specific binding was detected using intact cells. The specific binding to permeable cells was reversible (t/sup 1/2/ approx. 60s) and could be inhibited in a dose-dependent manner by (1,4,5)IP/sub 3/ (EC/sub 50/ = 30 nM) and by other calcium mobilizing inositol phosphates ((2,4,5)IP/sub 3/) but not by inactive analogs ((1,4)IP/sub 2/, (4,5)IP/sub 2/, (1)IP). The dose-responses of (1,4,5)IP/sub 3/ and (2,4,5)IP/sub 3/ in inhibiting (/sup 32/P)(1,4,5)IP/sub 3/ specific binding correlated well with their abilities to release Ca/sup 2 +/ from nonmitochondrial vesicular stores in the same preparation of cells, suggesting that the authors have identified the physiological receptor for (1,4,5)IP/sub 3/.

  8. Inositol tetrakisphosphate (IP4)- and inositol triphosphate (IP3)-dependent Ca2+ influx in cortical neuronal nuclei of newborn piglets following graded hypoxia.

    PubMed

    Mishra, Om Prakash; Delivoria-Papadopoulos, Maria

    2004-02-01

    Previous studies have shown that hypoxia results in a modification of the binding characteristics of the neuronal nuclear membrane inositol tetrakisphosphate (IP4) and inositol triphosphate (IP3) receptors. The present study tests the hypothesis that hypoxia-induced modification of the IP4 and IP3 receptors results in increased IP4 and IP3 dependent Ca2+ influx in neuronal nuclei as a function of the degree of cerebral tissue hypoxia in newborn piglets. Studies were performed in piglets, 3-5 days old, divided into normoxic (N = 5) and hypoxic (N = 6) groups. The hypoxic group was exposed to decreased FiO2 ranging from 0.15 to 0.05 for 1 h. Brain tissue hypoxia was documented biochemically by determining ATP and phosphocreatine (PCr) levels. Neuronal nuclei were isolated and 45Ca2+ influx was determined in a medium containing 50 mM Tris buffer (pH 7.4), neuronal nuclei (150 microg protein), 1 microM 45Ca2+, with or without 10 microM IP4 or IP3. In normoxic and hypoxic groups, ATP levels were 4.27 +/- 0.80 and 1.40 +/- 0.69 micromoles/g brain, respectively, P < .001 (ranging from 4.78 to 0.82). PCr levels were 3.40 +/- 0.99 and 0.91 +/- 0.57 micromoles/g brain, respectively, P < .001 (raning from 4.07 to 0.60). During hypoxia, IP4-dependent intranuclear 45Ca2+ influx increased from 3.39 +/- 0.64 in normoxic nuclei to 13.30 +/- 2.18 pM/mg protein in hypoxic nuclei (P < .01). There was an inverse correlation between the 45Ca2+ influx in neuronal nuclei and the levels of cerebral tissue ATP (r = 0.83) and PCr (r = 0.85). Similarly, IP3-dependent intranuclear 45Ca2+ influx increased from 2.26 +/- 0.38 pmoles/mg protein in normoxic nuclei to 11.12 +/- 1.65 pmoles/mg protein in hypoxic nuclei and showed an inverse correlation between 45Ca2+ influx in neuronal nuclei and the levels of cerebral tissue ATP (r = 0.86) and PCr (r = 0.71). The data demonstrate that there is an IP4- as well as IP3-dependent increase in nuclear Ca2+ influx with increasing cerebral tissue hypoxia

  9. Comparative mechanistic and substrate specificity study of inositol polyphosphate 5-phosphatase Schizosaccharomyces pombe Synaptojanin and SHIP2.

    PubMed

    Chi, Yuling; Zhou, Bo; Wang, Wei-Qing; Chung, Sung-Kee; Kwon, Yong-Uk; Ahn, Young-Hoon; Chang, Young-Tae; Tsujishita, Yosuke; Hurley, James H; Zhang, Zhong-Yin

    2004-10-22

    Inositol-5-phosphatases are important enzymes involved in the regulation of diverse cellular processes from synaptic vesicle recycling to insulin signaling. We describe a comparative study of two representative inositol-5-phosphatases, Schizosaccharomyces pombe synaptojanin (SPsynaptojanin) and human SH2 domain-containing inositol-5-phosphatase SHIP2. We show that in addition to Mg2+, transition metals such as Mn2+, Co2+, and Ni2+ are also effective activators of SPsynaptojanin. In contrast, Ca2+ and Cu2+ are inhibitory. We provide evidence that Mg2+ binds the same site occupied by Ca2+ observed in the crystal structure of SPsynaptojanin complexed with inositol 1,4-bisphosphate (Ins(1,4)P2). Ionizations important for substrate binding and catalysis are defined for the SPsynaptojanin-catalyzed Ins(1,4,5)P3 reaction. Kinetic analysis with four phosphatidylinositol lipids bearing a 5-phosphate and 54 water-soluble inositol phosphates reveals that SP-synaptojanin and SHIP2 possess much broader substrate specificity than previously appreciated. The rank order for SPsynaptojanin is Ins(2,4,5)P3 > phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) approximately Ins(4,5)P2 approximately Ins(1,4,5)P3 approximately Ins(4,5,6)P3 > PtdIns(3,5)P2 approximately PtdIns(3,4,5)P3 approximately Ins(1,2,4,5)P4 approximately Ins(1,3,4,5)P4 approximately Ins-(2,4,5,6)P4 approximately Ins(1,2,4,5,6)P5. The rank order for SHIP2 is Ins(1,2,3,4,5)P5 > Ins(1,3,4,5)P4 > PtdIns(3,4,5)P4 approximately PtdIns(3,5)P2 approximately Ins(1,4,5,6)P4 approximately Ins(2,4,5,6)P4. Because inositol phosphate isomers elicit different biological activities, the extended substrate specificity for SPsynaptojanin and SHIP2 suggest that these enzymes likely have multiple roles in cell signaling and may regulate distinct pathways. The unique substrate specificity profiles and the importance of 2-position phosphate in binding also have important implications for the design of potent and selective

  10. The protective effect of myo-inositol on hippocamal cell loss and structural alterations in neurons and synapses triggered by kainic acid-induced status epilepticus.

    PubMed

    Kotaria, Nato; Kiladze, Maia; Zhvania, Mzia G; Japaridze, Nadezhda J; Bikashvili, Tamar; Solomonia, Revaz O; Bolkvadze, Tamar

    2013-07-01

    It is known that myo-inositol pretreatment attenuates the seizure severity and several biochemical changes provoked by experimentally induced status epilepticus. However, it remains unidentified whether such properties of myo-inositol influence the structure of epileptic brain. In the present light and electron microscopic research we elucidate if pretreatment with myo-inositol has positive effect on hippocampal cell loss, and cell and synapses damage provoked by kainic acid-induced status epilepticus. Adult male Wistar rats were treated with (i) saline, (ii) saline + kainic acid, (iii) myo-inositol + kainic acid. Assessment of cell loss at 2, 14, and 30 days after treatment demonstrate cytoprotective effect of myo-inositol in CA1 and CA3 areas. It was strongly expressed in pyramidal layer of CA1, radial and oriental layers of CA3 and in less degree-in other layers of both fields. Ultrastructural alterations were described in CA1, 14 days after treatment. The structure of neurons, synapses, and porosomes are well preserved in the rats pretreated with myo-inositol in comparing with rats treated with only kainic acid.

  11. VIH2 Regulates the Synthesis of Inositol Pyrophosphate InsP8 and Jasmonate-Dependent Defenses in Arabidopsis[OPEN

    PubMed Central

    Laha, Debabrata; Johnen, Philipp; Azevedo, Cristina; Dynowski, Marek; Weiß, Michael; Capolicchio, Samanta; Mao, Haibin; Iven, Tim; Steenbergen, Merel; Freyer, Marc; Gaugler, Philipp; de Campos, Marília K.F.; Zheng, Ning; Feussner, Ivo; Jessen, Henning J.; Van Wees, Saskia C.M.; Saiardi, Adolfo; Schaaf, Gabriel

    2015-01-01

    Diphosphorylated inositol polyphosphates, also referred to as inositol pyrophosphates, are important signaling molecules that regulate critical cellular activities in many eukaryotic organisms, such as membrane trafficking, telomere maintenance, ribosome biogenesis, and apoptosis. In mammals and fungi, two distinct classes of inositol phosphate kinases mediate biosynthesis of inositol pyrophosphates: Kcs1/IP6K- and Vip1/PPIP5K-like proteins. Here, we report that PPIP5K homologs are widely distributed in plants and that Arabidopsis thaliana VIH1 and VIH2 are functional PPIP5K enzymes. We show a specific induction of inositol pyrophosphate InsP8 by jasmonate and demonstrate that steady state and jasmonate-induced pools of InsP8 in Arabidopsis seedlings depend on VIH2. We identify a role of VIH2 in regulating jasmonate perception and plant defenses against herbivorous insects and necrotrophic fungi. In silico docking experiments and radioligand binding-based reconstitution assays show high-affinity binding of inositol pyrophosphates to the F-box protein COI1-JAZ jasmonate coreceptor complex and suggest that coincidence detection of jasmonate and InsP8 by COI1-JAZ is a critical component in jasmonate-regulated defenses. PMID:25901085

  12. [The dynamics of the domains of the IP3-binding site of the inositol-1,4,5-triphosphate-sensitive calcium channel, induced by inositol-1,4,5-triphosphate and calcium].

    PubMed

    Veresov, V G; Konev, S V

    2005-01-01

    The dynamics of the inositol-1,4,5-triphosphate-sensitive calcium channel after binding of inositol-1,4,5-triphosphate and Ca2+ was analyzed by the Monte Carlo minimization technique. It was shown that the binding of Ca2+ with the unliganded receptor (channel) leads to a turning of the beta-sheet domain relative to the alpha-helical domain with the formation of the receptor conformation that is open for the entry of ions into the cytoplasmic channel vestibule, sterically closed for their passage through the vestibule in the part adjacent to the alpha-helical domains, and unfavourable for subsequent binding of inositol-1,4,5-triphosphate with the receptor. When both co-agonists bind to the receptor, the structure rearrangements induced eliminate both these steric obstacles for the passage of ions through the IP3-binding domain: one at the entrance of the channel cytoplasmic vestibule and the other that is placed deeper in the vestibule near the alpha-domains. The role of the dynamics of the receptor binding core in the IP3-sensitive channel gating is discussed.

  13. Synthesis of an inositol hexakisphosphate (IP6) affinity probe to study the interactome from a colon cancer cell line.

    PubMed

    Yin, Meng-Xin; Catimel, Bruno; Gregory, Mark; Condron, Melanie; Kapp, Eugene; Holmes, Andrew B; Burgess, Antony W

    2016-03-14

    Inositol hexakisphosphate (InsP6 or IP6) is an important signalling molecule in vesicular trafficking, neurotransmission, immune responses, regulation of protein kinases and phosphatases, activation of ion channels, antioxidant functions and anticancer activities. An IP6 probe was synthesised from myo-inositol via a derivatised analogue, which was immobilised through a terminal amino group onto Dynabeads. Systematic analysis of the IP6 interactome has been performed using the IP6 affinity probe using cytosolic extracts from the LIM1215 colonic carcinoma cell line. LC/MS/MS analysis identified 77 proteins or protein complexes that bind to IP6 specifically, including AP-2 complex proteins and β-arrestins as well as a number of novel potential IP6 interacting proteins. Bioinformatic enrichment analysis of the IP6 interactome reinforced the concept that IP6 regulates a number of biological processes including cell cycle and division, signal transduction, intracellular protein transport, vesicle-mediated transport and RNA splicing. PMID:26840369

  14. Metabolism and possible compartmentalization of inositol lipids in isolated rat-liver nuclei.

    PubMed Central

    Vann, L R; Wooding, F B; Irvine, R F; Divecha, N

    1997-01-01

    (1) The removal of the nuclear envelope from isolated rat-liver nuclei by washing with Triton X-100 (TX-100) was assessed by electron microscopy. All the envelope was removed by 0.04% (w/v) TX-100. (2) After this removal, phosphorylation of inositol lipids and diacylglycerol (DAG) from [gamma-32P]ATP still occurs, despite the near complete absence of detectable (by mass assay) DAG and PtdIns. This suggests that the majority of these two lipids in nuclei are present in the nuclear membrane, but the small amounts remaining after extraction, defined as intranuclear, are available for phosphorylation by lipid kinases (36% for DAG and 24% for PtdIns respectively, when expressed as a percentage of incorporation of intact nuclei). (3) PtdIns(4,5)P2 did not follow the same pattern as PtdIns and DAG; after removal of the nuclear membrane, 40% of the mass of this lipid was left in the nucleus. Moreover, a similar amount of PtdIns(4,5)P2 was also resistant to extraction with even higher concentrations of detergent, suggesting that PtdIns(4,5)P2 has a discrete intranuclear location, probably bound to nuclear proteins. (4) Addition of exogenous substrates, PtdIns, PtdIns(4)P and DAG, to membrane-depleted nuclei resulted in reconstitution of the majority of lipid phosphorylations from [gamma-32P]ATP (70%, 90% and 94% of intact nuclei respectively), suggesting a predominantly intranuclear location for the respective kinases. (5) Nuclei also showed phosphomonoesterase and phosphatidic acid hydrolase activity; dephosphorylation of pre-radiolabelled PtdIns(4)P, PtdIns(4,5)P2 and phosphatidic acid was observed when [gamma-32P]ATP was removed. However, some of the radioactivity was apparently resistant to these enzymes, suggesting the existence of multiple pools of these lipids. (6) Addition of excess non-radiolabelled ATP to nuclei pre-labelled with [gamma-32P]ATP resulted in an initial increase in the label in PtdIns(4,5)P2, implying a precursor-product relationship between the

  15. Effect of inositol and tri-iodothyronine on the hormonal responsiveness of hepatocytes obtained from partially hepatectomized rats.

    PubMed Central

    Huerta-Bahena, J; García-Sáinz, J A

    1984-01-01

    Hepatocytes obtained from animals partially hepatectomized (72 h before the experiment) have a diminished responsiveness to alpha 1-adrenergic amines, vasopressin, angiotensin and glucagon and an increased responsiveness to beta-adrenergic amines. Administration of inositol or tri-iodothyronine to the hepatectomized animals induced a recovery in the hepatocyte responsiveness to the Ca2+-dependent hormones and abolished that to beta-adrenergic amines; the response to glucagon was not improved. PMID:6508748

  16. Differential methylation of the gene encoding myo-inositol 3-phosphate synthase (Isyna1) in rat tissues

    PubMed Central

    Seelan, Ratnam S; Pisano, M Michele; Greene, Robert M; Casanova, Manuel F; Parthasarathy, Ranga N

    2011-01-01

    Aims Myo-inositol levels are frequently altered in several brain disorders. Myo-inositol 3-phosphate synthase, encoded by the Isyna1 gene, catalyzes the synthesis of myo-inositol in cells. Very little is known about the mechanisms regulating Isyna1 expression in brain and other tissues. In this study, we have examined the role of DNA methylation in regulating Isyna1 expression in rat tissues. Materials & methods Transfection analysis using in vitro methylated promoter constructs, Southern blot analysis of genomic DNA from various tissues digested with a methylation-sensitive enzyme and CpG methylation profiling of genomic DNA from different tissues were used to determine differential methylation of Isyna1 in tissues. Transfection analysis using plasmids harboring mutated CpG residues in the 5’-upstream region of Isyna1 was used to identify critical residues mediating promoter activity. Results The −700 bp to −500 bp region (region 1) of Isyna1 exhibited increased methylation in brain cortex compared with other tissues; it also exhibited sex-specific methylation differences between matched male and female brain cortices. Mutation analysis identified one CpG residue in region 1 necessary for promoter activity in neuronal cells. A tissue-specific differentially methylated region (T-DMR) was found to be localized between +450 bp and +650 bp (region 3). This DMR was comparatively highly methylated in spleen, moderately methylated in brain cortex and poorly methylated in testis, consistent with mRNA levels observed in these tissues. Conclusion Rat Isyna1 exhibits tissue-specific DNA methylation. Brain DNA was uniquely methylated in the 5’-upstream region and displayed gender specificity. A T-DMR was identified within the gene body of Isyna1. These findings suggest that Isyna1 is regulated, in part, by DNA methylation and that significant alterations in methylation patterns during development could have a major impact on inositol phosphate synthase expression in

  17. Dual functions for the Schizosaccharomyces pombe inositol kinase Ipk1 in nuclear mRNA export and polarized cell growth.

    PubMed

    Sarmah, Bhaskarjyoti; Wente, Susan R

    2009-02-01

    The inositol 1,3,4,5,6-pentakisphosphate (IP(5)) 2-kinase (Ipk1) catalyzes the production of inositol hexakisphosphate (IP(6)) in eukaryotic cells. Previous studies have shown that IP(6) is required for efficient nuclear mRNA export in the budding yeast Saccharomyces cerevisiae. Here, we report the first functional analysis of ipk1(+) in Schizosaccharomyces pombe. S. pombe Ipk1 (SpIpk1) is unique among Ipk1 orthologues in that it harbors a novel amino (N)-terminal domain with coiled-coil structural motifs similar to those of BAR (Bin-amphiphysin-Rvs) domain proteins. Mutants with ipk1(+) deleted (ipk1Delta) had mRNA export defects as well as pleiotropic defects in polarized growth, cell morphology, endocytosis, and cell separation. The SpIpk1 catalytic carboxy-terminal domain was required to rescue these defects, and the mRNA export block was genetically linked to SpDbp5 function and, likely, IP(6) production. However, the overexpression of the N-terminal domain alone also inhibited these functions in wild-type cells. This revealed a distinct noncatalytic function for the N-terminal domain. To test for connections with other inositol polyphosphates, we also analyzed whether the loss of asp1(+) function, encoding an IP(6) kinase downstream of Ipk1, had an effect on ipk1Delta cells. The asp1Delta mutant alone did not block mRNA export, and its cell morphology, polarized growth, and endocytosis defects were less severe than those of ipk1Delta cells. Moreover, ipk1Delta asp1Delta double mutants had altered inositol polyphosphate levels distinct from those of the ipk1Delta mutant. This suggested novel roles for asp1(+) upstream of ipk1(+). We propose that IP(6) production is a key signaling linchpin for regulating multiple essential cellular processes. PMID:19047361

  18. Inositol phosphates influence the membrane bound Ca/sup 2 +//Mg/sup 2 +/ stimulated ATPase from human erythrocyte membranes

    SciTech Connect

    Kester, M.; Ekholm, J.; Kumar, R.; Hanahan, D.J.

    1986-03-01

    The modulation by exogenous inositol phosphates of the membrane Ca/sup 2 +//Mg/sup 2 +/ ATPase from saponin/EGTA lysed human erythrocytes was determined in a buffer (pH 7.6) containing histidine, 80 mM, MgCl/sub 2/, 3.3 mM, NaCl, 74 mM, KCl, 30 mM, Na/sub 2/ATP, 2.3 mM, ouabain, 0.83 mM, with variable amounts of CaCl/sub 2/ and EGTA. The ATPase assay was linear with time at 44/sup 0/C. The inositol phosphates were commercially obtained and were also prepared from /sup 32/P labeled rabbit platelet inositol phospholipids. Inositol triphosphate (IP/sub 3/) elevated the Ca/sup 2 +//Mg/sup 2 +/ ATPase activity over basal levels in a dose, time, and calcium dependent manner and were increased up to 85% of control values. Activities for the Na/sup +//K/sup +/-ATPase and a Mg/sup 2 +/ ATPase were not effected by IP/sub 3/. Ca/sup 2 +//Mg/sup 2 +/APTase activity with IP/sub 2/ or IP/sub 3/ could be synergistically elevated with calmodulin addition. The activation of the ATPase with IP/sub 3/ was calcium dependent in a range from .001 to .02 mM. The apparent Km and Vmax values were determined for IP/sub 3/ stimulated Ca/sup 2 +//Mg/sup 2 +/ ATPase.

  19. L-myo-inosose-1 as a probable intermediate in the reaction catalyzed by myo-inositol oxygenase

    SciTech Connect

    Naber, N.I.; Swan, J.S.; Hamilton, G.A.

    1986-11-04

    In previous investigations, it was necessary to have Fe(II) and cysteine present in order to assay the catalytic activity of purified hog kidney myo-inositol oxygenase. In the present study it was found that, if this purified nonheme iron enzyme is slowly frozen in solution with glutathione and stored at -20 degrees C, it is fully active in the absence of activators if catalase is present to remove adventitious H/sub 2/O/sub 2/. With this simpler assay system it was possible to clarify the effects of several variables on the enzymic reaction. Thus, the maximum velocity is pH-dependent with a maximum around pH 9.5, but the apparent Km for myo-inositol (air atmosphere) remains constant at 5.0 mM throughout a broad pH range. The enzyme is quite specific for its substrate myo-inositol, is very sensitive to oxidants and reductants, but is not affected by a variety of complexing agents, nucleotides, sulfhydryl reagents, etc. In other experiments it was found that L-myo-inosose-1, a potential intermediate in the enzymic reaction, is a potent competitive inhibitor (Ki = 62 microM), while other inososes and a solution thought to contain D-glucodialdehyde, another potential intermediate, are weak inhibitors. Also, both a kinetic deuterium isotope effect (kH/kD = 2.1) and a tritium isotope effect (kH/kT = 7.5) are observed for the enzymic reaction when (1-2H)- and (1-3H)-myo-inositol are used as reactants. These latter results are considered strong evidence that the oxygenase reaction proceeds by a pathway involving L-myo-inosose-1 as an intermediate rather than by an alternative pathway that would have D-glucodialdehyde as the intermediate.

  20. A Mycobacterium smegmatis mutant with a defective inositol monophosphate phosphatase gene homolog has altered cell envelope permeability.

    PubMed Central

    Parish, T; Liu, J; Nikaido, H; Stoker, N G

    1997-01-01

    A bacteriophage infection mutant (strain LIMP7) of Mycobacterium smegmatis was isolated following transposon mutagenesis. The mutant showed an unusual phenotype, in that all phages tested produced larger plaques on this strain compared to the parent strain. Other phenotypic characteristics of the mutant were slower growth, increased clumping in liquid culture, increased resistance to chloramphenicol and erythromycin, and increased sensitivity to isoniazid and several beta-lactam antibiotics. Permeability studies showed decreases in the accumulation of lipophilic molecules (norfloxacin and chenodeoxycholate) and a small increase with hydrophilic molecules (cephaloridine); taken together, these characteristics indicate an altered cell envelope. The DNA adjacent to the transposon in LIMP7 was cloned and was shown to be highly similar to genes encoding bacterial and mammalian inositol monophosphate phosphatases. Inositol is important in mycobacteria as a component of the major thiol mycothiol and also in the cell wall, with phosphatidylinositol anchoring lipoarabinomannan (LAM) in the cell envelope. In LIMP7, levels of phosphatidylinositol dimannoside, the precursor of LAM, were less than half of those in the wild-type strain, confirming that the mutation had affected the synthesis of inositol-containing molecules. The impA gene is located within the histidine biosynthesis operon in both M. smegmatis and Mycobacterium tuberculosis, lying between the hisA and hisF genes. PMID:9401044

  1. Building blocks for the synthesis of glycosyl-myo-inositols involved in the insulin intracellular signalling process.

    PubMed

    Zapata, A; Martín-Lomas, M

    1992-10-01

    Glycosylation of (+/- )-1-O-benzyl-2,3:5,6-di-O-isopropylidene-myo-inositol (4) with 6-O-acetyl-4-O-allyl-2-azido-3-O-benzyl-2-deoxy-beta-D-glucopyranosyl trichloroacetimidate (6) gave the 4-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)- myo-inositol derivative (9) as a mixture of diastereoisomers which could be resolved by chromatography. Likewise alpha-glycosylation of 4 with 6-O-acetyl-2-azido-3-O-benzoyl-2-deoxy-4-O-(2,3,4,6-tetra-O-acetyl-beta- D- galactopyranosyl)-D-glucopyranosyl trichloroacetimidate (10) gave the corresponding pseudotrisaccharide derivative 16 as a mixture of diastereomers which could be resolved partially by chromatography. alpha-Glycosylation of enantiomerically pure 2,3:5,6- (18) and 2,3:4,5-di-O-isopropylidene-1-O-menthoxycarbonyl-myo-inositol (19) with 3,4,6-tri-O-acetyl-2-azido-2-deoxy-D-glucopyranosyl trichloroacetimidate (20) gave the pseudodisaccharide derivatives 21 and 22, respectively. Likewise, alpha-glycosylation of 18 with 10 afforded a pseudotrisaccharide derivative (23). PMID:1468083

  2. Renal depletion of myo-inositol is associated with its increased degradation in animal models of metabolic disease.

    PubMed

    Chang, H-H; Chao, H-N; Walker, C S; Choong, S-Y; Phillips, A; Loomes, K M

    2015-11-01

    Renal depletion of myo-inositol (MI) is associated with the pathogenesis of diabetic nephropathy in animal models, but the underlying mechanisms involved are unclear. We hypothesized that MI depletion was due to changes in inositol metabolism and therefore examined the expression of genes regulating de novo biosynthesis, reabsorption, and catabolism of MI. We also extended the analyses from diabetes mellitus to animal models of dietary-induced obesity and hypertension. We found that renal MI depletion was pervasive across these three distinct disease states in the relative order: hypertension (-51%)>diabetes mellitus (-35%)>dietary-induced obesity (-19%). In 4-wk diabetic kidneys and in kidneys derived from insulin-resistant and hypertensive rats, MI depletion was correlated with activity of the MI-degrading enzyme myo-inositol oxygenase (MIOX). By contrast, there was decreased MIOX expression in 8-wk diabetic kidneys. Immunohistochemistry localized the MI-degrading pathway comprising MIOX and the glucuronate-xylulose (GX) pathway to the proximal tubules within the renal cortex. These findings indicate that MI depletion could reflect increased catabolism through MIOX and the GX pathway and implicate a common pathological mechanism contributing to renal oxidative stress in metabolic disease.

  3. Activation of frog (Xenopus laevis) eggs by inositol trisphosphate. I. Characterization of Ca2+ release from intracellular stores

    PubMed Central

    1985-01-01

    Iontophoresis of inositol 1, 4, 5-triphosphate into frog (Xenopus laevis) eggs activated early developmental events such as membrane depolarization, cortical contraction, cortical granule exocytosis, and abortive cleavage furrow formation (pseudocleavage). Inositol 1, 4- bisphosphate also triggered these events, but only at doses approximately 100-fold higher, whereas no level of fructose-1, 6- bisphosphate tested activated eggs. Using Ca2+-selective microelectrodes, we observed that activating doses of inositol 1, 4, 5- trisphosphate triggered a Ca2+ release from intracellular stores that was indistinguishable from that previously observed at fertilization (Busa, W. B., and R. Nuccitelli, 1985, J. Cell Biol., 100:1325-1329), whereas subthreshold doses triggered only a localized Ca2+ release at the site of injection. The subthreshold IP3 response could be distinguished from the major Ca2+ release at activation with respect to their dose-response characteristics, relative timing, sensitivity to external Ca2+ levels, additivity, and behavior in the activated egg, suggesting that the Xenopus egg may possess two functionally distinct Ca2+ pools mobilized by different effectors. In light of these differences, we suggest a model for intracellular Ca2+ mobilization by sperm-egg interaction. PMID:3874873

  4. /sup 2/H-NMR studies of hypocotyl cell walls of germinating beams supplied with perdeuterated myo-inositol

    SciTech Connect

    Sasaki, K.; Wallace, J.C.; MacKay, A.L.; Balza, F.; Taylor, I.E.P.

    1987-04-01

    When myo-(2-/sup 3/H) inositol (MI) was supplied to bean seeds by imbibition, only uronic acid, arabinose and xylose residues of cell wall polysaccharides were labeled. To study the structural mobility of the uronic acid- and/or pentose-rich polysaccharides in cell wall using /sup 2/H-NMR, the authors supplied perdeuterated MI with (2-/sup 3/H) MI to germinating bean seeds. Perdeuterated MI was prepared by the /sup 1/H-/sup 2/H exchange reaction of MI in deuterium oxide with Raney nickel. During the exchange reaction, extensive epimerization occurred and at least 6 inositol epimers in addition to MI were identified in the reaction mixture of GC/MS. The perdeuterated MI was completely resolved from other inositol epimers and purified by anion-exchange chromatography using Dowex 1 (borate form) and by crystallization. The /sup 2/H-NMR analysis resolved the /sup 2/H-labeled hypocotyl cell walls into two components (rigid and mobile components). They also report the distribution of /sup 2/H and /sup 3/H from perdeuterated and (2-/sup 3/H) MI in the cell wall sugar residues.

  5. High-performance ion chromatography method for separation and quantification of inositol phosphates in diets and digesta.

    PubMed

    Blaabjerg, K; Hansen-Møller, J; Poulsen, H D

    2010-02-01

    A gradient high-performance ion chromatographic method for separation and quantification of inositol phosphates (InsP(2)-InsP(6)) in feedstuffs, diets, gastric and ileal digesta from pigs was developed and validated. The InsP(2)-InsP(6) were separated on a Dionex CarboPac PA1 column using a gradient with 1.5 mol L(-1) methanesulfonic acid and water. The exchange of the commonly used HCl with methanesulfonic acid has two advantages: (i) the obtained baseline during the separation is almost horizontal and (ii) it is not necessary to use an inert HPIC equipment as the methanesulfonic acid is not as aggressive as HCl. Twenty-three of the 27 separated inositol phosphate isomers were isolated. ICP-MS was used for quantification of phosphorus in the isolated isomers and used for calculation of correction factors for each isomer allowing InsP(6) to be used as calibration standard. The detection limits for InsP(2)-InsP(6) were in the range of 0.9-4.4 mg phosphorus L(-1). The recovery of the major part of the inositol phosphates was 80-100%, and the CV for repeatability and reproducibility were 1-17% and 1-14%, respectively. PMID:20022822

  6. Phorbol 12,13-dibutyrate and 1-oleyl-2-acetyldiacylglycerol stimulate inositol trisphosphate dephosphorylation in human platelets

    SciTech Connect

    Molina y Vedia, L.M.; Lapetina, E.G.

    1986-08-15

    Inositol trisphosphate (IP3) is formed in response to specific agonists that cause activation of phospholipase C and degradation of phosphatidylinositol bisphosphate. IP3 is a second messenger that releases Ca/sup 2 +/ from the dense tubular system to the cytosol in stimulated platelets. Our present information indicates that (/sup 3/H)IP3 is dephosphorylated to (/sup 3/H)inositol bisphosphate (IP2) and (/sup 3/H)inositol monophosphate (IP) by human platelets treated with 0.05-0.10% Triton X-100. This dephosphorylation of (/sup 3/H)IP3 to (/sup 3/H)IP2 and (/sup 3/H)IP is also observed when platelets are permeabilized by electrical stimulation or by 20 micrograms/ml saponin. These detergents or electropermeabilization allow IP3 to access cytosolic IP3 phosphatase. Pretreatment of intact platelets with phorbol dibutyrate and 1-oleyl-2-acetyldiacylglycerol for 30 s, at concentrations that maximally activate protein kinase C, stimulates the conversion of IP3 to IP2 and IP. This suggests a role for protein kinase C in the regulation of IP3 degradation.

  7. Dietary arginine silicate inositol complex during the late laying period of quail at different environmental temperatures.

    PubMed

    Onderci, M; Sahin, N; Sahin, K; Balci, T A; Gursu, M F; Juturu, V; Kucuk, O

    2006-04-01

    Arginine silicate inositol complex (ASIdagger; arginine 49.5%, silicon 8.2%, inositol 25%) is a novel material which is a bioavailable source of silicon and arginine. ASI offers potential benefits for vascular and bone health. Poor eggshell quality has been a major economic concern to commercial egg producers. Poor egg quality, skeletal abnormalities and architectural deterioration of bone tissue are common problems under hot conditions and in older birds. The effects of ASI supplementation on egg production, egg quality, levels of osteocalcin (OC) and bone mineral content were investigated in heat-stressed Japanese quail during the later part of the laying period. The birds were randomly assigned to six treatment groups consisting of six replicates of five birds each in a 2 x 3 factorial arrangement of treatments (temperatures, ASI levels). The birds were kept in wire cages in a temperature-controlled room at either 22 degrees C (TN) or 34 degrees C (HS) for 8 h/d and fed either a basal (control) diet or the basal diet supplemented with either 500 or 1000 g of ASI/kg. Heat exposure reduced egg production, egg quality and bone mineralisation when the basal diet was fed. ASI supplementation had no effect on feed intake or egg production under TN or HS conditions. However, ASI supplementation increased egg weight, shell thickness, shell weight and Haugh unit in both TN and HS groups during the late laying period. Bone mineral density (BMD) was significantly improved by ASI supplementation in both TN and HS groups. Serum osteocalcin (OC) concentrations and alkaline phosphatase (ALP) activity increased linearly with dietary ASI supplementation during the late laying period. The amount of calcium and phosphorus in the excreta decreased, while ash, mineral content, calcium and phosphorus concentrations in tibia increased in ASI-supplemented quail in both TN and HS groups during the late laying period. ASI supplementation significantly improved egg quality and bone

  8. Metabolic Studies on Intermediates in the myo-Inositol Oxidation Pathway in Lilium longiflorum Pollen: II. Evidence for the Participation of Uridine Diphosphoxylose and Free Xylose as Intermediates.

    PubMed

    Rosenfield, C L; Loewus, F A

    1978-01-01

    myo-Inositol-linked glucogenesis in germinated lily (Lilium longiflorum Thunb., cv. Ace) pollen was investigated by studying the effects of added l-arabinose or d-xylose on metabolism of myo-[2-(3)H]inositol and by determining the distribution of radioisotope in pentosyl and hexosyl residues of polysaccharides from pollen labeled with myo-[2-(14)C]inositol, myo-[2-(3)H]inositol, l-[5-(14)C]arabinose, and d-[5R,5S-(3)H]xylose.myo-[2-(14)C]Inositol and l-[5-(14)C]arabinose produced labeled glucose with similar patterns of distribution of (14)C, 35% in C1, and 55% in C6. Arabinosyl units were labeled exclusively in C5. Incorporation of (3)H into arabinosyl and xylosyl units in pollen labeled with myo-[2-(3)H]inositol was repressed when unlabeled l-arabinose was included in the germination medium and a related (3)H exchange with water was stimulated. Results are consistent with a process of glucogenesis in which the myo-inositol oxidation pathway furnishes UDP-d-xylose as a key intermediate for conversion to hexose via free d-xylose and the pentose phosphate pathway.Additional evidence for this process was obtained from pollen labeled with d-[5R,5S-(3)H]xylose or myo-[2-(3)H]inositol which produces d-[5R-(3)H]xylose. Glucosyl units from polysaccharides in the former had 11% of the (3)H in C1 and 78% in C6 while glucosyl units in the latter had only 4% in C1 and 78% in C6. Stereochemical considerations involving selective exchange with water of prochiral-R (3)H in C1 of fructose-6-P during conversion to glucose provide explanation for observed differences in the metabolism of these 5-labeled xyloses.Incorporation of (3)H from myo-[2-(3)H]inositol into arabinosyl and xylosyl units of pollen polysaccharides was unaffected by the presence of unlabeled d-xylose in the medium. Exchange of (3)H with water was greatly affected, decreasing from a value of 21% exchange in the absence of unlabeled d-xylose to 5% in the presence of 6.7 mmd-xylose.d-Xylose was rapidly utilized for

  9. Membrane processes associated with the osmotic-pulse incorporation of inositol hexaphosphate.

    PubMed

    Wyse, J W; Franco, R S; Barker, R; Yacko, M A; Butterfield, D A

    1990-02-16

    In previous studies (Biochem. Biophys. Res. Commun. 144, 779-786 (1987); Prog. Clin. Biol. Res. 292, 65-75 (1989)), we showed that inositol hexaphosphate (IHP), when added to erythrocyte membrane ghosts in the range 0.6-2.5 mM, caused a large disruption of skeletal protein-protein interactions as monitored by electron paramagnetic resonance techniques. IHP incorporated into intact cells by an osmotic-pulse method (J. Cell. Physiol. 129, 221-229 (1986)) leads to cells with markedly decreased oxygen affinity. Exposure of the red cells to higher levels of IHP during the osmotic pulse leads to less lysis and more normal cellular indices after healing of the transiently-disrupted membrane (J. Lab. Clin. Med. 113, 58-66 (1989)). In order to determine what effect higher levels of IHP had on skeletal proteins and bilayer lipids of membrane ghosts, spin labeling studies were performed. The main findings were: (a) There was a concentration-dependent alteration in skeletal protein interactions. At concentrations greater than 25 mM IHP, the effectiveness of IHP to disrupt skeletal protein interactions was diminished. (b) No apparent alteration of the motion or order of phospholipids or the lipid water interface of intact cells into which IHP was incorporated occurred, suggesting that higher levels of IHP do not alter the physical state of the lipid bilayer. PMID:2154262

  10. Multibody cofactor and substrate molecular recognition in the myo-inositol monophosphatase enzyme

    PubMed Central

    Ferruz, Noelia; Tresadern, Gary; Pineda-Lucena, Antonio; De Fabritiis, Gianni

    2016-01-01

    Molecular recognition is rarely a two-body protein-ligand problem, as it often involves the dynamic interplay of multiple molecules that together control the binding process. Myo-inositol monophosphatase (IMPase), a drug target for bipolar disorder, depends on 3 Mg2+ ions as cofactor for its catalytic activity. Although the crystallographic pose of the pre-catalytic complex is well characterized, the binding process by which substrate, cofactor and protein cooperate is essentially unknown. Here, we have characterized cofactor and substrate cooperative binding by means of large-scale molecular dynamics. Our study showed the first and second Mg2+ ions identify the binding pocket with fast kinetics whereas the third ion presents a much higher energy barrier. Substrate binding can occur in cooperation with cofactor, or alone to a binary or ternary cofactor-IMPase complex, although the last scenario occurs several orders of magnitude faster. Our atomic description of the three-body mechanism offers a particularly challenging example of pathway reconstruction, and may prove particularly useful in realistic contexts where water, ions, cofactors or other entities cooperate and modulate the binding process. PMID:27440438

  11. Insulin stimulates the generation from hepatic plasma membranes of modulators derived from an inositol glycolipid.

    PubMed Central

    Saltiel, A R; Cuatrecasas, P

    1986-01-01

    Insulin binding to plasma membrane receptors results in the generation of substances that acutely mimic the actions of the hormone on certain target enzymes. Two such substances, which modulate the activity of the high-affinity cAMP phosphodiesterase (EC 3.1.4.17), have been purified from hepatic plasma membranes. The two have similar properties and activities but can be resolved by ion-exchange chromatography and high-voltage electrophoresis. They exhibit a net negative charge, even at pH 1.9, and an apparent molecular weight of approximately 1400. The generation of these substances from membranes by insulin can be reproduced by addition of a phosphatidylinositol-specific phospholipase C purified from Staphylococcus aureus. This enzyme is known to selectively hydrolyze phosphatidylinositol and release from membranes several proteins that are covalently linked to phosphatidylinositol by a glycan anchor. Both enzyme-modulating substances appear to be generated by the phosphodiesterase cleavage of a phosphatidylinositol-containing glycolipid precursor that has been characterized by thin-layer chromatography. Some of the chemical properties of these substances have been examined. They appear to be related complex carbohydrate-phosphate substances containing glucosamine and inositol. These findings suggest that insulin may activate a selective phospholipase activity that hydrolyzes a membrane phospholipid, releasing a carbohydrate-containing molecule that regulates cAMP phosphodiesterase and perhaps other insulin-sensitive enzymes. PMID:3016721

  12. Multibody cofactor and substrate molecular recognition in the myo-inositol monophosphatase enzyme.

    PubMed

    Ferruz, Noelia; Tresadern, Gary; Pineda-Lucena, Antonio; De Fabritiis, Gianni

    2016-07-21

    Molecular recognition is rarely a two-body protein-ligand problem, as it often involves the dynamic interplay of multiple molecules that together control the binding process. Myo-inositol monophosphatase (IMPase), a drug target for bipolar disorder, depends on 3 Mg(2+) ions as cofactor for its catalytic activity. Although the crystallographic pose of the pre-catalytic complex is well characterized, the binding process by which substrate, cofactor and protein cooperate is essentially unknown. Here, we have characterized cofactor and substrate cooperative binding by means of large-scale molecular dynamics. Our study showed the first and second Mg(2+) ions identify the binding pocket with fast kinetics whereas the third ion presents a much higher energy barrier. Substrate binding can occur in cooperation with cofactor, or alone to a binary or ternary cofactor-IMPase complex, although the last scenario occurs several orders of magnitude faster. Our atomic description of the three-body mechanism offers a particularly challenging example of pathway reconstruction, and may prove particularly useful in realistic contexts where water, ions, cofactors or other entities cooperate and modulate the binding process.

  13. Isolation and characterization of the inositol trisphosphate receptor from smooth muscle

    SciTech Connect

    Chadwick, C.C.; Saito, A.; Fleischer, S. )

    1990-03-01

    The release of Ca{sup 2+} from internal stores is requisite to muscle contraction. In skeletal muscle and heart, the Ca{sup 2+} release channels (ryanodine receptor) of sarcoplasmic reticulum, involved in excitation-contraction coupling, have recently been isolated and characterized. In smooth muscle, inositol 1,4,5-trisphosphate (IP{sub 3}) is believed to mobilize Ca{sup 2+} from internal stores and thereby modulate contraction. The authors describe the isolation of an IP{sub 3} receptor from smooth muscle. Bovine aorta smooth muscle microsomes were solubilized with 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate, and the IP{sub 3} receptor was purified by sucrose gradient centrifugation and column chromatography with heparin-agarose and wheat germ agglutinin-agarose. The receptor is an oligomer of a single polypeptide with a M{sub r} of 224,000 as determined by SDS/PAGE. Negative-staining electron microscopy reveals that the receptor is a large pinwheel-like structure having surface dimensions of {approx}250 {times} 250 {angstrom} with fourfold symmetry. The IP{sub 3} receptor from smooth muscle is similar to the ryanodine receptor with regard to its large size and fourfold symmetry, albeit distinct with regard to appearance, protomer size, and ligand binding.

  14. The menstrual cycle regularization following D-chiro-inositol treatment in PCOS women: a retrospective study.

    PubMed

    La Marca, Antonio; Grisendi, Valentina; Dondi, Giulia; Sighinolfi, Giovanna; Cianci, Antonio

    2015-01-01

    Polycystic ovary syndrome is characterized by irregular cycles, hyperandrogenism, polycystic ovary at ultrasound and insulin resistance. The effectiveness of D-chiro-inositol (DCI) treatment in improving insulin resistance in PCOS patients has been confirmed in several reports. The objective of this study was to retrospectively analyze the effect of DCI on menstrual cycle regularity in PCOS women. This was a retrospective study of patients with irregular cycles who were treated with DCI. Of all PCOS women admitted to our centre, 47 were treated with DCI and had complete medical charts. The percentage of women reporting regular menstrual cycles significantly increased with increasing duration of DCI treatment (24% and 51.6% at a mean of 6 and 15 months of treatment, respectively). Serum AMH levels and indexes of insulin resistance significantly decreased during the treatment. Low AMH levels, high HOMA index, and the presence of oligomenorrhea at the first visit were the independent predictors of obtaining regular menstrual cycle with DCI. In conclusion, the use of DCI is associated to clinical benefits for many women affected by PCOS including the improvement in insulin resistance and menstrual cycle regularity. Responders to the treatment may be identified on the basis of menstrual irregularity and hormonal or metabolic markers.

  15. Synthesis and biological activities of d-chiro-inositol analogues with insulin-like actions.

    PubMed

    Rendle, P M; Kassibawi, F; Johnston, K A; Hart, J B; Cameron, S A; Falshaw, A; Painter, G F; Loomes, K M

    2016-10-21

    d-chiro-inositol (DCI, 1) evokes therapeutic actions in diabetes and insulin resistance but has sub-optimal pharmacokinetic profiles. To investigate what positions on the DCI cyclohexanol ring may be amenable to modification to improve pharmaceutical formulations, a series of analogues based on DCI were synthesised. These compounds were then evaluated for their ability to stimulate glucose transport using 3T3-L1 adipocytes as a model system. Positional analyses indicate that the hydroxyl group at position 1 is not essential for activity and can be modified without affecting glucose uptake. Removal of the hydroxyl at position 3 also had minimal effect on activity but this group is sensitive to modification. By comparison, the oxygen at position 2 is crucial to the potency of DCI, although this group can withstand modification without fundamentally affecting activity. These data reveal that positions 1 and 2 on the cyclohexanol ring of DCI offer further scope for modification to develop DCI analogues with desirable pharmacokinetic profiles for the potential treatment of metabolic disease. PMID:27410479

  16. Inositol 1,4,5-trisphosphate mass changes from fertilization through first cleavage in Xenopus laevis.

    PubMed Central

    Stith, B J; Goalstone, M; Silva, S; Jaynes, C

    1993-01-01

    After fertilization in Xenopus laevis, inositol 1,4,5-trisphosphate (IP3) mass increased from 53 to 261 fmol/cell and returned to near basal by 10 min after insemination. IP3 was also elevated over control egg levels during first mitosis and first cleavage. Because IP3 levels and the fertilization calcium wave decline at about the same time and because calcium ionophore or pricking the egg increased IP3, the fertilization calcium wave may be due to calcium-induced IP3 production. In addition, the onset of sperm motility was associated with an increase, whereas the acrosomal reaction was accompanied by a decrease in IP3 mass. Combining our published data with this report, the first chronology of the levels of IP3 from the induction of meiosis (maturation) through fertilization and cleavage in one cellular system is summarized. These data suggest an in vivo dose response for IP3 and calcium release. A small (17 fmol/cell) IP3 change during the induction of meiosis may not be associated with a calcium change. Larger IP3 changes at cleavage (40 fmol/cell) and mitosis (125 fmol/cell) are associated with localized small calcium increases, whereas the largest IP3 change (208 fmol/cell) is associated with the large calcium increase at fertilization. PMID:8507898

  17. Inositol-Requiring Enzyme 1-Dependent Activation of AMPK Promotes Brucella abortus Intracellular Growth

    PubMed Central

    Liu, Ning; Li, Yingying; Dong, Chunyan; Xu, Xiaohan; Wei, Pan; Sun, Wanchun

    2016-01-01

    ABSTRACT AMP-activated protein kinase (AMPK) is a serine/threonine kinase that is well conserved during evolution. AMPK activation inhibits production of reactive oxygen species (ROS) in cells via suppression of NADPH oxidase. However, the role of AMPK during the process of Brucella infection remains unknown. Our data demonstrate that B. abortus infection induces AMPK activation in HeLa cells in a time-dependent manner. The known AMPK kinases LKB1, CAMKKβ, and TAK1 are not required for the activation of AMPK by B. abortus infection. Instead, this activation is dependent on the RNase activity of inositol-requiring enzyme 1 (IRE1). Moreover, we also found that B. abortus infection-induced IRE1-dependent activation of AMPK promotes B. abortus intracellular growth with peritoneal macrophages via suppression of NADPH-derived ROS production. IMPORTANCE Previous studies showed that B. abortus infection does not promote any oxidative burst regulated by NADPH oxidase. However, the underlying mechanism remains elusive. We report for the first time that AMPK activation caused by B. abortus infection plays important role in NADPH oxidase-derived ROS production. PMID:26755628

  18. (1) H NMR analysis of O-methyl-inositol isomers: a joint experimental and theoretical study.

    PubMed

    De Almeida, Mauro V; Couri, Mara Rubia C; De Assis, João Vitor; Anconi, Cleber P A; Dos Santos, Hélio F; De Almeida, Wagner B

    2012-09-01

    Density functional theory (DFT) calculations of (1) H NMR chemical shifts for l-quebrachitol isomers were performed using the B3LYP functional employing the 6-31G(d,p) and 6-311 + G(2d,p) basis sets. The effect of the solvent on the B3LYP-calculated NMR spectrum was accounted for using the polarizable continuum model. Comparison is made with experimental (1) H NMR spectroscopic data, which shed light on the average uncertainty present in DFT calculations of chemical shifts and showed that the best match between experimental and theoretical B3LYP (1) H NMR profiles is a good strategy to assign the molecular structure present in the sample handled in the experimental measurements. Among four plausible O-methyl-inositol isomers, the l-quebrachitol 2a structure was unambiguously assigned based only on the comparative analysis of experimental and theoretical (1) H NMR chemical shift data. The B3LYP infrared (IR) spectrum was also calculated for the four isomers and compared with the experimental data, with analysis of the theoretical IR profiles corroborating assignment of the 2a structure. Therefore, it is confirmed in this study that a combined experimental/DFT spectroscopic investigation is a powerful tool in structural/conformational analysis studies. PMID:22865668

  19. Age-associated repression of type 1 inositol 1, 4, 5-triphosphate receptor impairs muscle regeneration

    PubMed Central

    Lee, Bora; Lee, Seung-Min; Bahn, Young Jae; Lee, Kwang-Pyo; Kang, Moonkyung; Kim, Yeon-Soo; Woo, Sun-Hee; Lim, Jae-Young; Kim, Eunhee; Kwon, Ki-Sun

    2016-01-01

    Skeletal muscle mass and power decrease with age, leading to impairment of mobility and metabolism in the elderly. Ca2+ signaling is crucial for myoblast differentiation as well as muscle contraction through activation of transcription factors and Ca2+-dependent kinases and phosphatases. Ca2+ channels, such as dihydropyridine receptor (DHPR), two-pore channel (TPC) and inositol 1,4,5-triphosphate receptor (ITPR), function to maintain Ca2+ homeostasis in myoblasts. Here, we observed a significant decrease in expression of type 1 IP3 receptor (ITPR1), but not types 2 and 3, in aged mice skeletal muscle and isolated myoblasts, compared with those of young mice. ITPR1 knockdown using shRNA-expressing viruses in C2C12 myoblasts and tibialis anterior muscle of mice inhibited myotube formation and muscle regeneration after injury, respectively, a typical phenotype of aged muscle. This aging phenotype was associated with repression of muscle-specific genes and activation of the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) pathway. ERK inhibition by U0126 not only induced recovery of myotube formation in old myoblasts but also facilitated muscle regeneration after injury in aged muscle. The conserved decline in ITPR1 expression in aged human skeletal muscle suggests utility as a potential therapeutic target for sarcopenia, which can be treated using ERK inhibition strategies. PMID:27658230

  20. Which pulse sequence is optimal for myo-Inositol detection at 3T?

    PubMed Central

    Hancu, Ileana

    2010-01-01

    Optimized myo-Inositol (mI) detection is important for diagnosing and monitoring a multitude of pathological conditions of the brain. Simulations are presented in this work, performed to decide which pulse sequence has the most significant advantage in terms of improving repeatability and accuracy of mI measurements at 3T over the pulse sequence used typically in the clinic, a TE=35ms PRESS sequence. Five classes of pulse sequences, four previously suggested for optimized mI detection (a short TE PRESS, a Carr-Purcell PRESS sequence, an optimized STEAM sequence, an optimized zero quantum filter), and one optimized for mI detection in this work (a single quantum filter) were compared to a standard, TE=35ms pulse sequence. While limiting the SNR of an acquisition to the equivalent SNR of a spectrum acquired in 5min from a 8cc voxel, it was found through simulations that the most repeatable mI measurements would be obtained with a Carr-Purcell sequence. This sequence was implemented in a clinical scanner, and improved mI measurements were demonstrated in vivo. PMID:19006101

  1. 5'-Inositol phosphatase SHIP2 recruits Mena to stabilize invadopodia for cancer cell invasion.

    PubMed

    Rajadurai, Charles V; Havrylov, Serhiy; Coelho, Paula P; Ratcliffe, Colin D H; Zaoui, Kossay; Huang, Bruce H; Monast, Anie; Chughtai, Naila; Sangwan, Veena; Gertler, Frank B; Siegel, Peter M; Park, Morag

    2016-09-12

    Invadopodia are specialized membrane protrusions that support degradation of extracellular matrix (ECM) by cancer cells, allowing invasion and metastatic spread. Although early stages of invadopodia assembly have been elucidated, little is known about maturation of invadopodia into structures competent for ECM proteolysis. The localized conversion of phosphatidylinositol(3,4,5)-triphosphate and accumulation of phosphatidylinositol(3,4)-bisphosphate at invadopodia is a key determinant for invadopodia maturation. Here we investigate the role of the 5'-inositol phosphatase, SHIP2, and reveal an unexpected scaffold function of SHIP2 as a prerequisite for invadopodia-mediated ECM degradation. Through biochemical and structure-function analyses, we identify specific interactions between SHIP2 and Mena, an Ena/VASP-family actin regulatory protein. We demonstrate that SHIP2 recruits Mena, but not VASP, to invadopodia and that disruption of SHIP2-Mena interaction in cancer cells leads to attenuated capacity for ECM degradation and invasion in vitro, as well as reduced metastasis in vivo. Together, these findings identify SHIP2 as a key modulator of carcinoma invasiveness and a target for metastatic disease.

  2. Bovine inositol monophosphatase. Modification, identification and mutagenesis of reactive cysteine residues.

    PubMed Central

    Knowles, M R; Gee, N; McAllister, G; Ragan, C I; Greasley, P J; Gore, M G

    1992-01-01

    1. Bovine inositol monophosphatase reacts with thiol reagents such as 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), N-ethylmaleimide (NEM) and iodoacetic acid (IAA). 2. Modification by NEM results in nearly total loss of enzyme activity, whereas modification by IAA causes a slight increase in activity. 3. The loss of activity caused by NEM can be prevented by the inclusion of Ins1P, or better Ins1P and LiCl in the reaction mixture. 4. Two equivalents of p-nitrothiobenzoate (NTB2-) are released from the native enzyme on reaction with DTNB, and six equivalents of NTB2- are released from the SDS-denatured enzyme, suggesting that none of the six cysteine residues per molecule of enzyme is involved in intra- or inter-molecular disulphide bridges. 5. Both NEM and IAA react with two cysteine residues (residues 141 and 184 in the sequence) in a mutually exclusive manner. 6. NEM also reacts stoichiometrically with residue 218. 7. The NEM-induced loss of enzyme activity is accompanied by a 15% decrease in protein fluorescence. 8. A mutant of the enzyme which has an Ala-218 replacement for Cys-218 has full activity and is not sensitive to NEM, showing that the modification of this cysteine by NEM causes inhibition of the native protein by steric effects and that Cys-218 is not essential for activity. PMID:1322134

  3. Long-term physiological effects of enhanced O2 release by inositol hexaphosphate-loaded erythrocytes.

    PubMed Central

    Teisseire, B; Ropars, C; Villeréal, M C; Nicolau, C

    1987-01-01

    A continuous lysing and resealing procedure with erythrocytes permitted incorporation in these cells of inositol hexaphosphate (InsP6), a strong allosteric effector of Hb. This leads to significant rightward shifts of the HbO2 dissociation curves with in vitro P50 (partial pressure of O2 at 50% Hb saturation), values increasing from 32.2 +/- 1.8 torr for control erythrocytes to 86 +/- 60 torr (pH 7.40; PCO2 40 torr at 37 degrees C; 1 torr = 1.333 X 10(2) Pa). The shape of the dissociation curve was still sigmoidal, although the Hill coefficient was decreased. The life span of InsP6-loaded erythrocytes equaled that of control erythrocytes. The long-term physiological effects of the InsP6-loaded erythrocytes on piglets were increased O2 release and reduced cardiac output. The reduced O2 affinity of the InsP6-loaded erythrocytes was still effective 20 days after transfusion in awake piglets. The electrolyte concentration appeared stable over the 5-day observation period except for a transient, but significant, hyperkalemia immediately after transfusion. The reductions in the O2 affinity of Hb reported here are large compared with previously reported values. Introduction of InsP6 into viable erythrocytes improves tissue oxygenation when, for any reason, normal blood flow is impaired. PMID:3116545

  4. Jasmonate perception by inositol-phosphate-potentiated COI1-JAZ co-receptor

    SciTech Connect

    Sheard, Laura B; Tan, Xu; Mao, Haibin; Withers, John; Ben-Nissan, Gili; Hinds, Thomas R; Kobayashi, Yuichi; Hsu, Fong-Fu; Sharon, Michal; Browse, John; He, Sheng Yang; Rizo, Josep; Howe, Gregg A; Zheng, Ning

    2011-11-07

    Jasmonates are a family of plant hormones that regulate plant growth, development and responses to stress. The F-box protein CORONATINE INSENSITIVE 1 (COI1) mediates jasmonate signalling by promoting hormone-dependent ubiquitylation and degradation of transcriptional repressor JAZ proteins. Despite its importance, the mechanism of jasmonate perception remains unclear. Here we present structural and pharmacological data to show that the true Arabidopsis jasmonate receptor is a complex of both COI1 and JAZ. COI1 contains an open pocket that recognizes the bioactive hormone (3R,7S)-jasmonoyl-l-isoleucine (JA-Ile) with high specificity. High-affinity hormone binding requires a bipartite JAZ degron sequence consisting of a conserved {alpha}-helix for COI1 docking and a loop region to trap the hormone in its binding pocket. In addition, we identify a third critical component of the jasmonate co-receptor complex, inositol pentakisphosphate, which interacts with both COI1 and JAZ adjacent to the ligand. Our results unravel the mechanism of jasmonate perception and highlight the ability of F-box proteins to evolve as multi-component signalling hubs.

  5. An allosteric model of the inositol trisphosphate receptor with nonequilibrium binding

    NASA Astrophysics Data System (ADS)

    Jia, Chen; Jiang, Daquan; Qian, Minping

    2014-10-01

    The inositol trisphosphate receptor (IPR) is a crucial ion channel that regulates the Ca2+ influx from the endoplasmic reticulum (ER) to the cytoplasm. A thorough study of the IPR channel contributes to a better understanding of calcium oscillations and waves. It has long been observed that the IPR channel is a typical biological system which performs adaptation. However, recent advances on the physical essence of adaptation show that adaptation systems with a negative feedback mechanism, such as the IPR channel, must break detailed balance and always operate out of equilibrium with energy dissipation. Almost all previous IPR models are equilibrium models assuming detailed balance and thus violate the dissipative nature of adaptation. In this article, we constructed a nonequilibrium allosteric model of single IPR channels based on the patch-clamp experimental data obtained from the IPR in the outer membranes of isolated nuclei of the Xenopus oocyte. It turns out that our model reproduces the patch-clamp experimental data reasonably well and produces both the correct steady-state and dynamic properties of the channel. Particularly, our model successfully describes the complicated bimodal [Ca2+] dependence of the mean open duration at high [IP3], a steady-state behavior which fails to be correctly described in previous IPR models. Finally, we used the patch-clamp experimental data to validate that the IPR channel indeed breaks detailed balance and thus is a nonequilibrium system which consumes energy.

  6. Single-Molecule Tracking of Inositol Trisphosphate Receptors Reveals Different Motilities and Distributions

    PubMed Central

    Smith, Ian F.; Swaminathan, Divya; Dickinson, George D.; Parker, Ian

    2014-01-01

    Puffs are local Ca2+ signals that arise by Ca2+ liberation from the endoplasmic reticulum through the concerted opening of tightly clustered inositol trisphosphate receptors/channels (IP3Rs). The locations of puff sites observed by Ca2+ imaging remain static over several minutes, whereas fluorescence recovery after photobleaching (FRAP) experiments employing overexpression of fluorescently tagged IP3Rs have shown that the majority of IP3Rs are freely motile. To address this discrepancy, we applied single-molecule imaging to locate and track type 1 IP3Rs tagged with a photoswitchable fluorescent protein and expressed in COS-7 cells. We found that ∼70% of the IP3R1 molecules were freely motile, undergoing random walk motility with an apparent diffusion coefficient of ∼0.095 μm s−1, whereas the remaining molecules were essentially immotile. A fraction of the immotile IP3Rs were organized in clusters, with dimensions (a few hundred nanometers across) comparable to those previously estimated for the IP3R clusters underlying functional puff sites. No short-term (seconds) changes in overall motility or in clustering of immotile IP3Rs were apparent following activation of IP3/Ca2+ signaling. We conclude that stable clusters of small numbers of immotile IP3Rs may underlie local Ca2+ release sites, whereas the more numerous motile IP3Rs appear to be functionally silent. PMID:25140418

  7. Role of inositol 1,4,5-triphosphate signalling in gravitropic and phototropic gene expression.

    PubMed

    Salinas-Mondragon, Raul E; Kajla, Jyoti D; Perera, Imara Y; Brown, Christopher S; Sederoff, Heike Winter

    2010-12-01

    Plants sense light and gravity to orient their direction of growth. One common component in the early events of both phototropic and gravitropic signal transduction is activation of phospholipase C (PLC), which leads to an increase in inositol 1,4,5-triphosphate (InsP(3)) levels. The InsP(3) signal is terminated by hydrolysis of InsP(3) through inositolpolyphosphate-5-phosphatases (InsP 5-ptases). Arabidopsis plants expressing a heterologous InsP 5-ptase have low basal InsP(3) levels and exhibit reduced gravitropic and phototropic bending. Downstream effects of InsP(3)-mediated signalling are not understood. We used comparative transcript profiling to characterize gene expression changes in gravity- or light-stimulated Arabidopsis root apices that were manipulated in their InsP(3) metabolism either through inhibition of PLC activity or expression of InsP 5-ptase. We identified InsP(3)-dependent and InsP(3)-independent co-regulated gene sets in response to gravity or light stimulation. Inhibition of PLC activity in wild-type plants caused similar changes in transcript abundance in response to gravitropic and phototropic stimulation as in the transgenic lines. Therefore, we conclude that changes in gene expression in response to gravitropic and phototropic stimulation are mediated by two signal transduction pathways that vary in their dependence on changes in InsP(3).

  8. 2-Aminoethoxydiphenyl borate, a inositol 1,4,5-triphosphate receptor inhibitor, prevents atrial fibrillation.

    PubMed

    Xiao, Junjie; Liang, Dandan; Zhao, Hong; Liu, Ying; Zhang, Hong; Lu, Xiaowei; Liu, Yi; Li, Jun; Peng, Luying; Chen, Yi-Han

    2010-07-01

    The expression of the inositol 1,4,5-triphosphate receptor (IP3R) is upregulated and the function of IP3R also increases during atrial fibrillation (AF). 2-Aminoethoxydiphenyl borate (2-APB) is a membrane-permeable inhibitor of IP3R. However, the effect of 2-APB on AF is unknown. The aim of the present study is to explore the effects of 2-APB on AF. In vitro rabbit heart models of ischemia-, stretch- and cholinergic agitation-induced AF were developed. Fura-2-acetoxymethyl (Fura-2-AM) and Mg2+-Fura-2-AM were used to monitor alterations of intracellular Ca2+ and ATP, respectively, in HL-1 cells, an atrial muscle cell line, under chemical ischemia or cholinergic agitation. The results showed that inhibition of IP3R significantly reduced the incidence and its probability of being sustained in all three types of AF. IP3R inhibition ameliorated the cytoplasmic Ca2+ overload and energy compromise resulting from chemical ischemia or cholinergic agitation. Thus, IP3R inhibition may be a novel target for AF treatment, and IP3R may be an important molecule in the context of different kinds of AF.

  9. HLH-29 regulates ovulation in C. elegans by targeting genes in the inositol triphosphate signaling pathway.

    PubMed

    White, Ana; Fearon, Abegail; Johnson, Casonya M

    2012-03-15

    The reproductive cycle in the nematode Caenorhabditis elegans depends in part on the ability of the mature oocyte to ovulate into the spermatheca, fuse with the sperm during fertilization, and then exit the spermatheca as a fertilized egg. This cycle requires the integration of signals between the germ cells and the somatic gonad and relies heavily on the precise control of inositol 1,4,5 triphosphate (IP(3))levels. The HLH-29 protein, one of five Hairy/Enhancer of Split (HES) homologs in C. elegans, was previously shown to affect development of the somatic gonad. Here we show that HLH-29 expression in the adult spermatheca is strongly localized to the distal spermatheca valve and to the spermatheca-uterine valve, and that loss of hlh-29 activity interferes with oocyte entry into and egg exit from the spermatheca. We show that HLH-29 can regulate the transcriptional activity of the IP(3) signaling pathway genes ppk-1, ipp-5, and plc-1 and provide evidence that hlh-29 acts in a genetic pathway with each of these genes. We propose that the HES-like protein HLH-29 acts in the spermatheca of larval and adult animals to effectively increase IP(3) levels during the reproductive cycle.

  10. Structure-Function Analysis of Inositol Hexakisphosphate-induced Autoprocessing in Clostridium difficile Toxin A

    SciTech Connect

    Pruitt, Rory N.; Chagot, Benjamin; Cover, Michael; Chazin, Walter J.; Spiller, Ben; Lacy, D. Borden

    2009-09-25

    The action of Clostridium difficile toxins A and B depends on inactivation of host small G-proteins by glucosylation. Cellular inositol hexakisphosphate (InsP6) induces an autocatalytic cleavage of the toxins, releasing an N-terminal glucosyltransferase domain into the host cell cytosol. We have defined the cysteine protease domain (CPD) responsible for autoprocessing within toxin A (TcdA) and report the 1.6 {angstrom} x-ray crystal structure of the domain bound to InsP6. InsP6 is bound in a highly basic pocket that is separated from an unusual active site by a {beta}-flap structure. Functional studies confirm an intramolecular mechanism of cleavage and highlight specific residues required for InsP6-induced TcdA processing. Analysis of the structural and functional data in the context of sequences from similar and diverse origins highlights a C-terminal extension and a {pi}-cation interaction within the {beta}-flap that appear to be unique among the large clostridial cytotoxins.

  11. Molecular and functional characterization of inositol trisphosphate receptors during early zebrafish development.

    PubMed

    Ashworth, Rachel; Devogelaere, Benoit; Fabes, Jez; Tunwell, Richard E; Koh, Kevin R; De Smedt, Humbert; Patel, Sandip

    2007-05-11

    Fluctuations in cytosolic Ca(2+) are crucial for a variety of cellular processes including many aspects of development. Mobilization of intracellular Ca(2+) stores via the production of inositol trisphosphate (IP(3)) and the consequent activation of IP(3)-sensitive Ca(2+) channels is a ubiquitous means by which diverse stimuli mediate their cellular effects. Although IP(3) receptors have been well studied at fertilization, information regarding their possible involvement during subsequent development is scant. In the present study we examined the role of IP(3) receptors in early development of the zebrafish. We report the first molecular analysis of zebrafish IP(3) receptors which indicates that, like mammals, the zebrafish genome contains three distinct IP(3) receptor genes. mRNA for all isoforms was detectable at differing levels by the 64 cell stage, and IP(3)-induced Ca(2+) transients could be readily generated (by flash photolysis) in a controlled fashion throughout the cleavage period in vivo. Furthermore, we show that early blastula formation was disrupted by pharmacological blockade of IP(3) receptors or phospholipase C, by molecular inhibition of the former by injection of IRBIT (IP(3) receptor-binding protein released with IP(3)) and by depletion of thapsigargin-sensitive Ca(2+) stores after completion of the second cell cycle. Inhibition of Ca(2+) entry or ryanodine receptors, however, had little effect. Our work defines the importance of IP(3) receptors during early development of a genetically and optically tractable model vertebrate organism.

  12. Myo-Inositol Safety in Pregnancy: From Preimplantation Development to Newborn Animals

    PubMed Central

    Kuşcu, Nilay

    2016-01-01

    Myo-inositol (myo-Ins) has a physiological role in mammalian gametogenesis and embryonic development and a positive clinical impact on human medically assisted reproduction. We have previously shown that mouse embryo exposure to myo-Ins through preimplantation development in vitro increases proliferation activity and blastocyst production, representing an improvement in culture conditions. We have herein investigated biochemical mechanisms elicited by myo-Ins in preimplantation embryos and evaluated myo-Ins effects on postimplantation/postnatal development. To this end naturally fertilized embryos were cultured in vitro to blastocyst in the presence or absence of myo-Ins and analyzed for activation of the PKB/Akt pathway, known to modulate proliferation/survival cellular processes. In parallel, blastocyst-stage embryos were transferred into pseudopregnant females and allowed to develop to term and until weaning. Results obtained provide evidence that myo-Ins induces cellular pathways involving Akt and show that (a) exposure of preimplantation embryos to myo-Ins increases the number of blastocysts available for uterine transfer and of delivered animals and (b) the developmental patterns of mice obtained from embryos cultured in the presence or absence of myo-Ins, up to three weeks of age, overlap. These data further identify myo-Ins as a possibly important supplement for human preimplantation embryo culture in assisted reproduction technology. PMID:27698667

  13. The inositol phosphatase SHIP controls Salmonella enterica serovar Typhimurium infection in vivo.

    PubMed

    Bishop, Jennifer L; Sly, Laura M; Krystal, Gerald; Finlay, B Brett

    2008-07-01

    The SH2 domain-containing inositol 5'-phosphatase, SHIP, negatively regulates various hematopoietic cell functions and is critical for maintaining immune homeostasis. However, whether SHIP plays a role in controlling bacterial infections in vivo remains unknown. Salmonella enterica causes human salmonellosis, a disease that ranges in severity from mild gastroenteritis to severe systemic illness, resulting in significant morbidity and mortality worldwide. The susceptibility of ship(+/+) and ship(-/-) mice and bone marrow-derived macrophages to S. enterica serovar Typhimurium infection was compared. ship(-/-) mice displayed an increased susceptibility to both oral and intraperitoneal serovar Typhimurium infection and had significantly higher bacterial loads in intestinal and systemic sites than ship(+/+) mice, indicating a role for SHIP in the gut-associated and systemic pathogenesis of serovar Typhimurium in vivo. Cytokine analysis of serum from orally infected mice showed that ship(-/-) mice produce lower levels of Th1 cytokines than do ship(+/+) animals at 2 days postinfection, and in vitro analysis of supernatants taken from infected bone marrow-derived macrophages derived to mimic the in vivo ship(-/-) alternatively activated (M2) macrophage phenotype correlated with these data. M2 macrophages were the predominant population in vivo in both oral and intraperitoneal infections, since tissue macrophages within the small intestine and peritoneal macrophages from ship(-/-) mice showed elevated levels of the M2 macrophage markers Ym1 and Arginase 1 compared to ship(+/+) cells. Based on these data, we propose that M2 macrophage skewing in ship(-/-) mice contributes to ineffective clearance of Salmonella in vivo.

  14. Myo-Inositol Safety in Pregnancy: From Preimplantation Development to Newborn Animals

    PubMed Central

    Kuşcu, Nilay

    2016-01-01

    Myo-inositol (myo-Ins) has a physiological role in mammalian gametogenesis and embryonic development and a positive clinical impact on human medically assisted reproduction. We have previously shown that mouse embryo exposure to myo-Ins through preimplantation development in vitro increases proliferation activity and blastocyst production, representing an improvement in culture conditions. We have herein investigated biochemical mechanisms elicited by myo-Ins in preimplantation embryos and evaluated myo-Ins effects on postimplantation/postnatal development. To this end naturally fertilized embryos were cultured in vitro to blastocyst in the presence or absence of myo-Ins and analyzed for activation of the PKB/Akt pathway, known to modulate proliferation/survival cellular processes. In parallel, blastocyst-stage embryos were transferred into pseudopregnant females and allowed to develop to term and until weaning. Results obtained provide evidence that myo-Ins induces cellular pathways involving Akt and show that (a) exposure of preimplantation embryos to myo-Ins increases the number of blastocysts available for uterine transfer and of delivered animals and (b) the developmental patterns of mice obtained from embryos cultured in the presence or absence of myo-Ins, up to three weeks of age, overlap. These data further identify myo-Ins as a possibly important supplement for human preimplantation embryo culture in assisted reproduction technology.

  15. Molecular basis of cyclin-CDK-CKI regulation by reversible binding of an inositol pyrophosphate

    PubMed Central

    Lee, Young-Sam; Huang, Kexin; Quiocho, Florante A; O’Shea, Erin K

    2008-01-01

    When Saccharomyces cerevisiae cells are starved of inorganic phosphate, the Pho80-Pho85 cyclin–cyclin-dependent kinase (CDK) is inactivated by the Pho81 CDK inhibitor (CKI). The regulation of Pho80-Pho85 is distinct from previously characterized mechanisms of CDK regulation: the Pho81 CKI is constitutively associated with Pho80-Pho85, and a small-molecule ligand, inositol heptakisphosphate (IP7), is required for kinase inactivation. We investigated the molecular basis of the IP7- and Pho81-dependent Pho80-Pho85 inactivation using electrophoretic mobility shift assays, enzyme kinetics and fluorescence spectroscopy. We found that IP7 interacts noncovalently with Pho80-Pho85-Pho81 and induces additional interactions between Pho81 and Pho80-Pho85 that prevent substrates from accessing the kinase active site. Using synthetic peptides corresponding to Pho81, we define regions of Pho81 responsible for constitutive Pho80-Pho85 binding and IP7-regulated interaction and inhibition. These findings expand our understanding of the mechanisms of cyclin-CDK regulation and of the biochemical mechanisms of IP7 action. PMID:18059263

  16. (1) H NMR analysis of O-methyl-inositol isomers: a joint experimental and theoretical study.

    PubMed

    De Almeida, Mauro V; Couri, Mara Rubia C; De Assis, João Vitor; Anconi, Cleber P A; Dos Santos, Hélio F; De Almeida, Wagner B

    2012-09-01

    Density functional theory (DFT) calculations of (1) H NMR chemical shifts for l-quebrachitol isomers were performed using the B3LYP functional employing the 6-31G(d,p) and 6-311 + G(2d,p) basis sets. The effect of the solvent on the B3LYP-calculated NMR spectrum was accounted for using the polarizable continuum model. Comparison is made with experimental (1) H NMR spectroscopic data, which shed light on the average uncertainty present in DFT calculations of chemical shifts and showed that the best match between experimental and theoretical B3LYP (1) H NMR profiles is a good strategy to assign the molecular structure present in the sample handled in the experimental measurements. Among four plausible O-methyl-inositol isomers, the l-quebrachitol 2a structure was unambiguously assigned based only on the comparative analysis of experimental and theoretical (1) H NMR chemical shift data. The B3LYP infrared (IR) spectrum was also calculated for the four isomers and compared with the experimental data, with analysis of the theoretical IR profiles corroborating assignment of the 2a structure. Therefore, it is confirmed in this study that a combined experimental/DFT spectroscopic investigation is a powerful tool in structural/conformational analysis studies.

  17. The quantitative spectrum of inositol phosphate metabolites in avian erythrocytes, analysed by proton n.m.r. and h.p.l.c. with direct isomer detection.

    PubMed Central

    Radenberg, T; Scholz, P; Bergmann, G; Mayr, G W

    1989-01-01

    The spectrum of inositol phosphate isomers present in avian erythrocytes was investigated in qualitative and quantitative terms. Inositol phosphates were isolated in micromolar quantities from turkey blood by anion-exchange chromatography on Q-Sepharose and subjected to proton n.m.r. and h.p.l.c. analysis. We employed a h.p.l.c. technique with a novel, recently described complexometric post-column detection system, called 'metal-dye detection' [Mayr (1988) Biochem. J. 254, 585-591], which enabled us to identify non-radioactively labelled inositol phosphate isomers and to determine their masses. The results indicate that avian erythrocytes contain the same inositol phosphate isomers as mammalian cells. Denoted by the 'lowest-locant rule' [NC-IUB Recommendations (1988) Biochem. J. 258, 1-2] irrespective of true enantiomerism, these are Ins(1,4)P2, Ins(1,6)P2, Ins(1,3,4)P3, Ins(1,4,5)P3, Ins(1,3,4,5)P4, Ins(1,3,4,6)P4, Ins(1,4,5,6)P4, Ins(1,3,4,5,6)P5, and InsP6. Furthermore, we identified two inositol trisphosphate isomers hitherto not described for mammalian cells, namely Ins(1,5,6)P3 and Ins(2,4,5)P3. The possible position of these two isomers in inositol phosphate metabolism and implications resulting from absolute abundances of inositol phosphates are discussed. PMID:2604720

  18. Structural analysis of the carboxy terminal PH domain of pleckstrin bound to D-myo-inositol 1,2,3,5,6-pentakisphosphate

    PubMed Central

    Jackson, Sean G; Zhang, Yi; Haslam, Richard J; Junop, Murray S

    2007-01-01

    Background Pleckstrin homology (PH) domains are one of the most prevalent domains in the human proteome and represent the major phosphoinositide-binding module. These domains are often found in signaling proteins and function predominately by targeting their host proteins to the cell membrane. Inositol phosphates, which are structurally similar to phosphoinositides, are not only known to play a role as signaling molecules but are also capable of being bound by PH domains. Results In the work presented here it is shown that the addition of commercial myo-inositol hexakisphosphate (IP6) inhibited the binding of the carboxy terminal PH domain of pleckstrin (C-PH) to phosphatidylinositol 3,4-bisphosphate with an IC50 of 7.5 μM. In an attempt to characterize this binding structurally, C-PH was crystallized in the presence of IP6 and the structure was determined to 1.35 Å. Examination of the resulting electron density unexpectedly revealed the bound ligand to be D-myo-inositol 1,2,3,5,6-pentakisphosphate. Conclusion The discovery of D-myo-inositol 1,2,3,5,6-pentakisphosphate in the crystal structure suggests that the inhibitory effects observed in the binding studies may be due to this ligand rather than IP6. Analysis of the protein-ligand interaction demonstrated that this myo-inositol pentakisphosphate isomer interacts specifically with protein residues known to be involved in phosphoinositide binding. In addition to this, a structural alignment of other PH domains bound to inositol phosphates containing either four or five phosphate groups revealed that the majority of phosphate groups occupy conserved locations in the binding pockets of PH domains. These findings, taken together with other recently reported studies suggest that myo-inositol pentakisphosphates could act to regulate PH domain-phosphoinositide interactions by directly competing for binding, thus playing an important role as signaling molecules. PMID:18034889

  19. A Functional myo-Inositol Dehydrogenase Gene Is Required for Efficient Nitrogen Fixation and Competitiveness of Sinorhizobium fredii USDA191 To Nodulate Soybean (Glycine max [L.] Merr.)

    PubMed Central

    Jiang, Guoqiao; Krishnan, Ammulu Hari; Kim, Yong-Woong; Wacek, Thomas J.; Krishnan, Hari B.

    2001-01-01

    Inositol derivative compounds provide a nutrient source for soil bacteria that possess the ability to degrade such compounds. Rhizobium strains that are capable of utilizing certain inositol derivatives are better colonizers of their host plants. We have cloned and determined the nucleotide sequence of the myo-inositol dehydrogenase gene (idhA) of Sinorhizobium fredii USDA191, the first enzyme responsible for inositol catabolism. The deduced IdhA protein has a molecular mass of 34,648 Da and shows significant sequence similarity with protein sequences of Sinorhizobium meliloti IdhA and MocA; Bacillus subtilis IolG, YrbE, and YucG; and Streptomyces griseus StrI. S. fredii USDA191 idhA mutants revealed no detectable myo-inositol dehydrogenase activity and failed to grow on myo-inositol as a sole carbon source. Northern blot analysis and idhA-lacZ fusion expression studies indicate that idhA is inducible by myo-inositol. S. fredii USDA191 idhA mutant was drastically affected in its ability to reduce nitrogen and revealed deteriorating bacteroids inside the nodules. The number of bacteria recovered from such nodules was about threefold lower than the number of bacteria isolated from nodules initiated by S. fredii USDA191. In addition, the idhA mutant was also severely affected in its ability to compete with the wild-type strain in nodulating soybean. Under competitive conditions, nodules induced on soybean roots were predominantly occupied by the parent strain, even when the idhA mutant was applied at a 10-fold numerical advantage. Thus, we conclude that a functional idhA gene is required for efficient nitrogen fixation and for competitive nodulation of soybeans by S. fredii USDA191. PMID:11274120

  20. Thyrotropin-induced elevation of 1,2-diacylglycerol and stimulation of growth of FRTL-5 cells are not dependent on inositol lipid hydrolysis.

    PubMed

    Brenner-Gati, L; Trowbridge, J M; Moucha, C S; Gershengorn, M C

    1990-03-01

    We reported that TSH, which stimulates cAMP accumulation and proliferation of FRTL-5 thyroid cells, chronically increases the 1,2-diacylglycerol (1,2-DG) content of FRTL-5 cells. Because activation of inositol lipid hydrolysis by a phospholipase-C enzyme would generate 1,2-DG, we compared the effects of TSH on inositol lipid metabolism to TSH-induced increases in 1,2-DG content and stimulation of cAMP accumulation and cell growth. Acute stimulation of inositol lipid hydrolysis did not occur with doses of 1000 microU/ml or lower, but did occur with TSH doses of 3000 microU/ml and higher, with rates between 1-4%/h. More importantly, in cells chronically exposed to TSH, the rate of inositol lipid hydrolysis was increased only at TSH doses of 10,000 microU/ml or greater, and the maximum rate was 4-5%/h. When cells were growth arrested by TSH deprivation, there was no change in the content of inositol phosphates or polyphosphoinositides. In contrast to the high doses of TSH required to stimulate inositol lipid hydrolysis, TSH-induced elevation of 1,2-DG content and stimulation of cAMP accumulation and growth occurred at physiological TSH concentrations, with minimal effective doses in the range of 1-10 microU TSH/ml, and half-maximally effective doses between 50-200 microU TSH/ml. These data suggest that inositol lipid hydrolysis does not mediate the proliferative response to TSH in FRTL-5 cells and is not the mechanism by which increases in 1,2-DG content occur at physiological TSH concentrations.

  1. Inositol 1,4,5-trisphosphate receptor subtypes differentially recognize regioisomers of D-myo-inositol 1,4,5-trisphosphate.

    PubMed Central

    Hirata, M; Takeuchi, H; Riley, A M; Mills, S J; Watanabe, Y; Potter, B V

    1997-01-01

    The Ins(1,4,5)P3 regioisomers, Ins(1,4,6)P3 and Ins(1,3,6)P3, which can mimic the 1,4,5-arrangement on the inositol ring of Ins(1,4,5)P3, were examined for Ca2+ release by using four types of saponin-permeabilized cell possessing various abundances of receptor subtypes, with special reference to the relation of potency to receptor subtype. Ins(1,4,6)P3 and Ins(1,3,6)P3 were weak agonists in rat basophilic leukaemic cells (RBL cells), which possess predominantly subtype II receptors, with respective potencies of 1/200 and less than 1/500 that of Ins(1,4,5)P3 [the EC50 values were 0.2, 45 and more than 100 microM for Ins(1,4,5)P3, Ins(1,4,6)P3 and Ins(1,3,6)P3 respectively]. Similar rank order potencies were also evaluated for the displacement of [3H]Ins(1,4,5)P3 bound to RBL cell membranes by these regioisomers. However, they caused Ca2+ release from GH3 rat pituitary cells possessing predominantly subtype I receptors more potently; Ins(1,4,6)P3 and Ins(1,3,6)P3 evoked release at respective concentrations of only one-third and one-twentieth that of Ins(1,4,5)P3 (the EC50 values were 0.4, 1.2 and 8 microM for Ins(1,4,5)P3, Ins(1,4,6)P3 and Ins(1,3,6)P3 respectively). In COS-1 African green-monkey kidney cells, with the relative abundances of 37% of the subtype II and of 62% of the subtype III receptor, potencies of 1/40 and approx. 1/200 for Ins(1, 4,6)P3 and Ins(1,3,6)P3 respectively were exhibited relative to Ins(1,4,5)P3 (the EC50 values were 0.4, 15 and approx. 80 microM for Ins(1,4,5)P3, Ins(1,4,6)P3 and Ins(1,3,6)P3 respectively). In HL-60 human leukaemic cells, in spite of the dominant presence of subtype I receptors (71%), similar respective potencies to those seen with COS-1 cells were exhibited (the EC50 values were 0.3, 15 and approx. 100 microM for Ins(1,4,5)P3, Ins(1,4,6)P3 and Ins(1,3,6)P3 respectively). These results indicate that these regioisomers are the first ligands that distinguish between receptor subtypes; the present observations are of

  2. Ectopic Expression of a Glycine soja myo-Inositol Oxygenase Gene (GsMIOX1a) in Arabidopsis Enhances Tolerance to Alkaline Stress

    PubMed Central

    Chen, Chen; Sun, Xiaoli; Duanmu, Huizi; Yu, Yang; Liu, Ailin; Xiao, Jialei; Zhu, Yanming

    2015-01-01

    Myo-inositol participates in various aspects of plant physiology, and myo-inositol oxygenase is the key enzyme of the myo-inositol oxygenation pathway. Previous studies indicated that myo-inositol oxygenase may play a role in plant responses to abiotic stresses. In this study, we focused on the functional characterization of GsMIOX1a, a remarkable alkaline stress-responsive gene of Glycine soja 07256, based on RNA-seq data. Using quantitative real-time PCR, we demonstrated that GsMIOX1a is rapidly induced by alkaline stress and expressed predominantly in flowers. We also elucidated the positive function of GsMIOX1a in the alkaline response in the wild type, atmiox1 mutant as well as GsMIOX1a-overexpressing Arabidopsis. We determined that atmiox1 mutant decreased Arabidopsis tolerance to alkaline stress, whereas GsMIOX1a overexpression increased tolerance. Moreover, the expression levels of some alkaline stress-responsive and inducible marker genes, including H+-Ppase, NADP-ME, KIN1 and RD29B, were also up-regulated in GsMIOX1a overexpression lines compared with the wild type and atmiox1 mutant. Together, these results suggest that the GsMIOX1a gene positively regulates plant tolerance to alkaline stress. This is the first report to demonstrate that ectopic expression of myo-inositol oxygenase improves alkaline tolerance in plants. PMID:26091094

  3. Iron absorption from bread in humans: inhibiting effects of cereal fiber, phytate and inositol phosphates with different numbers of phosphate groups.

    PubMed

    Brune, M; Rossander-Hultén, L; Hallberg, L; Gleerup, A; Sandberg, A S

    1992-03-01

    Iron absorption was measured from five kinds of bread made from various types of flour and fermented in different ways in order to obtain a wide variation in the content of fiber, phytate (inositol hexaphosphate) and its degradation products, inorganic phosphate and inositol phosphates with fewer numbers of phosphate groups (inositol pentaphosphate through monophosphate). Each experiment had 9-10 subjects and, in each subject, iron absorption was measured from control rolls made from low extraction wheat flour and one kind of test roll using two different radioiron tracers: 55Fe and 59Fe. The inhibition of iron absorption was closely related to the content of phytate-phosphorous as determined using the AOAC method, and to the sum of the tri- through hexaphosphate groups as determined using the HPLC method. As an example, prolonged fermentation of whole-rye bread reduced total inositol phosphates to the same amount as in the control rolls and increased fractional iron absorption to the same high level, in spite of a fiber content five times as great. The results strongly suggest that the inhibitory effect of bran on iron absorption is due to its content of phytate and other inositol phosphates present after fermentation, rather than to its content of fiber or other constituents. Thus, effective fermentation will increase the bioavailability of iron in whole-meal bread.

  4. The Composition of Stigmatic Exudate from Lilium longiflorum: Labeling Studies with Myo-inositol, d-Glucose, and l-Proline.

    PubMed

    Labarca, C; Kroh, M; Loewus, F

    1970-07-01

    Stigmatic exudate, a secretion product recovered from the upper surface of Lilium longiflorum pistils, has been examined. Over 99% of the exudate is accounted for as water, carbohydrate, and protein. Exclusive of water, 95% is a high molecular weight, protein-containing polysaccharide composed of galactose, arabinose, rhamnose, glucuronic acid, and galacturonic acid.Detached pistils supplied with myo-inositol-U-(14)C, myo-inositol-2-(3)H, d-glucose-1-(14)C, or l-proline-U-(14)C produce labeled stigmatic exudate. When myo-inositol is supplied, the exudate is rich in labeled arabinose and uronic acids, but some label also recycles through the hexose phosphate pool of secreting cells, causing label to appear in galactose and rhamnose residues. When glucose is provided, galactose is the major constituent labeled but all of the other carbohydrate constituents are also labeled. Proline produces a pattern very similar to that obtained with glucose.Stigmatic exudate also contains a small amount of low molecular weight carbohydrate. If myo-inositol is used to label exudate, free labeled myo-inositol cannot be detected in the low molecular weight fraction until it has been subjected to acid hydrolysis. Similarly, if d-glucose is the source of label, free labeled glucose is found in the low molecular weight fraction only after acid hydrolysis.

  5. Disruption of Renal Tubular Mitochondrial Quality Control by Myo-Inositol Oxygenase in Diabetic Kidney Disease

    PubMed Central

    Zhan, Ming; Usman, Irtaza M.; Sun, Lin

    2015-01-01

    Diabetic kidney disease (DKD) is associated with oxidative stress and mitochondrial injury. Myo-inositol oxygenase (MIOX), a tubular-specific enzyme, modulates redox imbalance and apoptosis in tubular cells in diabetes, but these mechanisms remain unclear. We investigated the role of MIOX in perturbation of mitochondrial quality control, including mitochondrial dynamics and autophagy/mitophagy, under high-glucose (HG) ambience or a diabetic state. HK-2 or LLC-PK1 cells subjected to HG exhibited an upregulation of MIOX accompanied by mitochondrial fragmentation and depolarization, inhibition of autophagy/mitophagy, and altered expression of mitochondrial dynamic and mitophagic proteins. Furthermore, dysfunctional mitochondria accumulated in the cytoplasm, which coincided with increased reactive oxygen species generation, Bax activation, cytochrome C release, and apoptosis. Overexpression of MIOX in LLC-PK1 cells enhanced the effects of HG, whereas MIOX siRNA or d-glucarate, an inhibitor of MIOX, partially reversed these perturbations. Moreover, decreasing the expression of MIOX under HG ambience increased PTEN-induced putative kinase 1 expression and the dependent mitofusin-2–Parkin interaction. In tubules of diabetic mice, increased MIOX expression and mitochondrial fragmentation and defective autophagy were observed. Dietary supplementation of d-glucarate in diabetic mice decreased MIOX expression, attenuated tubular damage, and improved renal functions. Notably, d-glucarate administration also partially attenuated mitochondrial fragmentation, oxidative stress, and apoptosis and restored autophagy/mitophagy in the tubular cells of these mice. These results suggest a novel mechanism linking MIOX to impaired mitochondrial quality control during tubular injury in the pathogenesis of DKD and suggest d-glucarate as a potential therapeutic agent for the amelioration of DKD. PMID:25270067

  6. Type 1 and 3 inositol trisphosphate receptors are required for extra-embryonic vascular development.

    PubMed

    Uchida, Keiko; Nakazawa, Maki; Yamagishi, Chihiro; Mikoshiba, Katsuhiko; Yamagishi, Hiroyuki

    2016-10-01

    The embryonic-maternal interface of the placental labyrinth, allantois, and yolk sac are vital during embryogenesis; however, the precise mechanism underlying the vascularization of these structures remains unknown. Herein we focus on the role of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R), which are intracellular Ca(2+) release channels, in placentation. Double knockout (DKO) of type 1 and 3 IP3Rs (IP3R1 and IP3R3, respectively) in mice resulted in embryonic lethality around embryonic day (E) 11.5. Because IP3R1 and IP3R3 were co-expressed in endothelial cells in the labyrinth, allantois, and yolk sac, we investigated extra-embryonic vascular development in IP3R1- and IP3R3-DKO mice. The formation of chorionic plates and yolk sac vessels seemed dysregulated around the timing of the chorio-allantoic attachment, immediately followed by the disorganization of allantoic vessels, the decreased expression of the spongiotrophoblast cell marker Tpbpa and the growth retardation of the embryos in DKO mice. Fluorescent immunohistochemistry demonstrated downregulation of a vascular endothelial marker, CD31, in labyrinth embryonic vessels and poor elongation of extra-embryonic mesoderm into the labyrinth layer in DKO placenta, whereas the branching of the DKO chorionic trophoblast was initiated. In addition, allantoic and yolk sac vessels in extra-embryonic tissues were less remodeled in DKO mice. In vitro endothelial cord formation and migration activities of cultured vascular endothelial cells derived from human umbilical vein were downregulated under the inhibition of IP3R. Our results suggest that IP3R1 and IP3R3 are required for extra-embryonic vascularization in the placenta, allantois, and yolk sac. This is the first demonstration of the essential role of IP3/IP3Rs signaling in the development of the vasculature at the embryonic-maternal interface. PMID:27514653

  7. ANTIPROLIFERATIVE EFFECT OF INOSITOL HEXAPHOSPHATE ON HUMAN SKIN MELANOMA CELLS IN VITRO.

    PubMed

    Wawszczyk, Joanna; Kapral, Małgorzata; Lodowska, Jolanta; Jesse, Katarzyna; Hollek, Andrzej; Węglarz, Ludmiła

    2015-01-01

    Human malignant melanoma is a highly metastatic tumor with poor prognosis. The majority of metastatic melanomas are resistant to diverse chemotherapeutic agents. Consequently, the search for novel antimelanoma agents continues. In recent years, the interest in plants and their biologically active constituents as a source of novel potential drugs significantly increased. Inositol hexaphosphate (IP6) is a naturally occurring compound that has been shown to inhibit the growth of a wide variety of tumor cells in multiple experimental model systems. The aim of this study was to evaluate the antiproliferative and cytotoxic influence of IP6 on melanotic melanoma cells in vitro. The A2058 cells used as a model of human skin melanoma malignum were exposed to different concentrations of IP6 (0.1-5 mM) for a various period of time and their growth was determined by sulforhodamine B assay after 24, 48 and 72 h. The cytotoxicity of IP6 was measured at 24 and 72 h by XTT assay. IP6 has been found to cause dose-dependent growth suppression of A2058 melanoma cells. At low concentrations (0.1 and 0.5 mM) it did not exert any influence on the cell proliferation as compared to control cultures. Higher concentrations of IP6 (from 1 to 5 mM) had a statistically significant, suppressive effect on cell proliferation after 24 h incubation. When the experimental time period was increased up to 72 h, statistically significant inhibition of cell proliferation was monitored at all IP6 concentrations used. Data obtained from XTT assay indicated that IP6 had dose- and time-dependent cytotoxic effect on melanoma cells. The results demonstrate the antiproliferative and cytotoxic properties of IP6 in a wide range of concentrations on human A2058 melanoma cells. Hence, it can be suggested that IP6 could have a promising therapeutic significance in treating cancer.

  8. Proteolytic fragmentation of inositol 1,4,5-trisphosphate receptors: a novel mechanism regulating channel activity?

    PubMed

    Wang, Liwei; Alzayady, Kamil J; Yule, David I

    2016-06-01

    Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are a family of ubiquitously expressed intracellular Ca(2+) release channels. Regulation of channel activity by Ca(2+) , nucleotides, phosphorylation, protein binding partners and other cellular factors is thought to play a major role in defining the specific spatiotemporal characteristics of intracellular Ca(2+) signals. These properties are, in turn, believed pivotal for the selective and specific physiological activation of Ca(2+) -dependent effectors. IP3 Rs are also substrates for the intracellular cysteine proteases, calpain and caspase. Cleavage of the IP3 R has been proposed to play a role in apoptotic cell death by uncoupling regions important for IP3 binding from the channel domain, leaving an unregulated leaky Ca(2+) pore. Contrary to this hypothesis, we demonstrate following proteolysis that N- and C-termini of IP3 R1 remain associated, presumably through non-covalent interactions. Further, we show that complementary fragments of IP3 R1 assemble into tetrameric structures and retain their ability to be regulated robustly by IP3 . While peptide continuity is clearly not necessary for IP3 -gating of the channel, we propose that cleavage of the IP3 R peptide chain may alter other important regulatory events to modulate channel activity. In this scenario, stimulation of the cleaved IP3 R may support distinct spatiotemporal Ca(2+) signals and activation of specific effectors. Notably, in many adaptive physiological events, the non-apoptotic activities of caspase and calpain are demonstrated to be important, but the substrates of the proteases are poorly defined. We speculate that proteolytic fragmentation may represent a novel form of IP3 R regulation, which plays a role in varied adaptive physiological processes.

  9. Functional inositol 1,4,5-trisphosphate receptors assembled from concatenated homo- and heteromeric subunits.

    PubMed

    Alzayady, Kamil J; Wagner, Larry E; Chandrasekhar, Rahul; Monteagudo, Alina; Godiska, Ronald; Tall, Gregory G; Joseph, Suresh K; Yule, David I

    2013-10-11

    Vertebrate genomes code for three subtypes of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R1, -2, and -3). Individual IP3R monomers are assembled to form homo- and heterotetrameric channels that mediate Ca(2+) release from intracellular stores. IP3R subtypes are regulated differentially by IP3, Ca(2+), ATP, and various other cellular factors and events. IP3R subtypes are seldom expressed in isolation in individual cell types, and cells often express different complements of IP3R subtypes. When multiple subtypes of IP3R are co-expressed, the subunit composition of channels cannot be specifically defined. Thus, how the subunit composition of heterotetrameric IP3R channels contributes to shaping the spatio-temporal properties of IP3-mediated Ca(2+) signals has been difficult to evaluate. To address this question, we created concatenated IP3R linked by short flexible linkers. Dimeric constructs were expressed in DT40-3KO cells, an IP3R null cell line. The dimeric proteins were localized to membranes, ran as intact dimeric proteins on SDS-PAGE, and migrated as an ∼1100-kDa band on blue native gels exactly as wild type IP3R. Importantly, IP3R channels formed from concatenated dimers were fully functional as indicated by agonist-induced Ca(2+) release. Using single channel "on-nucleus" patch clamp, the channels assembled from homodimers were essentially indistinguishable from those formed by the wild type receptor. However, the activity of channels formed from concatenated IP3R1 and IP3R2 heterodimers was dominated by IP3R2 in terms of the characteristics of regulation by ATP. These studies provide the first insight into the regulation of heterotetrameric IP3R of defined composition. Importantly, the results indicate that the properties of these channels are not simply a blend of those of the constituent IP3R monomers.

  10. Type 1 and 3 inositol trisphosphate receptors are required for extra-embryonic vascular development.

    PubMed

    Uchida, Keiko; Nakazawa, Maki; Yamagishi, Chihiro; Mikoshiba, Katsuhiko; Yamagishi, Hiroyuki

    2016-10-01

    The embryonic-maternal interface of the placental labyrinth, allantois, and yolk sac are vital during embryogenesis; however, the precise mechanism underlying the vascularization of these structures remains unknown. Herein we focus on the role of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R), which are intracellular Ca(2+) release channels, in placentation. Double knockout (DKO) of type 1 and 3 IP3Rs (IP3R1 and IP3R3, respectively) in mice resulted in embryonic lethality around embryonic day (E) 11.5. Because IP3R1 and IP3R3 were co-expressed in endothelial cells in the labyrinth, allantois, and yolk sac, we investigated extra-embryonic vascular development in IP3R1- and IP3R3-DKO mice. The formation of chorionic plates and yolk sac vessels seemed dysregulated around the timing of the chorio-allantoic attachment, immediately followed by the disorganization of allantoic vessels, the decreased expression of the spongiotrophoblast cell marker Tpbpa and the growth retardation of the embryos in DKO mice. Fluorescent immunohistochemistry demonstrated downregulation of a vascular endothelial marker, CD31, in labyrinth embryonic vessels and poor elongation of extra-embryonic mesoderm into the labyrinth layer in DKO placenta, whereas the branching of the DKO chorionic trophoblast was initiated. In addition, allantoic and yolk sac vessels in extra-embryonic tissues were less remodeled in DKO mice. In vitro endothelial cord formation and migration activities of cultured vascular endothelial cells derived from human umbilical vein were downregulated under the inhibition of IP3R. Our results suggest that IP3R1 and IP3R3 are required for extra-embryonic vascularization in the placenta, allantois, and yolk sac. This is the first demonstration of the essential role of IP3/IP3Rs signaling in the development of the vasculature at the embryonic-maternal interface.

  11. Structural Studies of Inositol 1,4,5-Trisphosphate Receptor COUPLING LIGAND BINDING TO CHANNEL GATING

    SciTech Connect

    Chan, Jenny; Yamazaki, Haruka; Ishiyama, Noboru; Seo, Min-Duk; Mal, Tapas K.; Michikawa, Takayuki; Mikoshiba, Katsuhiko; Ikura, Mitsuhiko

    2010-11-22

    The three isoforms of the inositol 1,4,5-trisphosphate receptor (IP{sub 3}R) exhibit distinct IP{sub 3} sensitivities and cooperativities in calcium (Ca{sup 2+}) channel function. The determinants underlying this isoform-specific channel gating mechanism have been localized to the N-terminal suppressor region of IP3R. We determined the 1.9 {angstrom} crystal structure of the suppressor domain from type 3 IP{sub 3}R (IP{sub 3}R3{sub SUP}, amino acids 1-224) and revealed structural features contributing to isoform-specific functionality of IP{sub 3}R by comparing it with our previously determined structure of the type 1 suppressor domain (IP{sub 3}R1{sub SUP}). The molecular surface known to associate with the ligand binding domain (amino acids 224-604) showed marked differences between IP{sub 3}R3{sub SUP} and IP{sub 3}R1{sub SUP}. Our NMR and biochemical studies showed that three spatially clustered residues (Glu-20, Tyr-167, and Ser-217 in IP{sub 3}R1 and Glu-19, Trp-168, and Ser-218 in IP{sub 3}R3) within the N-terminal suppressor domains of IP{sub 3}R1{sub SUP} and IP{sub 3}R3{sub SUP} interact directly with their respective C-terminal fragments. Together with the accompanying paper (Yamazaki, H., Chan, J., Ikura, M., Michikawa, T., and Mikoshiba, K. (2010) J. Biol. Chem. 285, 36081-36091), we demonstrate that the single aromatic residue in this region (Tyr-167 in IP{sub 3}R1 and Trp-168 in IP{sub 3}R3) plays a critical role in the coupling between ligand binding and channel gating.

  12. Enhanced Dissolution and Transformation of ZnO Nanoparticles: The Role of Inositol Hexakisphosphate.

    PubMed

    Feng, Xionghan; Yan, Yupeng; Wan, Biao; Li, Wei; Jaisi, Deb P; Zheng, Lirong; Zhang, Jing; Liu, Fan

    2016-06-01

    The toxicity, reactivity, and behavior of zinc oxide (ZnO) nanoparticles (NPs) released in the environment are highly dependent on environmental conditions. Myo-inositol hexakisphosphate (IHP), a common organic phosphate, may interact with NPs and generate new transformation products. In this study, the role of IHP in mediating the dissolution and transformation of ZnO NPs was investigated in the laboratory kinetic experiments using powder X-ray diffraction, attenuated total reflectance Fourier transform infrared spectroscopy, (31)P nuclear magnetic resonance spectroscopy, high-resolution transmission electronic microscopy, and synchrotron-based extended X-ray absorption fine structure spectroscopy. The results indicate that IHP shows a dissolution-precipitation effect, which is different from citrate and EDTA that only enhances Zn dissolution. The enhanced dissolution and transformation of ZnO NPs by IHP (<0.5 h) is found to be strikingly faster than that induced by inorganic phosphate (Pi, > 3.0 h) at pH 7.0, and the reaction rate increases with decreasing pH and increasing IHP concentration. Multitechnique analyses reveal that interaction of ZnO NPs with IHP induces rapid transformation of ZnO NPs into zinc phytate complexes initially and poorly crystalline zinc phytate-like (Zn-IHP) phase finally. Additionally, ZnO NPs preferentially react with IHP and transform to Zn-IHP when Pi and IHP concurrently coexist in a system. Overall, results from this study contribute to an improved understanding of the role of organic phosphates (e.g., IHP) in the speciation and structural transformation of ZnO NPs, which can be leveraged for remediation of ZnO-polluted water and soils. PMID:27159895

  13. Hydrogen peroxide down-regulates inositol 1,4,5-trisphosphate receptor content through proteasome activation.

    PubMed

    Martín-Garrido, A; Boyano-Adánez, M C; Alique, M; Calleros, L; Serrano, I; Griera, M; Rodríguez-Puyol, D; Griendling, K K; Rodríguez-Puyol, M

    2009-11-15

    Hydrogen peroxide (H(2)O(2)) is implicated in the regulation of signaling pathways leading to changes in vascular smooth muscle function. Contractile effects produced by H(2)O(2) are due to the phosphorylation of myosin light chain kinase triggered by increases in intracellular calcium (Ca(2+)) from intracellular stores or influx of extracellular Ca(2+). One mechanism for mobilizing such stores involves the phosphoinositide pathway. Inositol 1,4,5-trisphosphate (IP(3)) mobilizes intracellular Ca(2+) by binding to a family of receptors (IP(3)Rs) on the endoplasmic-sarcoplasmic reticulum that act as ligand-gated Ca(2+) channels. IP(3)Rs can be rapidly ubiquitinated and degraded by the proteasome, causing a decrease in cellular IP(3)R content. In this study we show that IP(3)R(1) and IP(3)R(3) are down-regulated when vascular smooth muscle cells (VSMC) are stimulated by H(2)O(2), through an increase in proteasome activity. Moreover, we demonstrate that the decrease in IP(3)R by H(2)O(2) is accompanied by a reduction in calcium efflux induced by IP(3) in VSMC. Also, we observed that angiotensin II (ANGII) induces a decrease in IP(3)R by activation of NADPH oxidase and that preincubation with H(2)O(2) decreases ANGII-mediated calcium efflux and planar cell surface area in VSMC. The decreased IP(3) receptor content observed in cells was also found in aortic rings, which exhibited a decreased ANGII-dependent contraction after treatment with H(2)O(2). Altogether, these results suggest that H(2)O(2) mediates IP(3)R down-regulation via proteasome activity.

  14. Effects of zonisamide on neurotransmitter release associated with inositol triphosphate receptors.

    PubMed

    Yamamura, Satoshi; Saito, Hiromitsu; Suzuki, Noboru; Kashimoto, Sanae; Hamaguchi, Tatsuya; Ohoyama, Keiko; Suzuki, Dai; Kanehara, Shinich; Nakagawa, Masanori; Shiroyama, Takashi; Okada, Motohiro

    2009-04-17

    To clarify the antiepileptic mechanisms of zonisamide (ZNS), we determined the interaction between ZNS and inositol-1,4,5-triphosphate receptor (IP3R) on exocytosis of GABA and glutamate in rat frontal cortex using microdialysis. ZNS increased basal GABA release, but not glutamate, concentration-dependently, and reduced concentration-dependently K(+)-evoked GABA and glutamate releases. Inhibition and activation of IP3R reduced and enhanced basal and K(+)-evoked GABA releases, respectively. The K(+)-evoked glutamate release was reduced and enhanced by IP3R antagonist and agonist, respectively, whereas basal glutamate release was increased by IP3R agonist but not affected by IP3R antagonist. Under extracellular Ca(2+) depletion, IP3R agonist increased basal GABA and glutamate releases. The latter effects of IP3R agonist were weakly enhanced by ZNS, but such stimulatory action of ZNS was abolished by extracellular Ca(2+) depletion. In contrast, ZNS inhibited the stimulatory effect of IP3R agonist on K(+)-evoked release. The stimulatory effect of IP3R agonist on basal release was regulated by N-type voltage-sensitive Ca(2+) channel (VSCC) rather than P- and L-type VSCCs, whereas the stimulatory effect of IP3R agonist on K(+)-evoked release was regulated by P- and L-type VSCCs rather than N-type VSCC. These results suggest that ZNS-activated N-type VSCC enhances IP3R-associated neurotransmitter release during resting stage, whereas ZNS-induced suppression of P- and L-type VSCCs possibly attenuates IP3R-associated neurotransmitter release during neuronal hyperexcitability. Therefore, the combination of both of these two actions of ZNS on IP3R-associated neurotransmitter release mechanism seems to be involved, at least in part, in the mechanisms of antiepileptic and neuroprotective actions of ZNS.

  15. Effects of Inositol 1,4,5-triphosphate on Osteoclast Differentiation in RANKL-induced Osteoclastogenesis.

    PubMed

    Son, Aran; Kim, Min Seuk; Jo, Hae; Byun, Hae Mi; Shin, Dong Min

    2012-02-01

    The receptor activator of NF-κB ligand (RANKL) signal is an activator of tumor necrosis factor receptor-associated factor 6 (TRAF6), which leads to the activation of NF-κB and other signal transduction pathways essential for osteoclastogenesis, such as Ca(2+) signaling. However, the intracellular levels of inositol 1,4,5-trisphosphate (IP(3)) and IP(3)-mediated cellular function of RANKL during osteoclastogenesis are not known. In the present study, we determined the levels of IP(3) and evaluated IP(3)-mediated osteoclast differentiation and osteoclast activity by RANKL treatment of mouse leukemic macrophage cells (RAW 264.7) and mouse bone marrow-derived monocyte/macrophage precursor cells (BMMs). During osteoclastogenesis, the expression levels of Ca(2+) signaling proteins such as IP(3) receptors (IP(3)Rs), plasma membrane Ca(2+) ATPase, and sarco/endoplasmic reticulum Ca(2+) ATPase type2 did not change by RANKL treatment for up to 6 days in both cell types. At 24 h after RANKL treatment, a higher steady-state level of IP(3) was observed in RAW264.7 cells transfected with green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains of phospholipase C (PLC) δ, a probe specifically detecting intracellular IP(3) levels. In BMMs, the inhibition of PLC with U73122 [a specific inhibitor of phospholipase C (PLC)] and of IP(3)Rs with 2-aminoethoxydiphenyl borate (2APB; a non-specific inhibitor of IP(3)Rs) inhibited the generation of RANKL-induced multinucleated cells and decreased the bone-resorption rate in dentin slice, respectively. These results suggest that intracellular IP(3) levels and the IP(3)-mediated signaling pathway play an important role in RANKL-induced osteoclastogenesis.

  16. Inositol 1,4,5-trisphosphate receptor-isoform diversity in cell death and survival.

    PubMed

    Ivanova, Hristina; Vervliet, Tim; Missiaen, Ludwig; Parys, Jan B; De Smedt, Humbert; Bultynck, Geert

    2014-10-01

    Cell-death and -survival decisions are critically controlled by intracellular Ca(2+) homeostasis and dynamics at the level of the endoplasmic reticulum (ER). Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) play a pivotal role in these processes by mediating Ca(2+) flux from the ER into the cytosol and mitochondria. Hence, it is clear that many pro-survival and pro-death signaling pathways and proteins affect Ca(2+) signaling by directly targeting IP3R channels, which can happen in an IP3R-isoform-dependent manner. In this review, we will focus on how the different IP3R isoforms (IP3R1, IP3R2 and IP3R3) control cell death and survival. First, we will present an overview of the isoform-specific regulation of IP3Rs by cellular factors like IP3, Ca(2+), Ca(2+)-binding proteins, adenosine triphosphate (ATP), thiol modification, phosphorylation and interacting proteins, and of IP3R-isoform specific expression patterns. Second, we will discuss the role of the ER as a Ca(2+) store in cell death and survival and how IP3Rs and pro-survival/pro-death proteins can modulate the basal ER Ca(2+) leak. Third, we will review the regulation of the Ca(2+)-flux properties of the IP3R isoforms by the ER-resident and by the cytoplasmic proteins involved in cell death and survival as well as by redox regulation. Hence, we aim to highlight the specific roles of the various IP3R isoforms in cell-death and -survival signaling. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

  17. CaMKII regulation of cardiac ryanodine receptors and inositol triphosphate receptors.

    PubMed

    Camors, Emmanuel; Valdivia, Héctor H

    2014-01-01

    Ryanodine receptors (RyRs) and inositol triphosphate receptors (InsP3Rs) are structurally related intracellular calcium release channels that participate in multiple primary or secondary amplified Ca(2+) signals, triggering muscle contraction and oscillatory Ca(2+) waves, or activating transcription factors. In the heart, RyRs play an indisputable role in the process of excitation-contraction coupling as the main pathway for Ca(2+) release from sarcoplasmic reticulum (SR), and a less prominent role in the process of excitation-transcription coupling. Conversely, InsP3Rs are believed to contribute in subtle ways, only, to contraction of the heart, and in more important ways to regulation of transcription factors. Because uncontrolled activity of either RyRs or InsP3Rs may elicit life-threatening arrhythmogenic and/or remodeling Ca(2+) signals, regulation of their activity is of paramount importance for normal cardiac function. Due to their structural similarity, many regulatory factors, accessory proteins, and post-translational processes are equivalent for RyRs and InsP3Rs. Here we discuss regulation of RyRs and InsP3Rs by CaMKII phosphorylation, but touch on other kinases whenever appropriate. CaMKII is emerging as a powerful modulator of RyR and InsP3R activity but interestingly, some of the complexities and controversies surrounding phosphorylation of RyRs also apply to InsP3Rs, and a clear-cut effect of CaMKII on either channel eludes investigators for now. Nevertheless, some effects of CaMKII on global cellular activity, such as SR Ca(2+) leak or force-frequency potentiation, appear clear now, and this constrains the limits of the controversies and permits a more tractable approach to elucidate the effects of phosphorylation at the single channel level.

  18. Desensitization of histamine H1 receptor-mediated inositol phosphate production in HeLa cells.

    PubMed Central

    Bristow, D. R.; Zamani, M. R.

    1993-01-01

    1. Histamine stimulated the accumulation of total [3H]-inositol phosphates (IPn) in control HeLa cells with an EC50 of 3.7 +/- 0.7 microM in the presence of 10 mM LiCl. The maximum response to histamine after 15 min incubation was 43 +/- 5% over basal accumulation and occurred at a concentration of 1 mM histamine. 2. The histamine-induced IPn production in HeLa cells was confirmed as H1 receptor-mediated, since the H1 antagonist mepyramine (10(-6) M) inhibited the histamine response (10(-4) M) by 83 +/- 7%, whereas the H2 antagonist, ranitidine (10(-4) M), and H3 antagonist, thioperamide (10(-6) M), were ineffective. 3. Histamine (10(-4) M) pretreatment of HeLa cells for 30 min desensitized the subsequent histamine-induced IPn accumulation. The desensitized cells accumulated IPn in response to histamine with an EC50 of 1.7 +/- 0.7 microM after 15 min incubation. The maximum histamine-induced IPn accumulation at 10(-4) M was 19 +/- 5% over basal and was significantly lower (P < 0.03) than the maximum response in control cells. 4. The desensitization of histamine-induced IPn accumulation was time-dependent and, at a desensitizing histamine concentration of 10(-4) M, the half-maximal attenuation occurred after approximately 9 min and maximum desensitization was achieved by 15-20 min. The desensitization of the IPn accumulation was a reversible phenomenon and full recovery of the response occurred 150 min after the removal of the desensitizing histamine-containing medium. The half-time for the recovery of the histamine-induced response was estimated at 120 min.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8358540

  19. Molecular target sizes of inositol 1,4,5-trisphosphate receptors in liver and cerebellum.

    PubMed Central

    Nunn, D L; Potter, B V; Taylor, C W

    1990-01-01

    Ins(1,4,5)P3 is the intracellular messenger that mediates the effects of many cell-surface receptors on intracellular Ca2+ stores. Although radioligand-binding studies have identified high-affinity Ins(1,4,5)P3-binding sites in many tissues, these have not yet been convincingly shown to be the receptors that mediate Ca2+ mobilization, nor is it clear whether there are differences in these binding sites between tissues. Here we report that Ins(1,4,5)P3 binds to a single class of high-affinity sites in both permeabilized hepatocytes (KD = 7.8 +/- 1.1 nM) and cerebellar membranes (KD = 6.5 +/- 2.4 nM), and provide evidence that these are unlikely to reflect binding to either of the enzymes known to metabolize Ins(1,4,5)P3. Furthermore, the rank order of potency of synthetic inositol phosphate analogues in displacing specifically bound Ins(1,4,5)P3 is the same as their rank order of potency in stimulating mobilization of intracellular Ca2+ stores, suggesting that the Ins(1,4,5)P3-binding site may be the physiological receptor. Radiation inactivation of the Ins(1,4,5)P3-binding sites of liver and cerebellum reveals that they have similar molecular target sizes: 257 +/- 36 kDa in liver and 258 +/- 20 kDa in cerebellum. We conclude that an Ins(1,4,5)P3-binding protein with a molecular target size of about 260 kDa is probably the receptor that mediates Ca2+ mobilization in hepatocytes, and our limited data provide no evidence to distinguish this from the cerebellar Ins(1,4,5)P3-binding protein. PMID:2154187

  20. Identification of a gene cluster enabling Lactobacillus casei BL23 to utilize myo-inositol.

    PubMed

    Yebra, María Jesús; Zúñiga, Manuel; Beaufils, Sophie; Pérez-Martínez, Gaspar; Deutscher, Josef; Monedero, Vicente

    2007-06-01

    Genome analysis of Lactobacillus casei BL23 revealed that, compared to L. casei ATCC 334, it carries a 12.8-kb DNA insertion containing genes involved in the catabolism of the cyclic polyol myo-inositol (MI). Indeed, L. casei ATCC 334 does not ferment MI, whereas strain BL23 is able to utilize this carbon source. The inserted DNA consists of an iolR gene encoding a DeoR family transcriptional repressor and a divergently transcribed iolTABCDG1G2EJK operon, encoding a complete MI catabolic pathway, in which the iolK gene probably codes for a malonate semialdehyde decarboxylase. The presence of iolK suggests that L. casei has two alternative pathways for the metabolism of malonic semialdehyde: (i) the classical MI catabolic pathway in which IolA (malonate semialdehyde dehydrogenase) catalyzes the formation of acetyl-coenzyme A from malonic semialdehyde and (ii) the conversion of malonic semialdehyde to acetaldehyde catalyzed by the product of iolK. The function of the iol genes was verified by the disruption of iolA, iolT, and iolD, which provided MI-negative strains. By contrast, the disruption of iolK resulted in a strain with no obvious defect in MI utilization. Transcriptional analyses conducted with different mutant strains showed that the iolTABCDG1G2EJK cluster is regulated by substrate-specific induction mediated by the inactivation of the transcriptional repressor IolR and by carbon catabolite repression mediated by the catabolite control protein A (CcpA). This is the first example of an operon for MI utilization in lactic acid bacteria and illustrates the versatility of carbohydrate utilization in L. casei BL23. PMID:17449687

  1. Neutrophil cathepsin G increases calcium flux and inositol polyphosphate production in cultured endothelial cells

    SciTech Connect

    Peterson, M.W.; Gruenhaupt, D.; Shasby, D.M. )

    1989-07-15

    Exposure of endothelial cells (ENDO) to human neutrophil cathepsin G (CG) increases albumin flux across the endothelial monolayer. Since calcium influences cell shape and barrier function of ENDO monolayers, the current study was designed to determine if CG acted through alterations in Ca2+ homeostasis in ENDO. The role of Ca2+ in the increased permeability of ENDO monolayers to albumin after exposure to CG was studied by using ENDO monolayers cultured on polycarbonate filters. Exposure of ENDO monolayers to CG in the presence of the Ca2+-antagonist lanthanum partially prevented the increase in albumin flux, but exposure in the presence of agents that block voltage-regulated calcium channels did not block the increase in albumin flux. To monitor the effect of CG on Ca2+-flux, ENDO were labeled with {sup 45}Ca2+ and changes in Ca2+ flux were monitored by the release of {sup 45}Ca2+. From 1 to 15 minutes after exposure of ENDO to CG, there was increased release of {sup 45}Ca2+ compared with control cells. Calcium channel blocking agents did not inhibit the increased release of {sup 45}Ca2+, but lanthanum partially blocked the increase. The increased release of Ca2+ appeared to be due, at least in part, to activation of phospholipase C because there was an increase both in inositol polyphosphate species and in diglycerides after incubation of ENDO with CG. These studies support the hypothesis that CG increases the flux of calcium in ENDO, that this increase in Ca2+ flux may result from activation of phospholipase C, and that this system may be involved in the decreased barrier properties of the ENDO after CG exposure.

  2. Matrix Domain Modulates HIV-1 Gag's Nucleic Acid Chaperone Activity via Inositol Phosphate Binding ▿

    PubMed Central

    Jones, Christopher P.; Datta, Siddhartha A. K.; Rein, Alan; Rouzina, Ioulia; Musier-Forsyth, Karin

    2011-01-01

    Retroviruses replicate by reverse transcribing their single-stranded RNA genomes into double-stranded DNA using specific cellular tRNAs to prime cDNA synthesis. In HIV-1, human tRNA3Lys serves as the primer and is packaged into virions during assembly. The viral Gag protein is believed to chaperone tRNA3Lys placement onto the genomic RNA primer binding site; however, the timing and possible regulation of this event are currently unknown. Composed of the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains, the multifunctional HIV-1 Gag polyprotein orchestrates the highly coordinated process of virion assembly, but the contribution of these domains to tRNA3Lys annealing is unclear. Here, we show that NC is absolutely essential for annealing and that the MA domain inhibits Gag's tRNA annealing capability. During assembly, MA specifically interacts with inositol phosphate (IP)-containing lipids in the plasma membrane (PM). Surprisingly, we find that IPs stimulate Gag-facilitated tRNA annealing but do not stimulate annealing in Gag variants lacking the MA domain or containing point mutations involved in PM binding. Moreover, we find that IPs prevent MA from binding to nucleic acids but have little effect on NC or Gag. We propose that Gag binds to RNA either with both NC and MA domains or with NC alone and that MA-IP interactions alter Gag's binding mode. We propose that MA's interactions with the PM trigger the switch between these two binding modes and stimulate Gag's chaperone function, which may be important for the regulation of events such as tRNA primer annealing. PMID:21123373

  3. Effects of Inositol 1,4,5-triphosphate on Osteoclast Differentiation in RANKL-induced Osteoclastogenesis

    PubMed Central

    Son, Aran; Kim, Min Seuk; Jo, Hae; Byun, Hae Mi

    2012-01-01

    The receptor activator of NF-κB ligand (RANKL) signal is an activator of tumor necrosis factor receptor-associated factor 6 (TRAF6), which leads to the activation of NF-κB and other signal transduction pathways essential for osteoclastogenesis, such as Ca2+ signaling. However, the intracellular levels of inositol 1,4,5-trisphosphate (IP3) and IP3-mediated cellular function of RANKL during osteoclastogenesis are not known. In the present study, we determined the levels of IP3 and evaluated IP3-mediated osteoclast differentiation and osteoclast activity by RANKL treatment of mouse leukemic macrophage cells (RAW 264.7) and mouse bone marrow-derived monocyte/macrophage precursor cells (BMMs). During osteoclastogenesis, the expression levels of Ca2+ signaling proteins such as IP3 receptors (IP3Rs), plasma membrane Ca2+ ATPase, and sarco/endoplasmic reticulum Ca2+ ATPase type2 did not change by RANKL treatment for up to 6 days in both cell types. At 24 h after RANKL treatment, a higher steady-state level of IP3 was observed in RAW264.7 cells transfected with green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains of phospholipase C (PLC) δ, a probe specifically detecting intracellular IP3 levels. In BMMs, the inhibition of PLC with U73122 [a specific inhibitor of phospholipase C (PLC)] and of IP3Rs with 2-aminoethoxydiphenyl borate (2APB; a non-specific inhibitor of IP3Rs) inhibited the generation of RANKL-induced multinucleated cells and decreased the bone-resorption rate in dentin slice, respectively. These results suggest that intracellular IP3 levels and the IP3-mediated signaling pathway play an important role in RANKL-induced osteoclastogenesis. PMID:22416217

  4. Crosstalks between Myo-Inositol Metabolism, Programmed Cell Death and Basal Immunity in Arabidopsis

    PubMed Central

    Tcherkez, Guillaume; Blanchet, Sophie; Massoud, Kamal; Domenichini, Séverine; Henry, Yves; Soubigou-Taconnat, Ludivine; Lelarge-Trouverie, Caroline; Saindrenan, Patrick; Renou, Jean Pierre; Bergounioux, Catherine

    2009-01-01

    Background Although it is a crucial cellular process required for both normal development and to face stress conditions, the control of programmed cell death in plants is not fully understood. We previously reported the isolation of ATXR5 and ATXR6, two PCNA-binding proteins that could be involved in the regulation of cell cycle or cell death. A yeast two-hybrid screen using ATXR5 as bait captured AtIPS1, an enzyme which catalyses the committed step of myo-inositol (MI) biosynthesis. atips1 mutants form spontaneous lesions on leaves, raising the possibility that MI metabolism may play a role in the control of PCD in plants. In this work, we have characterised atips1 mutants to gain insight regarding the role of MI in PCD regulation. Methodology/Principal Findings - lesion formation in atips1 mutants depends of light intensity, is due to PCD as evidenced by TUNEL labelling of nuclei, and is regulated by phytohormones such as salicylic acid - MI and galactinol are the only metabolites whose accumulation is significantly reduced in the mutant, and supplementation of the mutant with these compounds is sufficient to prevent PCD - the transcriptome profile of the mutant is extremely similar to that of lesion mimic mutants such as cpr5, or wild-type plants infected with pathogens. Conclusion/Significance Taken together, our results provide strong evidence for the role of MI or MI derivatives in the regulation of PCD. Interestingly, there are three isoforms of IPS in Arabidopsis, but AtIPS1 is the only one harbouring a nuclear localisation sequence, suggesting that nuclear pools of MI may play a specific role in PCD regulation and opening new research prospects regarding the role of MI in the prevention of tumorigenesis. Nevertheless, the significance of the interaction between AtIPS1 and ATXR5 remains to be established. PMID:19812700

  5. Raising the intracellular level of inositol 1,4,5-trisphosphate changes plasma membrane ion transport in characean algae.

    PubMed Central

    Thiel, G; MacRobbie, E A; Hanke, D E

    1990-01-01

    Inositol 1,4,5-trisphosphate (InsP3) was introduced into the cytoplasm of characean algae in two different ways: (i) by iontophoretic injection into cytoplasm-enriched fragments from Chara and (ii) by adding InsP3 to the permeabilization medium of locally permeabilized cells of Nitella. In both systems this operation induced a depolarization of the membrane potential, ranging from a few mV to sequences of action potentials. The effect of InsP3 on locally permeabilized Nitella cells was abolished when InsP3 was added together with 30 mM EGTA. When inositol 1,4-bisphosphate or myo-inositol were substituted for InsP3 in this system, there was no change in the membrane potential. On the other hand, increasing the free Ca2+ concentration in the permeabilization medium induced, in a similar fashion to InsP3, action potentials. Similarities between InsP3 and Ca2+ action were also observed upon injection into Chara fragments. Both injections increased an inward current. In the first few seconds after injection the current/voltage characteristics of the InsP3-induced current resembled those of the Ca2(+)-sensitive current. Subsequently, differences between the InsP3- and Ca2(+)-induced phenomena became apparent in that the InsP3-induced current continued to increase while the Ca2(+)-induced current declined, returning to the resting level. Our results suggest that these plant cells contain an InsP3 sensitive system that, under experimental conditions, is able to affect membrane transport via an increase in cytoplasmic free Ca2+. PMID:2112084

  6. Guanine nucleotide regulation of muscarinic receptor-mediated inositol phosphate formation in permeabilized 1321N1 cells

    SciTech Connect

    Orellana, S.A.; Trilivas, I.; Brown, J.H.

    1986-03-05

    Carbachol and guanine nucleotides stimulate formation of the (/sup 3/H)inositol phosphates IP, IP2, and IP3 in saponin-permeabilized monolayers labelled with (/sup 3/H) inositol. Carbachol alone has little effect on formation of the (/sup 3/H) inositol phosphates (IPs), but GTP..gamma..S causes synergistic accumulation of (/sup 3/H)IPs to levels similar to those seen in intact cells. GTP, GppNHp, and GTP..gamma..S all support formation of the (/sup 3/H)IPs, with or without hormone, but GTP..gamma..S is the most effective. In the presence of GTP..gamma..S, the effect of carbachol is dose-dependent. Half-maximal and maximal accumulation of the (/sup 3/H)IPs occur at approx. 5 ..mu..M and approx. 100 ..mu..M carbachol, respectively; values close to those seen in intact cells. GTP..gamma..S alone stimulates formation of the (/sup 3/H)IPs after a brief lag time. The combination of GTP..gamma..S and carbachol both increases the rate of, and decreases the lag in, formation of the (/sup 3/H)IPs. LiCl increases (/sup 3/H)IP and IP2, but not IP3, accumulation; while 2,3-diphosphoglycerate substantially increases that of (/sup 3/H)IP3. GTP..gamma..S and carbachol cause formation of (/sup 3/H)IPs in the absence of Ca/sup + +/, but formation induced by GTP..gamma..S with or without carbachol is Ca/sup + +/-sensitive over a range of physiological concentrations. Although carbachol, Ca/sup + +/, and GTP..gamma..S all have effects on formation of (/sup 3/H)IPs, GTP..gamma..S appears to be a primary and obligatory regulator of phosphoinositide hydrolysis in the permeabilized 1321N1 astrocytoma cell.

  7. Regioselective phosphorylation of myo-inositol with BINOL-derived phosphoramidites and its application for protozoan lysophosphatidylinositol.

    PubMed

    Aiba, Toshihiko; Sato, Masaki; Umegaki, Daichi; Iwasaki, Takanori; Kambe, Nobuaki; Fukase, Koichi; Fujimoto, Yukari

    2016-07-12

    A regioselective phosphorylation method for myo-inositol was developed by utilizing readily preparable BINOL-derived phosphoramidites. The method also facilitated the complete separation of the diastereomeric products by simple chromatography. Based on this phosphorylation and Ni-catalyzed alkyl-alkyl cross-coupling reaction for long fatty acids, we achieved the first synthesis of a lysophosphatidylinositol, EhPIa having long fatty acid C30:1, as a partial structure of glycosylphosphatidylinositol (GPI) anchor from the cell membrane of a protozoa, Entamoeba histolytica. PMID:27326923

  8. Haplofungins, novel inositol phosphorylceramide synthase inhibitors, from Lauriomyces bellulus SANK 26899 I. Taxonomy, fermentation, isolation and biological activities.

    PubMed

    Ohnuki, Takashi; Yano, Tatsuya; Ono, Yasunori; Kozuma, Shiho; Suzuki, Toshihiro; Ogawa, Yasumasa; Takatsu, Toshio

    2009-10-01

    In the course of screening for antifungal agents, we have discovered eight novel compounds, haplofungin A, B, C, D, E, F, G and H, from a culture broth of the fungus strain Lauriomyces bellulus SANK 26899. Haplofungins are composed of an arabinonic acid moiety linked through an ester to a modified long alkyl chain and show potent inhibitory activities against fungal inositol phosphorylceramide (IPC) synthase. Haplofungin A inhibited the activity of IPC synthase from Saccharomyces cerevisiae with an IC(50) value of 0.0015 microg ml(-1). This inhibitor also suppressed the growth of Candida glabrata at the MIC value of 0.5 microg ml(-1).

  9. A myo-inositol-1-phosphate synthase gene, IbMIPS1, enhances salt and drought tolerance and stem nematode resistance in transgenic sweet potato.

    PubMed

    Zhai, Hong; Wang, Feibing; Si, Zengzhi; Huo, Jinxi; Xing, Lei; An, Yanyan; He, Shaozhen; Liu, Qingchang

    2016-02-01

    Myo-inositol-1-phosphate synthase (MIPS) is a key rate limiting enzyme in myo-inositol biosynthesis. The MIPS gene has been shown to improve tolerance to abiotic stresses in several plant species. However, its role in resistance to biotic stresses has not been reported. In this study, we found that expression of the sweet potato IbMIPS1 gene was induced by NaCl, polyethylene glycol (PEG), abscisic acid (ABA) and stem nematodes. Its overexpression significantly enhanced stem nematode resistance as well as salt and drought tolerance in transgenic sweet potato under field conditions. Transcriptome and real-time quantitative PCR analyses showed that overexpression of IbMIPS1 up-regulated the genes involved in inositol biosynthesis, phosphatidylinositol (PI) and ABA signalling pathways, stress responses, photosynthesis and ROS-scavenging system under salt, drought and stem nematode stresses. Inositol, inositol-1,4,5-trisphosphate (IP3 ), phosphatidic acid (PA), Ca(2+) , ABA, K(+) , proline and trehalose content was significantly increased, whereas malonaldehyde (MDA), Na(+) and H2 O2 content was significantly decreased in the transgenic plants under salt and drought stresses. After stem nematode infection, the significant increase of inositol, IP3 , PA, Ca(2+) , ABA, callose and lignin content and significant reduction of MDA content were found, and a rapid increase of H2 O2 levels was observed, peaked at 1 to 2 days and thereafter declined in the transgenic plants. This study indicates that the IbMIPS1 gene has the potential to be used to improve the resistance to biotic and abiotic stresses in plants. PMID:26011089

  10. H+/myo-inositol transporter genes, hmit-1.1 and hmit-1.2, have roles in the osmoprotective response in Caenorhabditis elegans.

    PubMed

    Kage-Nakadai, Eriko; Uehara, Tomoko; Mitani, Shohei

    2011-07-01

    Myo-inositol is one of the major organic osmolytes in the brain and the kidney. The accumulation of intracellular organic osmolytes allows cells to regulate intracellular osmolality without altering cytoplasmic ionic strength and to adapt to hyperosmotic conditions. Two types of myo-inositol transporters, sodium/myo-inositol transporter and H(+)/myo-inositol transporter (HMIT), have been identified. Sodium/myo-inositol transporters are induced by osmotic stress and might be involved in the intracellular accumulation of myo-inositol in mammals. The role of HMIT, however, remains unknown. In the present study, we characterized three Caenorhabditis elegansHMIT genes, hmit-1.1, hmit-1.2, and hmit-1.3. hmit-1.1 was expressed in the intestine, and hmit-1.2 was expressed in the glia and the excretory canal, which is an osmotic regulatory organ that is functionally analogous to the kidney. hmit-1.3 was expressed in the intestine and the glia. The expression of hmit-1.1 and hmit-1.2 but not hmit-1.3, was markedly induced under hyperosmotic conditions. Animals with mutant hmit-1.1 and hmit-1.2 were hypersensitive to osmotic stress. The defects of hmit-1.1 and hmit-1.2 mutants were rescued by hmit-1.1 and hmit-1.2 transgenes, respectively, and by modified human HMIT. In human cell lines, HMIT expression was induced in hyperosmotic conditions. These findings indicate that the C. elegans HMIT family has a crucial role in the osmoprotective response.

  11. d-myo-Inositol-3-Phosphate Affects Phosphatidylinositol-Mediated Endomembrane Function in Arabidopsis and Is Essential for Auxin-Regulated Embryogenesis[W][OA

    PubMed Central

    Luo, Yu; Qin, Genji; Zhang, Jun; Liang, Yuan; Song, Yingqi; Zhao, Meiping; Tsuge, Tomohiko; Aoyama, Takashi; Liu, Jingjing; Gu, Hongya; Qu, Li-Jia

    2011-01-01

    In animal cells, myo-inositol is an important regulatory molecule in several physiological and biochemical processes, including signal transduction and membrane biogenesis. However, the fundamental biological functions of myo-inositol are still far from clear in plants. Here, we report the genetic characterization of three Arabidopsis thaliana genes encoding d-myo-inositol-3-phosphate synthase (MIPS), which catalyzes the rate-limiting step in de novo synthesis of myo-inositol. Each of the three MIPS genes rescued the yeast ino1 mutant, which is defective in yeast MIPS gene INO1, and they had different dynamic expression patterns during Arabidopsis embryo development. Although single mips mutants showed no obvious phenotypes, the mips1 mips2 double mutant and the mips1 mips2 mips3 triple mutant were embryo lethal, whereas the mips1 mips3 and mips1 mips2+/− double mutants had abnormal embryos. The mips phenotypes resembled those of auxin mutants. Indeed, the double and triple mips mutants displayed abnormal expression patterns of DR5:green fluorescent protein, an auxin-responsive fusion protein, and they had altered PIN1 subcellular localization. Also, membrane trafficking was affected in mips1 mips3. Interestingly, overexpression of PHOSPHATIDYLINOSITOL SYNTHASE2, which converts myo-inositol to membrane phosphatidylinositol (PtdIns), largely rescued the cotyledon and endomembrane defects in mips1 mips3. We conclude that myo-inositol serves as the main substrate for synthesizing PtdIns and phosphatidylinositides, which are essential for endomembrane structure and trafficking and thus for auxin-regulated embryogenesis. PMID:21505066

  12. Immobilization of Candida krusei cells producing phytase in alginate gel beads: an application of the preparation of myo-inositol phosphates.

    PubMed

    Quan, C S; Fan, S D; Ohta, Y

    2003-07-01

    Cells of Candida krusei capable of producing phytase were immobilized in Ca-alginate gel beads and used for the preparation of myo-inositol phosphates. The immobilization yield was increased about 5-fold after the beads were treated for 96 h at pH 4.0, 4 degrees C. The increased yield was retained, even after 1 month, when the cells were kept at this temperature and pH. No shift in the pH optima of phytase of the immobilized cells was observed, compared with that of free cells. However, the optimum temperature for the enzyme of the immobilized cells was 55 degrees C, which was 15 degrees C higher than that of free cells. The degradation characteristics of the phytate in immobilized cells packed in a glass column (i.d. 1.2 cm, length 20 cm) were investigated. The variation in the composition of the products results from a change in the flow rate of phytate solution (5 mM). At a flow rate of 1.30 ml/min, a mixture of myo-inositol-2-monophosphate, myo-inositol-1,2,5-triphosphate and myo-inositol-1,2,5,6-tetrakisphosphate was produced, in which the latter two were physiologically active. Also, it was found by NMR analysis that the enzyme of this strain produced only one isomer of each of the inositol phosphates, with the exception of myo-inositol pentakisphosphate. Therefore, the pure isomers were easily isolated using ion-exchange chromatography.

  13. Biosensors to measure inositol 1,4,5-trisphosphate concentration in living cells with spatiotemporal resolution.

    PubMed

    Remus, Timothy P; Zima, Aleksey V; Bossuyt, Julie; Bare, Dan J; Martin, Jody L; Blatter, Lothar A; Bers, Donald M; Mignery, Gregory A

    2006-01-01

    Phosphoinositides participate in many signaling cascades via phospholipase C stimulation, which hydrolyzes phosphatidylinositol 4,5-bisphosphate, producing second messengers diacylglycerol and inositol 1,4,5-trisphosphate (InsP3). Destructive chemical approaches required to measure [InsP3] limit spatiotemporal understanding of subcellular InsP3 signaling. We constructed novel fluorescence resonance energy transfer-based InsP3 biosensors called FIRE (fluorescent InsP3-responsive element) by fusing plasmids encoding the InsP3-binding domain of InsP3 receptors (types 1-3) between cyan fluorescent protein and yellow fluorescent protein sequences. FIRE was expressed and characterized in COS-1 cells, cultured neonatal cardiac myocytes, and incorporated into an adenoviral vector for expression in adult cardiac ventricular myocytes. FIRE-1 exhibits an approximately 11% increase in the fluorescence ratio (F530/F480) at saturating [InsP3] (apparent K(d) = 31.3 +/- 6.7 nm InsP3). In COS-1 cells, neonatal rat cardiac myocytes and adult cat ventricular myocytes FIRE-1 exhibited comparable dynamic range and a 10% increase in donor (cyan fluorescent protein) fluorescence upon bleach of yellow fluorescent protein, indicative of fluorescence resonance energy transfer. In FIRE-1 expressing ventricular myocytes endothelin-1, phenylephrine, and angiotensin II all produced rapid and spatially resolved increases in [InsP3] using confocal microscopy (with free [InsP3] rising to approximately 30 nm). Local entry of intracellular InsP3 via membrane rupture by a patch pipette (containing InsP3)in myocytes expressing FIRE-1 allowed detailed spatiotemporal monitoring of intracellular InsP3 diffusion. Both endothelin-1-induced and direct InsP3 application (via pipette rupture) revealed that InsP3 diffusion into the nucleus occurs with a delay and blunted rise of [InsP3] versus cytosolic [InsP3]. These new biosensors allow studying InsP3 dynamics at high temporal and spatial resolution that

  14. Maturational regulation of inositol 1,4,5-trisphosphate metabolism in rabbit airway smooth muscle.

    PubMed Central

    Rosenberg, S M; Berry, G T; Yandrasitz, J R; Grunstein, M M

    1991-01-01

    Airway reactivity has been shown to vary with age; however, the mechanism(s) underlying this process remain unidentified. To elucidate the role of ontogenetic changes in phosphoinositide-linked signal transduction, we examined whether age-related differences in tracheal smooth muscle (TSM) contractility to carbachol (CCh) are associated with developmental changes in the production and metabolism of the second messenger, inositol 1,4,5-trisphosphate (Ins (1,4,5)P3). In TSM segments isolated from 2-wk-old and adult rabbits, both the maximal isometric contractile force and sensitivity (i.e., -logED50) to CCh (10(-10)-10(-4) M) were significantly greater in the immature vs. adult tissues (P less than 0.001). Similarly, Ins(1,4,5)P3 accumulation elicited by either receptor-coupled stimulation with CCh (10(-10)-10(-4) M) or post-receptor-mediated guanine nucleotide binding protein activation of permeabilized TSM with GTP gamma S (100 microM) was also significantly enhanced in 2-wk-old vs. adult TSM. Measurement of the activities of the degradative enzymes for Ins(1,4,5)P3 demonstrated that: (a) mean +/- SE maximal Ins(1,4,5)P3 3'-kinase activity was significantly reduced in the immature vs. adult TSM (i.e., approximately 71.7 +/- 6.0 vs. 137.8 +/- 10.0 pmol/min per mg protein, respectively; P less than 0.005); (b) by contrast, maximal Ins(1,4,5)P3 5'-phosphatase activity was significantly increased in the immature vs. adult TSM (i.e., 27.9 +/- 1.2 vs. 15.6 +/- 1.5 nmol/min per mg protein, respectively; P less than 0.001); and (c) the Km values for Ins(1,4,5)P3 5'-phosphatase were 14- and 19-fold greater than those for Ins(1,4,5)P3 3'-kinase in the 2-wk-old and adult TSM, respectively. Collectively, the findings suggest that the age-related decrease in agonist-induced rabbit TSM contractility is associated with a diminution in Ins(1,4,5)P3 accumulation which is attributed, at least in part, to ontogenetic changes in the relative activities of the degradative enzymes for

  15. Differential regulation of types-1 and -3 inositol trisphosphate receptors by cytosolic Ca2+.

    PubMed Central

    Cardy, T J; Traynor, D; Taylor, C W

    1997-01-01

    Biphasic regulation of inositol trisphosphate (IP3)-stimulated Ca2+ mobilization by cytosolic Ca2+ is believed to contribute to regenerative intracellular Ca2+ signals. Since cells typically express several IP3 receptor isoforms and the effects of cytosolic Ca2+ are not mediated by a single mechanism, it is important to resolve the properties of each receptor subtype. Full-length rat types-1 and -3 IP3 receptors were expressed in insect Sf9 cells at levels 10-40-fold higher than the endogenous receptors. The expressed receptors were glycosylated and assembled into tetramers, and binding of [3H]IP3 to each subtype was regulated by cytosolic Ca2+. The effects of increased [Ca2+] on native cerebellar and type-1 receptors expressed in Sf9 cells were indistinguishable. A maximally effective increase in [Ca2+] reversibly inhibited [3H]IP3 binding by approx. 50% by decreasing the number of IP3-binding sites (Bmax) without affecting their affinity for IP3. The effects of Ca2+ on type-3 receptors were more complex: increasing [Ca2+] first stimulated [3H]IP3 binding by increasing Bmax, and then inhibited it by causing a substantial decrease in the affinity of the receptor for IP3. The different effects of Ca2+ on the receptor subtypes were not a consequence of limitations in the availability of accessory proteins or of artifactual effects of Ca2+ on membrane structure. We conclude that Ca2+ can inhibit IP3 binding to types-1 and -3 IP3 receptors although by different mechanisms, and that IP3 binding to type-3 receptors is stimulated at intermediate [Ca2+]. A consequence of these differences is that, at resting cytosolic [Ca2+], type-3 receptors are more sensitive than type-1 receptors to IP3, but the situation reverses at higher cytosolic [Ca2+]. Such differences may be important in generating the spatially and temporally complex changes in cytosolic [Ca2+] evoked by receptors linked to IP3 formation. PMID:9396721

  16. Quaternary structure of carbonmonoxyhemoglobins in solution: structural changes induced by the allosteric effector inositol hexaphosphate.

    PubMed

    Gong, Qingguo; Simplaceanu, Virgil; Lukin, Jonathan A; Giovannelli, Janel L; Ho, Nancy T; Ho, Chien

    2006-04-25

    We have applied the residual dipolar coupling (RDC) method to investigate the solution quaternary structures of (2)H- and (15)N-labeled human normal adult recombinant hemoglobin (rHb A) and a low-oxygen-affinity mutant recombinant hemoglobin, rHb(alpha96Val-->Trp), both in the carbonmonoxy form, in the absence and presence of an allosteric effector, inositol hexaphosphate (IHP), using a stretched polyacrylamide gel as the alignment medium. Our recent RDC results [Lukin, J. A., Kontaxis, G., Simplaceanu, V., Yuan, Y., Bax, A., and Ho, C. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 517-520] indicate that the quaternary structure of HbCO A in solution is a dynamic ensemble between two previously determined crystal structures, R (crystals grown under high-salt conditions) and R2 (crystals grown under low-salt conditions). On the basis of a comparison of the geometric coordinates of the T, R, and R2 structures, it has been suggested that the oxygenation of Hb A follows the transition pathway from T to R and then to R2, with R being the intermediate structure [Srinivasan, R., and Rose, G. D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11113-11117]. The results presented here suggest that IHP can shift the solution quaternary structure of HbCO A slightly toward the R structure. The solution quaternary structure of rHbCO(alpha96Val-->Trp) in the absence of IHP is similar to that of HbCO A in the presence of IHP, consistent with rHbCO(alpha96Val-->Trp) having an affinity for oxygen lower than that of Hb A. Moreover, IHP has a much stronger effect in shifting the solution quaternary structure of rHbCO(alpha96Val-->Trp) toward the R structure and toward the T structure, consistent with IHP causing a more pronounced decrease in its oxygen affinity. The results presented in this work, as well as other results recently reported in the literature, clearly indicate that there are multiple quaternary structures for the ligated form of hemoglobin. These results also provide new insights

  17. Myo-inositol changes precede amyloid pathology and relate to APOE genotype in Alzheimer disease

    PubMed Central

    Sundgren, Pia C.; Strandberg, Olof; Zetterberg, Henrik; Minthon, Lennart; Blennow, Kaj; Wahlund, Lars-Olof; Westman, Eric

    2016-01-01

    Objective: We aimed to test whether in vivo levels of magnetic resonance spectroscopy (MRS) metabolites myo-inositol (mI), N-acetylaspartate (NAA), and choline are abnormal already during preclinical Alzheimer disease (AD), relating these changes to amyloid or tau pathology, and functional connectivity. Methods: In this cross-sectional multicenter study (a subset of the prospective Swedish BioFINDER study), we included 4 groups, representing the different stages of predementia AD: (1) cognitively healthy elderly with normal CSF β-amyloid 42 (Aβ42), (2) cognitively healthy elderly with abnormal CSF Aβ42, (3) patients with subjective cognitive decline and abnormal CSF Aβ42, (4) patients with mild cognitive decline and abnormal CSF Aβ42 (Ntotal = 352). Spectroscopic markers measured in the posterior cingulate/precuneus were considered alongside known disease biomarkers: CSF Aβ42, phosphorylated tau, total tau, [18F]-flutemetamol PET, f-MRI, and the genetic risk factor APOE. Results: Amyloid-positive cognitively healthy participants showed a significant increase in mI/creatine and mI/NAA levels compared to amyloid-negative healthy elderly (p < 0.05). In amyloid-positive healthy elderly, mI/creatine and mI/NAA correlated with cortical retention of [18F] flutemetamol tracer ( = 0.44, p = 0.02 and = 0.51, p = 0.01, respectively). Healthy elderly APOE ε4 carriers with normal CSF Aβ42 levels had significantly higher mI/creatine levels (p < 0.001) than ε4 noncarriers. Finally, elevated mI/creatine was associated with decreased functional connectivity within the default mode network (rpearson = −0.16, p = 0.02), independently of amyloid pathology. Conclusions: mI levels are elevated already at asymptomatic stages of AD. Moreover, mI/creatine concentrations were increased in healthy APOE ε4 carriers with normal CSF Aβ42 levels, suggesting that mI levels may reveal regional brain consequences of APOE ε4 before detectable amyloid pathology. PMID:27164711

  18. Confocal microfluorimetry of Ca2+ signals evoked in Xenopus oocytes by photoreleased inositol trisphosphate.

    PubMed Central

    Parker, I; Ivorra, I

    1993-01-01

    1. The subcellular characteristics of inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ liberation were studied in Xenopus oocytes by the use of confocal microfluorimetry to monitor Ca2+ signals from minutely localized region of the cell in response to photorelease of InsP3 from a caged precursor. 2. Photorelease of increasing amounts of InsP3 by progressively longer light flashes evoked transient Ca2+ responses that appeared abruptly at a certain threshold duration, and then grew steeply over a narrow range of flash durations to reach a maximum. Further lengthening of flash duration gave no increase in size of the Ca2+ signals, but their rate of rise continued to increase and their duration became longer. Simultaneous measurements of Ca(2+)-activated Cl- currents showed a slightly higher threshold than the Ca2+ signal, and a more graded dependence upon flash duration. 3. The threshold flash durations required to evoke Ca2+ and membrane current signals grew by more than 100-fold as the area of the oocyte exposed to photolysis light was reduced from a square of 140 microns to 5 microns. 4. Ca2+ signals evoked by photoreleased InsP3 began following a dose-dependent latency that was as long as several seconds with low intensity light, but shortened to about 50 ms at maximum intensity. The extrapolated minimum latency with infinite photorelease of InsP3 was about 30 ms. 5. InsP3-evoked membrane currents began 30 ms or longer after the corresponding Ca2+ signals, whereas currents evoked by photorelease of Ca2+ from a caged precursor began within 5 ms of the onset of the light flash. 6. No differences in duration of InsP3-evoked Ca2+ signals were apparent when the confocal measuring spot was positioned close to the plasma membrane or about 10 microns more deeply into the oocyte. At both locations the Ca2+ signals were more prolonged than the associated membrane current signals. 7. Ca2+ signals to a test light flash were suppressed for about 2 s following a conditioning

  19. Characteristics of inositol trisphosphate mediated Ca/sup 2 +/ release from permeabilized hepatocytes

    SciTech Connect

    Joseph, S.K.; Williamson, J.R.

    1986-05-01

    Ca/sup 2 +/ release triggered by inositol trisphosphate (IP/sub 3/) has been measured in saponin-permeabilized hepatocytes with /sup 45/Ca/sup 2 +/ or Quin 2. The initial rate of Ca/sup 2 +/ release was not markedly affected by the incubation temperature (175 +/- 40 pmol/s/mg at 30/sup 0/C versus 133 +/- 24 pmol/s/mg at 4/sup 0/C). This result is consistent with the membrane translocation of Ca/sup 2 +/ occurring through an ion-channel rather than an ion-carrier. The amount of Ca/sup 2 +/ released by IP/sub 3/ was not affected by pH (6.5-8.0) or by compounds that inhibit voltage-gated Ca/sup 2 +/ channels. La/sup 3 +/ (100 ..mu..M) markedly inhibits the effect of 1 ..mu..M IP/sub 3/. The possibility that La/sup 3 +/ chelates IP/sub 3/ cannot be excluded since the effect of La/sup 3 +/ can be overcome by increasing the IP/sub 3/ concentration. IP/sub 3/-mediated Ca/sup 2 +/ release displays a requirement for a permeant cation in the incubation medium. Optimal release is observed with K/sup +/ gluconate. Other monovalent cations, with the exception of Li/sup +/, can substitute for K/sup +/. Permeant anions, at concentrations above 40 mM, inhibit Ca/sup 2 +/ release produced by IP/sub 3/. Cl/sup -/, Br/sup -/, I/sup -/, and SO/sub 4//sup 2 -/ were equally effective. Ca/sup 2 +/ release was not inhibited by DIDS or Furosemide. /sup 85/Sr/sup 2 +/ and /sup 54/Mn/sup 2 +/ fluxes were also stimulated by IP/sub 3/. These results suggest that IP/sub 3/ acts to gate a divalent cation channel. The translocation of positive charge through this channel is balanced by ancillary movements of monovalent cations and anions across the reticular membrane.

  20. Characteristics of inositol trisphosphate-mediated Ca/sup 2 +/ release from permeabilized hepatocytes

    SciTech Connect

    Joseph, S.K.; Williamson, J.R.

    1986-11-05

    Ca/sup 2 +/ release triggered by inositol trisphosphate (Ins(1,4,5)P/sub 3/) has been measured in saponin-permeabilized hepatocytes with /sup 45/Ca/sup 2 +/ or Quin 2. The initial rate of Ca/sup 2 +/ release was not greatly affected by the incubation temperature. The amount of Ca/sup 2 +/ released by Ins(1,4,5)P/sub 3/ was not affected by pH (6.5-8.0). La/sup 3 +/ (100 ..mu..M) markedly inhibited the effect of 1 ..mu..M Ins(1,4,5)P/sub 3/. The possibility that La/sup 3 +/ chelates Ins(1,4,5)P/sub 3/ cannot be excluded since the effect of La/sup 3 +/ could be overcome by increasing the Ins(1,4,5)P/sub 3/ concentration. Ins(1,4,5)P/sub 3/-mediated Ca/sup 2 +/ release showed a requirement for permeant cations in the incubation medium. Optimal release was observed with potassium gluconate. Other monovalent cations, with the exception of Li/sup +/, can substitute for K/sup +/. Permeant anions, at concentrations above 40 mM, inhibited Ca/sup 2 +/ release produced by Ins(1,4,5)P/sub 3/. Cl/sup -/, Br/sup -/, I/sup -/, and SO/sup 2 -//sub 4/ were equally effective as inhibitors. Ins(1,4,5)P/sub 3/ also caused the release of /sup 54/Mn/sup 2 +/ and /sup 85/Sr/sup 2 +/ accumulated by the permeabilized hepatocytes. The results are consistent with Ins(1,4,5)P/sub 3/ promoting the membrane translocation of divalent cations through an ion channel rather than an ion carrier. The translocation of positive charge through this channel is balanced by ancillary movements of monovalent cations and anions across the reticular membranes. The transport systems responsible for these compensatory ion movements may represent a potential site for the regulation of the hormone-mediated Ca/sup 2 +/ signal.

  1. Synergism between Inositol Polyphosphates and TOR Kinase Signaling in Nutrient Sensing, Growth Control, and Lipid Metabolism in Chlamydomonas[OPEN

    PubMed Central

    Evans, Bradley S.; Li, Jia; Liu, Yu; Diamond, Spencer

    2016-01-01

    The networks that govern carbon metabolism and control intracellular carbon partitioning in photosynthetic cells are poorly understood. Target of Rapamycin (TOR) kinase is a conserved growth regulator that integrates nutrient signals and modulates cell growth in eukaryotes, though the TOR signaling pathway in plants and algae has yet to be completely elucidated. We screened the unicellular green alga Chlamydomonas reinhardtii using insertional mutagenesis to find mutants that conferred hypersensitivity to the TOR inhibitor rapamycin. We characterized one mutant, vip1-1, that is predicted to encode a conserved inositol hexakisphosphate kinase from the VIP family that pyrophosphorylates phytic acid (InsP6) to produce the low abundance signaling molecules InsP7 and InsP8. Unexpectedly, the rapamycin hypersensitive growth arrest of vip1-1 cells was dependent on the presence of external acetate, which normally has a growth-stimulatory effect on Chlamydomonas. vip1-1 mutants also constitutively overaccumulated triacylglycerols (TAGs) in a manner that was synergistic with other TAG inducing stimuli such as starvation. vip1-1 cells had reduced InsP7 and InsP8, both of which are dynamically modulated in wild-type cells by TOR kinase activity and the presence of acetate. Our data uncover an interaction between the TOR kinase and inositol polyphosphate signaling systems that we propose governs carbon metabolism and intracellular pathways that lead to storage lipid accumulation. PMID:27600537

  2. Inositol transporters AtINT2 and AtINT4 regulate arsenic accumulation in Arabidopsis seeds.

    PubMed

    Duan, Gui-Lan; Hu, Ying; Schneider, Sabine; McDermott, Joseph; Chen, Jian; Sauer, Norbert; Rosen, Barry P; Daus, Birgit; Liu, Zijuan; Zhu, Yong-Guan

    2016-01-01

    Arsenic contamination of groundwater and soils threatens the health of tens of millions of people worldwide. Understanding the way in which arsenic is taken up by crops such as rice, which serve as a significant source of arsenic in the human diet, is therefore important. Membrane transport proteins that catalyse arsenic uptake by roots, and translocation through the xylem to shoots, have been characterized in a number of plants, including rice. The transporters responsible for loading arsenic from the xylem into the phloem and on into the seeds, however, are yet to be identified. Here, we show that transporters responsible for inositol uptake in the phloem in Arabidopsis also transport arsenic. Transformation of Saccharomyces cerevisiae with AtINT2 or AtINT4 led to increased arsenic accumulation and increased sensitivity to arsenite. Expression of AtINT2 in Xenopus laevis oocytes also induced arsenite import. Disruption of AtINT2 or AtINT4 in Arabidopsis thaliana led to a reduction in phloem, silique and seed arsenic concentrations in plants fed with arsenite through the roots, relative to wild-type plants. These plants also exhibited a large drop in silique and seed arsenic concentrations when fed with arsenite through the leaves. We conclude that in Arabidopsis, inositol transporters are responsible for arsenite loading into the phloem, the key source of arsenic in seeds. PMID:27004129

  3. Inositol hexakisphosphate (IP6) generated by IP5K mediates cullin-COP9 signalosome interactions and CRL function.

    PubMed

    Scherer, Paul C; Ding, Yan; Liu, Zhiqing; Xu, Jing; Mao, Haibin; Barrow, James C; Wei, Ning; Zheng, Ning; Snyder, Solomon H; Rao, Feng

    2016-03-29

    The family of cullin-RING E3 Ligases (CRLs) and the constitutive photomorphogenesis 9 (COP9) signalosome (CSN) form dynamic complexes that mediate ubiquitylation of 20% of the proteome, yet regulation of their assembly/disassembly remains poorly understood. Inositol polyphosphates are highly conserved signaling molecules implicated in diverse cellular processes. We now report that inositol hexakisphosphate (IP6) is a major physiologic determinant of the CRL-CSN interface, which includes a hitherto unidentified electrostatic interaction between the N-terminal acidic tail of CSN subunit 2 (CSN2) and a conserved basic canyon on cullins. IP6, with an EC50 of 20 nM, acts as an intermolecular "glue," increasing cullin-CSN2 binding affinity by 30-fold, thereby promoting assembly of the inactive CRL-CSN complexes. The IP6 synthase, Ins(1,3,4,5,6)P5 2-kinase (IPPK/IP5K) binds to cullins. Depleting IP5K increases the percentage of neddylated, active Cul1 and Cul4A, and decreases levels of the Cul1/4A substrates p27 and p21. Besides dysregulating CRL-mediated cell proliferation and UV-induced apoptosis, IP5K depletion potentiates by 28-fold the cytotoxic effect of the neddylation inhibitor MLN4924. Thus, IP5K and IP6 are evolutionarily conserved components of the CRL-CSN system and are potential targets for cancer therapy in conjunction with MLN4924. PMID:26976604

  4. Inositol transporters AtINT2 and AtINT4 regulate arsenic accumulation in Arabidopsis seeds

    PubMed Central

    Duan, Gui-Lan; Hu, Ying; Schneider, Sabine; McDermott, Joseph; Chen, Jian; Sauer, Norbert; Rosen, Barry P.; Daus, Birgit; Liu, Zijuan; Zhu, Yong-Guan

    2015-01-01

    Arsenic is a global environmental contaminant that threatens tens of millions people world-wide via food and water. Understanding how arsenic is accumulated in crop seeds is of critical importance. To date, membrane transport proteins catalyzing arsenic uptake by roots and translocation through xylem to shoots have been characterized. However, no transporters responsible for loading arsenic from xylem into phloem and further unloading into plant seeds have been identified. In this study we demonstrate that expressing the gene for either Arabidopsis thaliana inositol transporter AtINT2 or AtINT4 in Saccharomyces cerevisiae leads to increased arsenic accumulation and elevated sensitivity to arsenite [As(III)], and Xenopus laevis oocytes expressing AtINT2 import As(III). When A. thaliana plants with disruptions in either AtINT2 or AtINT4 were supplemented with As(III) through roots, there was a substantial decrease in both the arsenic content in the phloem extrude and in total arsenic accumulation in siliques and seeds. Similarly, when As(III) is fed through the leaves, there was a very large decrease in arsenic accumulation in siliques and seeds compared with wild-type plants. These results clearly demonstrate that inositol transporters are responsible for As(III) loading into phloem, the key step regulating arsenic accumulation in seeds. PMID:27004129

  5. Cellular delivery and photochemical release of a caged inositol-pyrophosphate induces PH-domain translocation in cellulo.

    PubMed

    Pavlovic, Igor; Thakor, Divyeshsinh T; Vargas, Jessica R; McKinlay, Colin J; Hauke, Sebastian; Anstaett, Philipp; Camuña, Rafael C; Bigler, Laurent; Gasser, Gilles; Schultz, Carsten; Wender, Paul A; Jessen, Henning J

    2016-01-01

    Inositol pyrophosphates, such as diphospho-myo-inositol pentakisphosphates (InsP7), are an important family of signalling molecules, implicated in many cellular processes and therapeutic indications including insulin secretion, glucose homeostasis and weight gain. To understand their cellular functions, chemical tools such as photocaged analogues for their real-time modulation in cells are required. Here we describe a concise, modular synthesis of InsP7 and caged InsP7. The caged molecule is stable and releases InsP7 only on irradiation. While photocaged InsP7 does not enter cells, its cellular uptake is achieved using nanoparticles formed by association with a guanidinium-rich molecular transporter. This novel synthesis and unprecedented polyphosphate delivery strategy enable the first studies required to understand InsP7 signalling in cells with controlled spatiotemporal resolution. It is shown herein that cytoplasmic photouncaging of InsP7 leads to translocation of the PH-domain of Akt, an important signalling-node kinase involved in glucose homeostasis, from the membrane into the cytoplasm. PMID:26842801

  6. Flow injection potentiometric system for the simultaneous determination of inositol phosphates and phosphate: phosphorus nutritional evaluation on seeds and grains.

    PubMed

    Parra, Aleix; Ramon, Meritxell; Alonso, Julián; Lemos, Sherlan G; Vieira, Edivan C; Nogueira, Ana R A

    2005-10-01

    A simple flow injection potentiometric (FIP) system, which uses a tubular cobalt electrode, has been developed for phosphorus nutritional evaluation of seeds and grains. Inorganic phosphorus, P(i), is determined using a 1 x 10(-2) mol.L(-1) potassium phthalate buffer solution adjusted at pH 4. A sensitivity of 47 mV/decade and an operating range from 10 to 1000 mg.L(-1) (1 x 10(-4)-1 x 10(-2) M) of dihydrogen phosphate are obtained. The inositol phosphates amount, which is referred to the organic phosphorus, P(org), is directly determined from extracts using a 1 x 10(-2) mol.L(-1) Tris-HCl buffer solution adjusted at pH 8. A sensitivity of 127 mV/decade and an operating range of 10-1000 mg.L(-1) (2.5 x 10(-4)-5 x 10(-3) M) of P(org) (expressed as inositol hexakisphosphoric acid monocalcium) are achieved. Some samples of seed and grain are analyzed by an ICP-OES and a spectrophotometric method to compare results to the developed flow system; no significant differences at the 95% confidence level are observed using a paired t test. Other samples such as animal nursing feed, soybean meal, and corn are also analyzed with the proposed FIP system, showing a good correlation to the ICP-OES values.

  7. Inositol hexaphosphate-induced enhancement of natural killer cell activity correlates with suppression of colon carcinogenesis in rats

    PubMed Central

    Zhang, Zheng; Song, Yang; Wang, Xiu-Li

    2005-01-01

    AIM: To investigate the anti-neoplastic effect of inositol hexaphosphate (InsP6 or phytic acid) on dimethylhydrazine (DMH)-induced colon tumor in rats and its effect on blood natural killer (NK) cell activity. METHODS: Healthy Wistar rats, 4 wk old, were divided into control group (fed with common food) and InsP6 group (fed with common food+2% sodium inositol hexaphosphate in the drinking water), 15 rats in each group. Both groups were injected with 1,2-dimethylhydrazine subcutaneously (20 mg/kg body weight) once a week for 20 wk. Rats were killed after 21 wk. The whole large intestine was isolated to determine the general condition of tumors and to test blood NK cell activity by lactate-dehydrogenase-release assay. RESULTS: Administration of InsP6 significantly increased blood NK cell activity in DMH-induced colorectal tumor in rats. InsP6 group had a smaller tumor size on average and a smaller number of tumors than the control group. Its mortality was also higher than that in control. However, the variables of body weight and tumor incidence were not significantly different between the two groups. CONCLUSION: InsP6 can increase blood NK cell activity in DMH-induced colon tumor in rats and inhibit tumor growth and metastasis in rats. PMID:16124063

  8. The role of inositol 1,4,5-trisphosphate 3-kinase A in regulating emotional behavior and amygdala function

    PubMed Central

    Chung, Sooyoung; Kim, Il Hwan; Lee, Dongmin; Park, Kyungjoon; Kim, Joo Yeon; Lee, Yeon Kyung; Kim, Eun Joo; Lee, Hyun Woo; Choi, June-seek; Son, Gi Hoon; Sun, Woong; Shin, Ki Soon; Kim, Hyun

    2016-01-01

    Inositol 1,4,5-trisphosphate 3-kinase A (IP3K-A) is a molecule enriched in the brain and neurons that regulates intracellular calcium levels via signaling through the inositol trisphosphate receptor. In the present study, we found that IP3K-A expression is highly enriched in the central nucleus of the amygdala (CeA), which plays a pivotal role in the processing and expression of emotional phenotypes in mammals. Genetic abrogation of IP3K-A altered amygdala gene expression, particularly in genes involved in key intracellular signaling pathways and genes mediating fear- and anxiety-related behaviors. In agreement with the changes in amygdala gene expression profiles, IP3K-A knockout (KO) mice displayed more robust responses to aversive stimuli and spent less time in the open arms of the elevated plus maze, indicating high levels of innate fear and anxiety. In addition to behavioral phenotypes, decreased excitatory and inhibitory postsynaptic current and reduced c-Fos immunoreactivity in the CeA of IP3K-A KO mice suggest that IP3K-A has a profound influence on the basal activities of fear- and anxiety-mediating amygdala circuitry. In conclusion, our findings collectively demonstrate that IP3K-A plays an important role in regulating affective states by modulating metabotropic receptor signaling pathways and neural activity in the amygdala. PMID:27053114

  9. Biological response of hepatomas to an extract of Fagopyrum esculentum M. (buckwheat) is not mediated by inositols or rutin.

    PubMed

    Curran, Julianne M; Stringer, Danielle M; Wright, Brenda; Taylor, Carla G; Przybylski, Roman; Zahradka, Peter

    2010-03-10

    Buckwheat contains d-chiro-inositol (D-CI) and myo-inositol (MI), possible insulin-mimetic compounds; thus, this study investigated the insulin-mimetic activities of a buckwheat concentrate (BWC), D-CI, and MI on insulin signal transduction pathways and glucose uptake with H4IIE rat hepatoma cells. BWC stimulated phosphorylation of p42/44 extracellular-related kinase (p42/44 ERK) and its downstream target, p70(S6K), on Thr(421). In contrast, D-CI, MI, rutin, or its agylcone form, quercetin, did not activate these signal transduction proteins. Phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), another target of insulin, was also up-regulated upon BWC treatment. The effects of BWC on glucose uptake were subsequently investigated using H4IIE cells. Insulin and D-CI stimulated glucose uptake, whereas BWC inhibited basal and insulin-stimulated glucose uptake. Although results from this work suggest that BWC has insulin-mimetic effects on select protein phosphorylation events in H4IIE cells, D-CI and MI were not the active components responsible for the observed effects. The inhibition of glucose uptake by BWC suggests that buckwheat may affect hepatic glucose metabolism, possibly by inhibiting glucose flux. Furthermore, the fact that D-CI and MI stimulated glucose uptake in H4IIE cells suggests that other compounds are responsible for inhibition of glucose uptake by BWC.

  10. Cellular delivery and photochemical release of a caged inositol-pyrophosphate induces PH-domain translocation in cellulo

    PubMed Central

    Pavlovic, Igor; Thakor, Divyeshsinh T.; Vargas, Jessica R.; McKinlay, Colin J.; Hauke, Sebastian; Anstaett, Philipp; Camuña, Rafael C.; Bigler, Laurent; Gasser, Gilles; Schultz, Carsten; Wender, Paul A.; Jessen, Henning J.

    2016-01-01

    Inositol pyrophosphates, such as diphospho-myo-inositol pentakisphosphates (InsP7), are an important family of signalling molecules, implicated in many cellular processes and therapeutic indications including insulin secretion, glucose homeostasis and weight gain. To understand their cellular functions, chemical tools such as photocaged analogues for their real-time modulation in cells are required. Here we describe a concise, modular synthesis of InsP7 and caged InsP7. The caged molecule is stable and releases InsP7 only on irradiation. While photocaged InsP7 does not enter cells, its cellular uptake is achieved using nanoparticles formed by association with a guanidinium-rich molecular transporter. This novel synthesis and unprecedented polyphosphate delivery strategy enable the first studies required to understand InsP7 signalling in cells with controlled spatiotemporal resolution. It is shown herein that cytoplasmic photouncaging of InsP7 leads to translocation of the PH-domain of Akt, an important signalling-node kinase involved in glucose homeostasis, from the membrane into the cytoplasm. PMID:26842801

  11. Inositol transporters AtINT2 and AtINT4 regulate arsenic accumulation in Arabidopsis seeds.

    PubMed

    Duan, Gui-Lan; Hu, Ying; Schneider, Sabine; McDermott, Joseph; Chen, Jian; Sauer, Norbert; Rosen, Barry P; Daus, Birgit; Liu, Zijuan; Zhu, Yong-Guan

    2016-01-01

    Arsenic contamination of groundwater and soils threatens the health of tens of millions of people worldwide. Understanding the way in which arsenic is taken up by crops such as rice, which serve as a significant source of arsenic in the human diet, is therefore important. Membrane transport proteins that catalyse arsenic uptake by roots, and translocation through the xylem to shoots, have been characterized in a number of plants, including rice. The transporters responsible for loading arsenic from the xylem into the phloem and on into the seeds, however, are yet to be identified. Here, we show that transporters responsible for inositol uptake in the phloem in Arabidopsis also transport arsenic. Transformation of Saccharomyces cerevisiae with AtINT2 or AtINT4 led to increased arsenic accumulation and increased sensitivity to arsenite. Expression of AtINT2 in Xenopus laevis oocytes also induced arsenite import. Disruption of AtINT2 or AtINT4 in Arabidopsis thaliana led to a reduction in phloem, silique and seed arsenic concentrations in plants fed with arsenite through the roots, relative to wild-type plants. These plants also exhibited a large drop in silique and seed arsenic concentrations when fed with arsenite through the leaves. We conclude that in Arabidopsis, inositol transporters are responsible for arsenite loading into the phloem, the key source of arsenic in seeds.

  12. Isolation and developmental expression analysis of L-myo-inositol-1-phosphate synthase in four Actinidia species.

    PubMed

    Cui, Meng; Liang, Dong; Wu, Shan; Ma, Fengwang; Lei, Yushan

    2013-12-01

    Myo-inositol (MI) is an important polyol involved in cellular signal transduction, auxin storage, osmotic regulation, and membrane formation. It also serves as a precursor for the production of pinitol, ascorbic acid, and members of the raffinose family. The first committed step for MI formation is catalyzed by L-myo-inositol-1-phosphate synthase (MIPS). We isolated MIPS cDNA sequences from Actinidia eriantha, Actinidia rufa, and Actinidia arguta and compared them with that of Actinidia deliciosa. Each comprised 1533 bp, encoding 510 amino acids with a predicted molecular weight of 56.5 KDa. The MIPS protein was highly conserved in Actinidia, sharing 98.94% identity among species. The MIPS gene was expressed in the flowers, leaves, petioles, and carpopodia. Similarly high levels of expression were detected in the young fruit of all four species. Overall activity of the enzyme was also maximal in young fruit, indicating that this developmental stage is the key point for MI synthesis in Actinidia. Among the four species, A. arguta had the greatest concentration of MI as well as the highest ratios of MI:sucrose and MI:glucose+fructose. This suggests that conversion to MI from carbohydrates was most efficient in A. arguta during early fruit development.

  13. Evidence for inositol triphosphate as a second messenger for glucose-induced calcium signalling in budding yeast.

    PubMed

    Tisi, Renata; Belotti, Fiorella; Wera, Stefaan; Winderickx, Joris; Thevelein, Johan M; Martegani, Enzo

    2004-02-01

    The Saccharomyces cerevisiae phospholipase C Plc1 is involved in cytosolic transient glucose-induced calcium increase, which also requires the Gpr1/Gpa2 receptor/G protein complex and glucose hexokinases. Differing from mammalian cells, this increase in cytosolic calcium concentration is mainly due to an influx from the external medium. No inositol triphosphate receptor homologue has been identified in the S. cerevisiae genome; and, therefore, the transduction mechanism from Plc1 activation to calcium flux generation still has to be identified. Inositol triphosphate (IP(3)) in yeast is rapidly transformed into IP(4) and IP(5) by a dual kinase, Arg82. Then another kinase, Ipk1, phosphorylates the IP(5) into IP(6). In mutant cells that do not express either of these kinases, the glucose-induced calcium signal was not only detectable but was even wider than in the wild-type strain. IP(3) accumulation upon glucose addition was completely absent in the plc1Delta strain and was amplified both by deletion of either ARG82 or IPK1 genes and by overexpression of PLC1. These results taken together suggest that Plc1p activation by glucose, leading to cleavage of PIP(2) and generation of IP(3), seems to be sufficient for raising the calcium level in the cytosol. This is the first indication for a physiological role of IP(3) signalling in S. cerevisiae. Many aspects about the signal transduction mechanism and the final effectors require further study.

  14. IP6K structure and the molecular determinants of catalytic specificity in an inositol phosphate kinase family.

    PubMed

    Wang, Huanchen; DeRose, Eugene F; London, Robert E; Shears, Stephen B

    2014-06-24

    Inositol trisphosphate kinases (IP3Ks) and inositol hexakisphosphate kinases (IP6Ks) each regulate specialized signalling activities by phosphorylating either InsP3 or InsP6 respectively. The molecular basis for these different kinase activities can be illuminated by a structural description of IP6K. Here we describe the crystal structure of an Entamoeba histolytica hybrid IP6K/IP3K, an enzymatic parallel to a 'living fossil'. Through molecular modelling and mutagenesis, we extrapolated our findings to human IP6K2, which retains vestigial IP3K activity. Two structural elements, an α-helical pair and a rare, two-turn 310 helix, together forge a substrate-binding pocket with an open clamshell geometry. InsP6 forms substantial contacts with both structural elements. Relative to InsP6, enzyme-bound InsP3 rotates 55° closer to the α-helices, which provide most of the protein's interactions with InsP3. These data reveal the molecular determinants of IP6K activity, and suggest an unusual evolutionary trajectory for a primordial kinase that could have favored efficient bifunctionality, before propagation of separate IP3Ks and IP6Ks.

  15. Influence of cyclic nucleotides (cAMP) on inositol phospholipid (InsPL) metabolism in cultured mesangial (MS) cells

    SciTech Connect

    Troyer, D.A.; Venkatachalam, M.A.; Bonventre, J.V.; Kreisberg, J.I.

    1986-03-01

    Elevation of cAMP inhibits hormone-induced contraction of MS cells, and in other cell types, reduces stimulated InsPL metabolism. The authors found that neither isobutylmethylxanthine (MIX, 0.5 mM), which increases MS cell cAMP levels 4-fold, nor forskolin (100 ..mu..M) altered vasopressin (VP, 10 nM) induced release of /sup 3/H-inositol trisphosphate from prelabelled MS cells. Also, maneuvers which elevated cAMP did not block the VP-induced rise of intracellular calcium as measured by quin-2. Further, neither MIX nor isoproterenol affected the stimulation of glycolysis by VP as measured by lactic acid production. MIX diminished VP stimulated /sup 32/P orthophosphate (/sup 32/P) incorporation into phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol 4-phosphate and phosphatidylinositol. The /sup 32/P content in phosphoinositides of cells treated with MIX and VP was 65% of that in cells treated with VP only. However, the authors found that the specific activity of /sup 32/P in ATP in the presence of MIX + VP was 74% of that with VP alone. Thus, the apparent suppression of /sup 32/P incorporation due to MIX was attributable to a concurrent diminution of the specific activity of /sup 32/P in ATP. The authors conclude that increases of cAMP interfere with contraction distal to PIP/sub 2/ hydrolysis, inositol phosphate release, calcium mobilization, and enhancement of glycolysis.

  16. Cellular delivery and photochemical release of a caged inositol-pyrophosphate induces PH-domain translocation in cellulo.

    PubMed

    Pavlovic, Igor; Thakor, Divyeshsinh T; Vargas, Jessica R; McKinlay, Colin J; Hauke, Sebastian; Anstaett, Philipp; Camuña, Rafael C; Bigler, Laurent; Gasser, Gilles; Schultz, Carsten; Wender, Paul A; Jessen, Henning J

    2016-02-04

    Inositol pyrophosphates, such as diphospho-myo-inositol pentakisphosphates (InsP7), are an important family of signalling molecules, implicated in many cellular processes and therapeutic indications including insulin secretion, glucose homeostasis and weight gain. To understand their cellular functions, chemical tools such as photocaged analogues for their real-time modulation in cells are required. Here we describe a concise, modular synthesis of InsP7 and caged InsP7. The caged molecule is stable and releases InsP7 only on irradiation. While photocaged InsP7 does not enter cells, its cellular uptake is achieved using nanoparticles formed by association with a guanidinium-rich molecular transporter. This novel synthesis and unprecedented polyphosphate delivery strategy enable the first studies required to understand InsP7 signalling in cells with controlled spatiotemporal resolution. It is shown herein that cytoplasmic photouncaging of InsP7 leads to translocation of the PH-domain of Akt, an important signalling-node kinase involved in glucose homeostasis, from the membrane into the cytoplasm.

  17. Relationship between stimulated phosphatidic acid production and inositol lipid hydrolysis in intestinal longitudinal smooth muscle from guinea pig.

    PubMed

    Mallows, R S; Bolton, T B

    1987-06-15

    Accumulation of [32P]phosphatidic acid (PA) and total [3H]inositol phosphates (IPs) was measured in the longitudinal smooth-muscle layer from guinea-pig small intestine. Stimulation with carbachol, histamine and substance P produced increases in accumulation of both [3H]IPs and [32P]PA over the same concentration range. The increase in [32P]PA accumulation in response to carbachol (1 microM-0.1 mM) was inhibited in the presence of atropine (0.5 microM). Buffering the external free [Ca2+] to 10 nM did not prevent the carbachol-stimulated increase in [32P]PA accumulation. Carbachol and Ca2+ appear to act synergistically to increase accumulation of [32P]PA. In contrast, although incubation with noradrenaline also increased accumulation of [3H]IPs, no increase in accumulation of [32P]PA could be detected. These results suggest that an increase in formation of IPs is not necessarily accompanied by an increase in PA formation, and imply the existence of receptor-modulated pathways regulating PA concentrations other than by phospholipase-C-catalysed inositol phospholipid hydrolysis.

  18. The effect of M & B 22948 on carbachol-induced inositol trisphosphate accumulation and contraction in iris sphincter smooth muscle.

    PubMed

    Akhtar, R A; Abdel-Latif, A A

    1991-04-25

    The effect of a cyclic GMP phosphodiesterase inhibitor, M & B 22948, on carbachol-induced phosphatidylinositol 4,5-bis-phosphate (PIP2) breakdown and phosphatidic acid labeling, 1,4,5-inositol trisphosphate (IP3) accumulation and muscle contraction was studied in bovine iris sphincter smooth muscle. Addition of carbachol (10 microM) to 32P-labeled tissue resulted in increased labeling of phosphatidic acid and hydrolysis of PIP2. In myo[3H]inositol labeled tissue, carbachol caused rapid accumulation of IP3 which reached its maximum at about 2 min. Under identical experimental conditions, carbachol initiated a rapid increase in muscle contraction (phasic component) which was followed by a slightly lower contractile response (tonic component) that lasted for several minutes. Pretreatment of the iris sphincter with M & B 22948 did not alter carbachol-stimulated PIP2 breakdown and phosphatidic acid labeling, IP3 accumulation, or phasic component of the contractile response. However, the tonic component of the contractile response was increasingly attenuated by increasing concentrations of the drug. In conclusion, the data presented demonstrate a close correlation between carbachol-induced IP3 accumulation and muscle contraction, and that M & B 22948 does not inhibit carbachol-induced responses in the iris sphincter.

  19. Inositol 1,4,5-trisphosphate 3-kinase B controls survival and prevents anergy in B cells.

    PubMed

    Maréchal, Yoann; Quéant, Séverine; Polizzi, Selena; Pouillon, Valérie; Schurmans, Stéphane

    2011-01-01

    Inositol 1,4,5-trisphosphate 3-kinase B (or Itpkb) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), its reaction product, play an important role in the control of B lymphocyte fate and function in vivo. In order to investigate the fine mechanisms of Itpkb and Ins(1,3,4,5)P4 action in B cells, we crossed Itpkb(-/-) mice with transgenic mice expressing a 3-83μδ B cell receptor (BCR) specific for membrane-bound MHC-I H2-K(b) and H2-K(k) molecules. On a non-deleting H2-K(d) genetic background, we show that Itpkb is important for the control of Bim protein expression and B cell survival rather than for the control of B cell development from one stage to another. Analyses of cell surface markers expression, proapoptotic Bim protein expression, in vitro survival and in vivo turnover demonstrated that BCR transgenic Itpkb(-/-) B cells exhibit an anergic phenotype with the notable exception of their enhanced antigen-induced calcium signalling. On a deleting H2-K(b) genetic background, we show that Itpkb is not essential for BCR editing or negative selection. These data establish Itpkb as an important regulator of B cell survival and anergy in vivo.

  20. Direct modification and regulation of a nuclear receptor-PIP2 complex by the nuclear inositol-lipid kinase IPMK

    PubMed Central

    Blind, Raymond D.; Suzawa, Miyuki; Ingraham, Holly A.

    2012-01-01

    Phosphatidylinositol (4,5)-bisphosphate (PIP2) is best known as a plasma membrane-bound regulatory lipid. While PIP2 and phosphoinositide-modifying enzymes coexist in the nucleus, their roles in the nucleus remain unclear. Here we show that the nuclear inositol polyphosphate multikinase (IPMK), which functions both as an inositol- and a PI3-kinase, interacts with the nuclear receptor SF-1 (NR5A1) and phosphorylates its bound ligand, PIP2. IPMK failed to recognize SF-1/PIP2 after blocking or displacing PIP2 from SF-1’s large hydrophobic pocket. In contrast to IPMK, p110 catalytic subunits of type 1 PI3-kinases were inactive on SF-1/PIP2. These and other in vitro analyses demonstrated specificity of IPMK for the SF-1/PIP2 protein/lipid complex. Once generated, SF-1/PIP3 is readily dephosphorylated by the lipid phosphatase PTEN. Importantly, decreasing IPMK or increasing PTEN expression greatly reduced SF-1 transcriptional activity. This ability of lipid kinases and phosphatases to alter the activity and directly remodel a non-membrane protein/lipid complex such SF-1/PIP2, establishes a new pathway for promoting lipid-mediated signaling in the nucleus. PMID:22715467

  1. The inhibition of phosphoinositide synthesis and muscarinic-receptor-mediated phospholipase C activity by Li+ as secondary, selective, consequences of inositol depletion in 1321N1 cells.

    PubMed Central

    Batty, I H; Downes, C P

    1994-01-01

    Conditions are described for culture of 1321N1 cells under which cellular inositol is decreased from approximately 20 mM to < 0.5 mM but phosphoinositide concentrations are unaffected. The effects of the muscarinic-receptor agonist carbachol (1 mM) and/or LiCl (10 mM) on phosphoinositide turnover in these or in inositol-replete cells was examined after steady-state [3H]inositol labelling of phospholipid pools. In both inositol-replete and -depleted cells, carbachol stimulated similar initial (0-15 min) rates of phospholipase C (PLC) activity, in the presence of Li+. Subsequently (> 30-60 min) stimulated PLC activity and [3H]PtdIns concentrations declined dramatically only in depleted cells. In inositol-depleted cells, carbachol alone evoked increased concentrations of [3H]inositol, [3H]InsP1, [3H]InsP2, [3H]InsP3 and [3H]InsP4, which were largely sustained over 90 min, and concentrations of [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 were decreased only to approximately 82, 84 and 93% of control respectively. In the presence of Li+ in these cells, the stimulated rise in [3H]inositol was prevented and, although accumulation of [3H]InsP1, [3H]InsP2 and [3H]InsP3 was initially (0-30 min) potentiated, rates of accumulation of [3H]InsP1 and concentrations of [3H]polyphosphates later (> 30-60 min) declined, and concentrations of [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 were decreased respectively to approximately 39, 48 and 81% of control. After 60 min in the presence of both carbachol and Li+, stimulated PLC activity was decreased by approximately 70% compared with the initial rate in depleted cells. This decreased PLC activity was reflected by changes in the stimulated concentrations of [3H]Ins(1,3,4)P3 but not of [3H]Ins(1,4,5)P3, but effects of Li+ on the latter may have been obscured by the demonstrated, concomitant and equal stimulated accumulation of [3H]inositol 1:2cyclic,4,5-trisphosphate. These data suggest that receptor-mediated PLC activity is selectively

  2. Ice nucleation of an insect lipoprotein ice nucleator (LPIN) correlates with retardation of the hydrogen bond dynamics at the myo-inositol ring.

    PubMed

    Bäumer, Alexander; Duman, John G; Havenith, Martina

    2016-07-28

    Remarkably little is known about the mechanism of action of ice nucleation proteins (INPs), although their ability to trigger ice nucleation could be used in a broad variety of applications. We present CD measurements of an insect lipoprotein ice nucleator (LPIN) which show that the lipoproteins consist of a high amount of β-structures (35%). Terahertz absorption spectroscopy is used to probe the influence of the LPIN on the H-bond network dynamics. We observe a small, but significant THz excess, as an indication of an influence on the H-bond network dynamics. When adding the ice nucleation inhibitor sodium borate, this effect is considerably reduced, similar to that observed before for antifreeze glycoproteins (AFGPs). We propose that myo-inositol, the functional group of phosphatidylinositols, is crucial for the observed change of the H-bond network dynamics of hydration water. This hypothesis is confirmed by additional THz experiments which revealed that the influence of myo-inositol on the hydrogen bond network can be blocked by sodium borate, similar to the case of LPINs. Interestingly, we find a less significant effect when myo-inositol is replaced for chiro- and allo-inositol which underlines the importance of the exact positioning of the OH groups for the interaction with the H-bond network. We propose that a local ordering of water molecules is supporting ice nucleation activity for the LPIN in a similar way to that found for AFP activity in the case of hyperactive insect AFPs. PMID:27373225

  3. Determination of the lithium binding site in inositol monophosphatase, the putative target for lithium therapy, by magic-angle-spinning solid-state NMR.

    PubMed

    Haimovich, Anat; Eliav, Uzi; Goldbourt, Amir

    2012-03-28

    Inositol monophosphatase (IMPase) catalyzes the hydrolysis of inositol monophosphate to inorganic phosphate and inositol. For this catalytic process to occur, Mg(2+) cations must exist in the active site. According to the inositol depletion hypothesis, IMPase activity is assumed to be higher than normal in patients suffering from bipolar disorder. Treatment with Li(+), an inhibitor of IMPase, reduces its activity, but the mechanism by which lithium exerts its therapeutic effects is still at a stage of conjecture. The Escherichia coli SuhB gene product possesses IMPase activity, which is also strongly inhibited by Li(+). It has significant sequence similarity to human IMPase and has most of its key active-site residues. Here we show that by using (7)Li magic-angle-spinning solid-state NMR spectroscopy, including {(13)C}(7)Li dipolar recoupling experiments, the bound form of lithium in the active site of wild-type E. coli SuhB can be unambiguously detected, and on the basis of our data and other biochemical data, lithium binds to site II, coupled to aspartate residues 84, 87, and 212.

  4. Translocation of radiolabeled indole-3-acetic acid and indole-3-acetyl-myo-inositol from kernel to shoot of Zea mays L

    NASA Technical Reports Server (NTRS)

    Chisnell, J. R.; Bandurski, R. S.

    1988-01-01

    Either 5-[3H]indole-3-acetic acid (IAA) or 5-[3H]indole-3-acetyl-myo-inositol was applied to the endosperm of kernels of dark-grown Zea mays seedlings. The distribution of total radioactivity, radiolabeled indole-3-acetic acid, and radiolabeled ester conjugated indole-3-acetic acid, in the shoots was then determined. Differences were found in the distribution and chemical form of the radiolabeled indole-3-acetic acid in the shoot depending upon whether 5-[3H]indole-3-acetic acid or 5-[3H]indole-3-acetyl-myo-inositol was applied to the endosperm. We demonstrated that indole-3-acetyl-myo-inositol applied to the endosperm provides both free and ester conjugated indole-3-acetic acid to the mesocotyl and coleoptile. Free indole-3-acetic acid applied to the endosperm supplies some of the indole-3-acetic acid in the mesocotyl but essentially no indole-3-acetic acid to the coleoptile or primary leaves. It is concluded that free IAA from the endosperm is not a source of IAA for the coleoptile. Neither radioactive indole-3-acetyl-myo-inositol nor IAA accumulates in the tip of the coleoptile or the mesocotyl node and thus these studies do not explain how the coleoptile tip controls the amount of IAA in the shoot.

  5. Thyroid-stimulating hormone stimulates increases in inositol phosphates as well as cyclic AMP in the FRTL-5 rat thyroid cell line.

    PubMed Central

    Field, J B; Ealey, P A; Marshall, N J; Cockcroft, S

    1987-01-01

    Studies were conducted to determine whether thyroid-stimulating hormone (TSH; thyrotropin), a hormone known to increase cytosol concentrations of cyclic AMP, also stimulates the formation of inositol phosphates in thyroid cells. TSH and noradrenaline both stimulated [3H]inositol phosphate formation in a concentration-dependent manner in the rat thyroid cell line, FRTL-5 cells, which had been prelabelled with [3H]inositol. The threshold concentration of TSH required to stimulate inositol phosphate formation was more than 20 munits/ml, which is approx. 10(3)-fold greater than that required for cyclic AMP accumulation and growth in these cells. We also demonstrate that membranes prepared from FRTL-5 cells possess a guanine nucleotide-activatable polyphosphoinositide phosphodiesterase, which suggests that activation of inositide metabolism in these cells may be coupled to receptors by the G-protein, Gp. Our findings suggest that two second-messenger systems exist to mediate the action of TSH in the thyroid. PMID:2827631

  6. Biosynthesis of glycosylphosphatidylinositols of Plasmodium falciparum in a cell-free incubation system: inositol acylation is needed for mannosylation of glycosylphosphatidylinositols.

    PubMed Central

    Gerold, P; Jung, N; Azzouz, N; Freiberg, N; Kobe, S; Schwarz, R T

    1999-01-01

    The structures of glycosylphosphatidylinositols (GPIs) in Plasmodium have been described [Gerold, Schuppert and Schwarz (1994) J. Biol. Chem. 269, 2597-2606]. A detailed understanding of GPI synthesis in Plasmodium is a prerequisite for identifying differences present in biosynthetic pathways of parasites and host cells. A comparison of the biosynthetic pathway of GPIs has revealed differences between mammalian cells and parasitic protozoans. A cell-free incubation system prepared from asexual erythrocytic stages of Plasmodium falciparum, the causative agent of malaria in humans, is capable of synthesizing the same spectrum of GPIs as that found in metabolically labelled parasites. The formation of mannosylated GPIs in the cell-free system is shown to be inhibited by GTP and, unexpectedly, micromolar concentrations of GDP-Man. Lower concentrations of GDP-Man affect the spectrum of GPIs synthesized. The inositol ring of GPIs of P. falciparum is modified by an acyl group. The preferred donor of this fatty acid at the inositol ring is myristoyl-CoA. Inositol acylation has to precede the mannosylation of GPIs because, in the absence of acyl-CoA or CoA, mannosylated GPIs were not detected. Inositol myristoylation is a unique feature of plasmodial GPIs and thus might provide a potential target for drug therapy. PMID:10585859

  7. Identification and quantification of inositol hexa-kis phosphate (IHP) in environmental samples at neutral pH using electro-spray ionization and raman spectroscopy

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phosphorous (P) is a major contributor to eutophication of surface waters, yet a complete understanding of the P cycle remains elusive. Inositol hexa-kis phosphate (IHP) is the primary form of organic P in the environment and has been implicated as an important sink in aquatic and terrestrial sampl...

  8. Prior Activation of Inositol 1,4,5-Trisphosphate Receptors Suppresses the Subsequent Induction of Long-Term Potentiation in Hippocampal CA1 Neurons

    ERIC Educational Resources Information Center

    Fujii, Satoshi; Yamazaki, Yoshihiko; Goto, Jun-Ichi; Fujiwara, Hiroki; Mikoshiba, Katsuhiko

    2016-01-01

    We investigated the role of inositol 1,4,5-trisphosphate receptors (IP3Rs) activated by preconditioning low-frequency afferent stimulation (LFS) in the subsequent induction of long-term potentiation (LTP) in CA1 neurons in hippocampal slices from mature guinea pigs. Induction of LTP in the field excitatory postsynaptic potential or the population…

  9. Hypotonic Activation of the Myo-Inositol Transporter SLC5A3 in HEK293 Cells Probed by Cell Volumetry, Confocal and Super-Resolution Microscopy

    PubMed Central

    Andronic, Joseph; Shirakashi, Ryo; Pickel, Simone U.; Westerling, Katherine M.; Klein, Teresa; Holm, Thorge; Sauer, Markus; Sukhorukov, Vladimir L.

    2015-01-01

    Swelling-activated pathways for myo-inositol, one of the most abundant organic osmolytes in mammalian cells, have not yet been identified. The present study explores the SLC5A3 protein as a possible transporter of myo-inositol in hyponically swollen HEK293 cells. To address this issue, we examined the relationship between the hypotonicity-induced changes in plasma membrane permeability to myo-inositol Pino [m/s] and expression/localization of SLC5A3. Pino values were determined by cell volumetry over a wide tonicity range (100–275 mOsm) in myo-inositol-substituted solutions. While being negligible under mild hypotonicity (200–275 mOsm), Pino grew rapidly at osmolalities below 200 mOsm to reach a maximum of ∼3 nm/s at 100–125 mOsm, as indicated by fast cell swelling due to myo-inositol influx. The increase in Pino resulted most likely from the hypotonicity-mediated incorporation of cytosolic SLC5A3 into the plasma membrane, as revealed by confocal fluorescence microscopy of cells expressing EGFP-tagged SLC5A3 and super-resolution imaging of immunostained SLC5A3 by direct stochastic optical reconstruction microscopy (dSTORM). dSTORM in hypotonic cells revealed a surface density of membrane-associated SLC5A3 proteins of 200–2000 localizations/μm2. Assuming SLC5A3 to be the major path for myo-inositol, a turnover rate of 80–800 myo-inositol molecules per second for a single transporter protein was estimated from combined volumetric and dSTORM data. Hypotonic stress also caused a significant upregulation of SLC5A3 gene expression as detected by semiquantitative RT-PCR and Western blot analysis. In summary, our data provide first evidence for swelling-mediated activation of SLC5A3 thus suggesting a functional role of this transporter in hypotonic volume regulation of mammalian cells. PMID:25756525

  10. Hypotonic activation of the myo-inositol transporter SLC5A3 in HEK293 cells probed by cell volumetry, confocal and super-resolution microscopy.

    PubMed

    Andronic, Joseph; Shirakashi, Ryo; Pickel, Simone U; Westerling, Katherine M; Klein, Teresa; Holm, Thorge; Sauer, Markus; Sukhorukov, Vladimir L

    2015-01-01

    Swelling-activated pathways for myo-inositol, one of the most abundant organic osmolytes in mammalian cells, have not yet been identified. The present study explores the SLC5A3 protein as a possible transporter of myo-inositol in hyponically swollen HEK293 cells. To address this issue, we examined the relationship between the hypotonicity-induced changes in plasma membrane permeability to myo-inositol P ino [m/s] and expression/localization of SLC5A3. P ino values were determined by cell volumetry over a wide tonicity range (100-275 mOsm) in myo-inositol-substituted solutions. While being negligible under mild hypotonicity (200-275 mOsm), P ino grew rapidly at osmolalities below 200 mOsm to reach a maximum of ∼ 3 nm/s at 100-125 mOsm, as indicated by fast cell swelling due to myo-inositol influx. The increase in P ino resulted most likely from the hypotonicity-mediated incorporation of cytosolic SLC5A3 into the plasma membrane, as revealed by confocal fluorescence microscopy of cells expressing EGFP-tagged SLC5A3 and super-resolution imaging of immunostained SLC5A3 by direct stochastic optical reconstruction microscopy (dSTORM). dSTORM in hypotonic cells revealed a surface density of membrane-associated SLC5A3 proteins of 200-2000 localizations/μm2. Assuming SLC5A3 to be the major path for myo-inositol, a turnover rate of 80-800 myo-inositol molecules per second for a single transporter protein was estimated from combined volumetric and dSTORM data. Hypotonic stress also caused a significant upregulation of SLC5A3 gene expression as detected by semiquantitative RT-PCR and Western blot analysis. In summary, our data provide first evidence for swelling-mediated activation of SLC5A3 thus suggesting a functional role of this transporter in hypotonic volume regulation of mammalian cells. PMID:25756525

  11. Hypotonic activation of the myo-inositol transporter SLC5A3 in HEK293 cells probed by cell volumetry, confocal and super-resolution microscopy.

    PubMed

    Andronic, Joseph; Shirakashi, Ryo; Pickel, Simone U; Westerling, Katherine M; Klein, Teresa; Holm, Thorge; Sauer, Markus; Sukhorukov, Vladimir L

    2015-01-01

    Swelling-activated pathways for myo-inositol, one of the most abundant organic osmolytes in mammalian cells, have not yet been identified. The present study explores the SLC5A3 protein as a possible transporter of myo-inositol in hyponically swollen HEK293 cells. To address this issue, we examined the relationship between the hypotonicity-induced changes in plasma membrane permeability to myo-inositol P ino [m/s] and expression/localization of SLC5A3. P ino values were determined by cell volumetry over a wide tonicity range (100-275 mOsm) in myo-inositol-substituted solutions. While being negligible under mild hypotonicity (200-275 mOsm), P ino grew rapidly at osmolalities below 200 mOsm to reach a maximum of ∼ 3 nm/s at 100-125 mOsm, as indicated by fast cell swelling due to myo-inositol influx. The increase in P ino resulted most likely from the hypotonicity-mediated incorporation of cytosolic SLC5A3 into the plasma membrane, as revealed by confocal fluorescence microscopy of cells expressing EGFP-tagged SLC5A3 and super-resolution imaging of immunostained SLC5A3 by direct stochastic optical reconstruction microscopy (dSTORM). dSTORM in hypotonic cells revealed a surface density of membrane-associated SLC5A3 proteins of 200-2000 localizations/μm2. Assuming SLC5A3 to be the major path for myo-inositol, a turnover rate of 80-800 myo-inositol molecules per second for a single transporter protein was estimated from combined volumetric and dSTORM data. Hypotonic stress also caused a significant upregulation of SLC5A3 gene expression as detected by semiquantitative RT-PCR and Western blot analysis. In summary, our data provide first evidence for swelling-mediated activation of SLC5A3 thus suggesting a functional role of this transporter in hypotonic volume regulation of mammalian cells.

  12. Characterization of an Arabidopsis inositol 1,3,4,5,6-pentakisphosphate 2-kinase (AtIPK1)

    PubMed Central

    Sweetman, Dylan; Johnson, Sue; Caddick, Samuel E. K.; Hanke, David E.; Brearley, Charles A.

    2005-01-01

    The metabolic pathway(s) by which plants synthesize InsP6 (inositol 1,2,3,4,5,6-hexakisphosphate) remains largely undefined [Shears (1998) Biochim. Biophys. Acta 1436, 49–67], while the identities of the genes that encode enzymes catalysing individual steps in these pathways are, with the notable exception of myo-inositol phosphate synthase and ZmIpk [Shi, Wang, Wu, Hazebroek, Meeley and Ertl (2003) Plant Physiol. 131, 507–515], unidentified. A yeast enzyme, ScIPK1, catalyses the synthesis of InsP6 by 2-phosphorylation of Ins(1,3,4,5,6)P5 (inositol 1,3,4,5,6-pentakisphosphate). A human orthologue, HsIPK1, is able to substitute for yeast ScIPK1, restoring InsP6 production in a Saccharomyces cerevisiae mutant strain lacking the ScIPK1 open reading frame (ScIpk1Δ). We have identified an Arabidopsis genomic sequence, AtIPK1, encoding an Ins(1,3,4,5,6)P5 2-kinase. Inclusion of the AtIPK1 protein in alignments of amino acid sequences reveals that human and Arabidopis kinases are more similar to each other than to the S. cerevisiae enzyme, and further identifies an additional motif. Recombinant AtIPK1 protein expressed in Escherichia coli catalysed the synthesis of InsP6 from Ins(1,3,4,5,6)P5. The enzyme obeyed Michaelis–Menten kinetics with an apparent Vmax of 35 nmol·min−1·(mg of protein)−1 and a Km for Ins(1,3,4,5,6)P5 of 22 μM at 0.4 mM ATP. RT (reverse transcriptase)–PCR analysis of AtIPK1 transcripts revealed that AtIPK1 is expressed in siliques, leaves and cauline leaves. In situ hybridization experiments further revealed strong expression of AtIPK1 in male and female organs of flower buds. Expression of AtIPK1 protein in an ScIpk1Δ mutant strain restored InsP6 production and rescued the temperature-sensitive growth phenotype of the yeast. PMID:16223361

  13. Alteration of membrane fatty acid composition and inositol phosphate metabolism in HT-29 human colon cancer cells.

    PubMed

    Awad, A B; Fink, C S; Horvath, P J

    1993-01-01

    The present study was designed to investigate the role of membrane fatty acid (FA) composition on inositol phosphate (InsP) release by a human colon tumor cell line. Cells were supplemented for five days in culture with 0, 10, 30, or 100 microM sodium stearate (18:0), linoleate [18:2(omega-6)], or linolineate [18:3(omega-3)]. These FAs were supplied as a complex with FA-free bovine serum albumin. InsP release was examined in these cells with or without stimulation with deoxycholic acid (DCA) after they were labeled with [3H]myoinositol. FA enrichment was found to influence inositol incorporation into membrane lipids. Although 18:0 had no effect, 18:2(omega-6) decreased the incorporation. On the other hand, 18:3(omega-3) increased the incorporation of inositol compared with the cells supplemented with the other FAs, but they were not different from control. Basal release of total InsP was elevated only with supplementation of 10 and 30 microM 18:3(omega-3). FA supplementation with 18:0 at 30 microM and 18:2 at 30 and 100 microM resulted in downregulation of bsal release of InsP. Enrichment of HT-29 cell membranes with polyunsaturated FAs resulted in a significant increase in stimulated release of InsP, but this was not seen with saturated FA supplementation. At 10 microM supplementation, 18:2 had the greatest effect on stimulated InsP release. This effect of 18:2 disappeared at 30 microM. However, the increase in the stimulated InsP release caused by 18:3 occurred at 10 and 30 microM. DCA-stimulated release of InsP was not downregulated by any FA supplementation. This study showed that enrichment of the membranes with polyunsaturated FAs increases the response of the phosphatidylinositol cycle to DCA stimulation. In addition, enrichment with 18:3(omega-3) increases the basal turnover of InsP. It is concluded that alteration of membrane FAs has a profound effect on the phosphatidylinositol cycle.

  14. Comparison of Three Amyloid Assembly Inhibitors: The Sugar scyllo-Inositol, the Polyphenol Epigallocatechin Gallate, and the Molecular Tweezer CLR01

    PubMed Central

    2012-01-01

    Many compounds have been tested as inhibitors or modulators of amyloid β-protein (Aβ) assembly in hope that they would lead to effective, disease-modifying therapy for Alzheimer’s disease (AD). These compounds typically were either designed to break apart β-sheets or selected empirically. Two such compounds, the natural inositol derivative scyllo-inositol and the green-tea-derived flavonoid epigallocatechin gallate (EGCG), currently are in clinical trials. Similar to most of the compounds tested thus far, the mechanism of action of scyllo-inositol and EGCG is not understood. Recently, we discovered a novel family of assembly modulators, Lys-specific molecular tweezers, which act by binding specifically to Lys residues and modulate the self-assembly of amyloid proteins, including Aβ, into formation of nontoxic oligomers by a process-specific mechanism (Sinha, S., Lopes, D. H., Du, Z., Pang, E. S., Shanmugam, A., Lomakin, A., Talbiersky, P., Tennstaedt, A., McDaniel, K., Bakshi, R., Kuo, P. Y., Ehrmann, M., Benedek, G. B., Loo, J. A., Klarner, F. G., Schrader, T., Wang, C., and Bitan, G. (2011) Lysine-specific molecular tweezers are broad-spectrum inhibitors of assembly and toxicity of amyloid proteins. J. Am. Chem. Soc.133, 16958–16969). Here, we compared side-by-side the capability of scyllo-inositol, EGCG, and the molecular tweezer CLR01 to inhibit Aβ aggregation and toxicity. We found that EGCG and CLR01 had comparable activity whereas scyllo-inositol was a weaker inhibitor. Exploration of the binding of EGCG and CLR01 to Aβ using heteronuclear solution-state NMR showed that whereas CLR01 bound to the two Lys and single Arg residues in Aβ monomers, only weak, nonspecific binding was detected for EGCG, leaving the binding mode of the latter unresolved. PMID:22860214

  15. Diminished agonist-stimulated inositol trisphosphate generation blocks stimulus-secretion coupling in mouse pancreatic acini during diet-induced experimental pancreatitis

    SciTech Connect

    Powers, R.E.; Saluja, A.K.; Houlihan, M.J.; Steer, M.L.

    1986-05-01

    Young female mice fed a choline-deficient, ethionine-supplemented (CDE) diet rapidly develop acute hemorrhagic pancreatitis. We have observed that pancreatic acini prepared from these mice are unable to secrete amylase in response to addition of the cholinergic agonist carbachol, although they retain the ability to secrete amylase in response to the Ca2+ ionophore A23187. The CDE diet does not alter the binding characteristics (Kd or the maximal number of binding sites) for muscarinic cholinergic receptors as tested using the antagonist (/sup 3/H)N-methylscopolamine nor the competition for this binding by carbachol. Addition of carbachol to acini prepared from mice fed the CDE diet does not result in as marked an increase in cytosolic free Ca2+ levels as that noted in control samples (evaluated using quin2 fluorescence). These observations indicate that the CDE diet interferes with stimulus-secretion coupling in mouse pancreatic acini at a step subsequent to hormone-receptor binding and prior to Ca2+ release. This conclusion is confirmed by our finding that the hormone-stimulated generation of (/sup 3/H)inositol phosphates (inositol trisphosphate, inositol bisphosphate, and inositol monophosphate) from acini labeled with (/sup 3/H)myoinositol is markedly reduced in acini prepared from mice fed the CDE diet. This reduction is not due to a decrease in phosphatidylinositol-4,5-bisphosphate. This communication represents the first report of a system in which a blockade of inositol phosphate generation can be related to a physiologic defect and pathologic lesion.

  16. Short-Term Treatment with Cell Wall Degrading Enzymes Increases the Activity of the Inositol Phospholipid Kinases and the Vanadate-Sensitive ATPase of Carrot Cells 1

    PubMed Central

    Chen, Qiuyun; Boss, Wendy F.

    1990-01-01

    Treating carrot (Daucus carota L.) suspension culture cells with a mixture of cell wall degrading enzymes, Driselase, resulted in an increase in the percentage of [3H]phosphatidylinositol bisphosphate. Analysis of the lipid kinase activities in the isolated plasma membranes after whole cell treatment indicated that treatment with Driselase (2% weight/volume; the equivalent of 340 units per milliliter of hemicellulase and 400 units per milliliter of cellulase activity) or treatment with hemicellulase (31.7% weight/volume, 20.7 units per milliliter) resulted in an increase in the inositol phospholipid kinase activity. However, treatment with cellulase alone had no effect at 0.5% (weight/volume, 17.2 units per milliliter) or inhibited the kinase activity at 1% (weight/volume, 34.4 units per milliliter). The active stimulus in Driselase was heat sensitive. The plasma membrane vanadate-sensitive ATPase activity also increased when the cells were treated with Driselase. A time course study indicated that both the inositol phospholipid kinases and the plasma membrane vanadate-sensitive ATPase responded to as little as 5 seconds of treatment with 2% Driselase. However, at the lowest concentration of Driselase (0.04%, weight/volume) that resulted in an increase in inositol phospholipid kinase activity, the ATPase activity was not affected. Because inositol phospholipids have been shown to activate the vanadate-sensitive ATPase from plants (AR Memon, Q Chen, WF Boss [1989] Biochem Biophys Res Commun 162: 1295-1301), a stimulus-response pathway involving both the inositol phospholipid kinases and the plasma membrane vanadate-sensitive ATPase activity is discussed. Images Figure 2 Figure 6 PMID:16667922

  17. Restoration of the di-myo-inositol-phosphate pathway in the piezo-hyperthermophilic archaeon Thermococcus barophilus.

    PubMed

    Cario, Anaïs; Mizgier, Alex; Thiel, Axel; Jebbar, Mohamed; Oger, Phil M

    2015-11-01

    Most Thermococcales accumulate di-myo-inositol-phosphate (DIP) as an organic solute as a response to heat stress. We have studied the accumulation of this osmolyte in the high-hydrostatic pressure adapted hyperthermophile Thermococcus barophilus. We found no accumulation of DIP under any of the stress conditions tested, although this archaeon harbors the 3 DIP synthesis genes. Lack of synthesis is due to the lack of expression of TERMP_01135 coding for the second step of DIP synthesis. In contrast to other species, the T. barophilus synthesis operon is interrupted by a four gene locus, in reverse orientation. Restoring an operon like structure at the DIP locus restored DIP synthesis, but did not have an impact on growth characteristics, suggesting that other mechanisms have evolved in this organism to cope with heat stress. PMID:26005095

  18. Synaptotagmin 1 causes phosphatidyl inositol lipid-dependent actin remodeling in cultured non-neuronal and neuronal cells

    SciTech Connect

    Johnsson, Anna-Karin; Karlsson, Roger

    2012-01-15

    Here we demonstrate that a dramatic actin polymerizing activity caused by ectopic expression of the synaptic vesicle protein synaptotagmin 1 that results in extensive filopodia formation is due to the presence of a lysine rich sequence motif immediately at the cytoplasmic side of the transmembrane domain of the protein. This polybasic sequence interacts with anionic phospholipids in vitro, and, consequently, the actin remodeling caused by this sequence is interfered with by expression of a phosphatidyl inositol (4,5)-bisphosphate (PIP2)-targeted phosphatase, suggesting that it intervenes with the function of PIP2-binding actin control proteins. The activity drastically alters the behavior of a range of cultured cells including the neuroblastoma cell line SH-SY5Y and primary cortical mouse neurons, and, since the sequence is conserved also in synaptotagmin 2, it may reflect an important fine-tuning role for these two proteins during synaptic vesicle fusion and neurotransmitter release.

  19. Inhibition of the Inositol Kinase Itpkb Augments Calcium Signaling in Lymphocytes and Reveals a Novel Strategy to Treat Autoimmune Disease

    PubMed Central

    Miller, Andrew T.; Dahlberg, Carol; Sandberg, Mark L.; Wen, Ben G.; Beisner, Daniel R.; Hoerter, John A. H.; Parker, Albert; Schmedt, Christian; Stinson, Monique; Avis, Jacqueline; Cienfuegos, Cynthia; McPate, Mark; Tranter, Pamela; Gosling, Martin; Groot-Kormelink, Paul J.; Dawson, Janet; Pan, Shifeng; Tian, Shin-Shay; Seidel, H. Martin; Cooke, Michael P.

    2015-01-01

    Emerging approaches to treat immune disorders target positive regulatory kinases downstream of antigen receptors with small molecule inhibitors. Here we provide evidence for an alternative approach in which inhibition of the negative regulatory inositol kinase Itpkb in mature T lymphocytes results in enhanced intracellular calcium levels following antigen receptor activation leading to T cell death. Using Itpkb conditional knockout mice and LMW Itpkb inhibitors these studies reveal that Itpkb through its product IP4 inhibits the Orai1/Stim1 calcium channel on lymphocytes. Pharmacological inhibition or genetic deletion of Itpkb results in elevated intracellular Ca2+ and induction of FasL and Bim resulting in T cell apoptosis. Deletion of Itpkb or treatment with Itpkb inhibitors blocks T-cell dependent antibody responses in vivo and prevents T cell driven arthritis in rats. These data identify Itpkb as an essential mediator of T cell activation and suggest Itpkb inhibition as a novel approach to treat autoimmune disease. PMID:26121493

  20. Regulation of inositol 1,4,5-trisphosphate-induced Ca2+ release by reversible phosphorylation and dephosphorylation

    PubMed Central

    VANDERHEYDEN, Veerle; DEVOGELAERE, Benoit; MISSIAEN, Ludwig; DE SMEDT, Humbert; BULTYNCK, Geert; PARYS, Jan B.

    2009-01-01

    Summary The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) is a universal intracellular Ca2+-release channel. It is activated after cell stimulation and plays a crucial role in the initiation and propagation of the complex spatio-temporal Ca2+ signals that control cellular processes as different as fertilization, cell division, cell migration, differentiation, metabolism, muscle contraction, secretion, neuronal processing, and ultimately cell death. To achieve these various functions, often in a single cell, exquisite control of the Ca2+ release is needed. This review aims to highlight how protein kinases and protein phosphatases can interact with the IP3R or with associated proteins and so provide a large potential for fine tuning the Ca2+-release activity and for creating efficient Ca2+ signals in subcellular microdomains. PMID:19133301

  1. Regulation of inositol 1,4,5-trisphosphate-induced Ca2+ release by reversible phosphorylation and dephosphorylation.

    PubMed

    Vanderheyden, Veerle; Devogelaere, Benoit; Missiaen, Ludwig; De Smedt, Humbert; Bultynck, Geert; Parys, Jan B

    2009-06-01

    The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) is a universal intracellular Ca2+-release channel. It is activated after cell stimulation and plays a crucial role in the initiation and propagation of the complex spatio-temporal Ca2+ signals that control cellular processes as different as fertilization, cell division, cell migration, differentiation, metabolism, muscle contraction, secretion, neuronal processing, and ultimately cell death. To achieve these various functions, often in a single cell, exquisite control of the Ca2+ release is needed. This review aims to highlight how protein kinases and protein phosphatases can interact with the IP3R or with associated proteins and so provide a large potential for fine tuning the Ca2+-release activity and for creating efficient Ca2+ signals in subcellular microdomains.

  2. Restoration of the di-myo-inositol-phosphate pathway in the piezo-hyperthermophilic archaeon Thermococcus barophilus.

    PubMed

    Cario, Anaïs; Mizgier, Alex; Thiel, Axel; Jebbar, Mohamed; Oger, Phil M

    2015-11-01

    Most Thermococcales accumulate di-myo-inositol-phosphate (DIP) as an organic solute as a response to heat stress. We have studied the accumulation of this osmolyte in the high-hydrostatic pressure adapted hyperthermophile Thermococcus barophilus. We found no accumulation of DIP under any of the stress conditions tested, although this archaeon harbors the 3 DIP synthesis genes. Lack of synthesis is due to the lack of expression of TERMP_01135 coding for the second step of DIP synthesis. In contrast to other species, the T. barophilus synthesis operon is interrupted by a four gene locus, in reverse orientation. Restoring an operon like structure at the DIP locus restored DIP synthesis, but did not have an impact on growth characteristics, suggesting that other mechanisms have evolved in this organism to cope with heat stress.

  3. A Universal Role for Inositol 1,4,5-Trisphosphate-Mediated Signaling in Plant Gravitropism1[W

    PubMed Central

    Perera, Imara Y.; Hung, Chiu-Yueh; Brady, Shari; Muday, Gloria K.; Boss, Wendy F.

    2006-01-01

    Inositol 1,4,5-trisphosphate (InsP3) has been implicated in the early signaling events of plants linking gravity sensing to the initiation of the gravitropic response. However, at present, the contribution of the phosphoinositide signaling pathway in plant gravitropism is not well understood. To delineate the role of InsP3 in plant gravitropism, we generated Arabidopsis (Arabidopsis thaliana) plants constitutively expressing the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme that specifically hydrolyzes InsP3. The transgenic plants show no significant differences in growth and life cycle compared to wild-type plants, although basal InsP3 levels are reduced by greater than 90% compared to wild-type plants. With gravistimulation, InsP3 levels in inflorescence stems of transgenic plants show no detectable change, whereas in wild-type plant inflorescences, InsP3 levels increase approximately 3-fold within the first 5 to 15 min of gravistimulation, preceding visible bending. Furthermore, gravitropic bending of the roots, hypocotyls, and inflorescence stems of the InsP 5-ptase transgenic plants is reduced by approximately 30% compared with the wild type. Additionally, the cold memory response of the transgenic plants is attenuated, indicating that InsP3 contributes to gravisignaling in the cold. The transgenic roots were shown to have altered calcium sensitivity in controlling gravitropic response, a reduction in basipetal indole-3-acetic acid transport, and a delay in the asymmetric auxin-induced β-glucuronidase expression with gravistimulation as compared to the controls. The compromised gravitropic response in all the major axes of growth in the transgenic Arabidopsis plants reveals a universal role for InsP3 in the gravity signal transduction cascade of plants. PMID:16384898

  4. Insulin-Mimicking Bioactivities of Acylated Inositol Glycans in Several Mouse Models of Diabetes with or without Obesity

    PubMed Central

    Suzuki, Susumu; Suzuki, Chitose; Hinokio, Yoshinori; Ishigaki, Yasushi; Katagiri, Hideki; Kanzaki, Makoto; Azev, Viatcheslav N.; Chakraborty, Nilanjana; d'Alarcao, Marc

    2014-01-01

    Insulin-mimetic species of low molecular weight are speculated to mediate some intracellular insulin actions. These inositol glycans, which are generated upon insulin stimulation from glycosylphosphatidylinositols, might control the activity of a multitude of insulin effector enzymes. Acylated inositol glycans (AIGs) are generated by cleavage of protein-free GPI precursors through the action of GPI-specific phospholipase C (GPI-PLC) and D (GPI-PLD). We synthesized AIGs (IG-1, IG-2, IG-13, IG-14, and IG-15) and then evaluated their insulin-mimicking bioactivities. IG-1 significantly stimulated glycogen synthesis and lipogenesis in 3T3-L1 adipocytes and rat isolated adipocytes dose-dependently. IG-2 significantly stimulated lipogenesis in rat isolated adipocytes dose-dependently. IG-15 also enhanced glycogen synthesis and lipogenesis in 3T3-L1 adipocytes. The administration of IG-1 decreased plasma glucose, increased glycogen content in liver and skeletal muscles and improved glucose tolerance in C57B6N mice with normal diets. The administration of IG-1 decreased plasma glucose in STZ-diabetic C57B6N mice. The treatment of IG-1 decreased plasma glucose, increased glycogen content in liver and skeletal muscles and improved glucose tolerance in C57B6N mice with high fat-diets and db/db mice. The long-term treatment of IG-1 decreased plasma glucose and reduced food intake and body weight in C57B6N mice with high fat-diets and ob/ob mice. Thus, IG-1 has insulin-mimicking bioactivities and improves glucose tolerance in mice models of diabetes with or without obesity. PMID:24971987

  5. Converting NAD-specific inositol dehydrogenase to an efficient NADP-selective catalyst, with a surprising twist.

    PubMed

    Zheng, Hongyan; Bertwistle, Drew; Sanders, David A R; Palmer, David R J

    2013-08-27

    myo-Inositol dehydrogenase (IDH, EC 1.1.1.18) from Bacillus subtilis converts myo-inositol to scyllo-inosose and is strictly dependent on NAD for activity. We sought to alter the coenzyme specificity to generate an NADP-dependent enzyme in order to enhance our understanding of coenzyme selectivity and to create an enzyme capable of recycling NADP in biocatalytic processes. Examination of available structural information related to the GFO/MocA/IDH family of dehydrogenases and precedents for altering coenzyme selectivity allowed us to select residues for substitution, and nine single, double, and triple mutants were constructed. Mutagenesis experiments with B. subtilis IDH proved extremely successful; the double mutant D35S/V36R preferred NADP to NAD by a factor of 5. This mutant is an excellent catalyst with a second-order rate constant with respect to NADP of 370 000 s⁻¹ M⁻¹, and the triple mutant A12K/D35S/V36R had a value of 570 000 s⁻¹ M⁻¹, higher than that of the wild-type IDH with NAD. The high-resolution X-ray crystal structure of the double mutant A12K/D35S was solved in complex with NADP. Surprisingly, the binding of the coenzyme is altered such that although the nicotinamide ring maintains the required position for catalysis, the coenzyme has twisted by nearly 90°, so the adenine moiety no longer binds to a hydrophobic cleft in the Rossmann fold as in the wild-type enzyme. This change in binding conformation has not previously been observed in mutated dehydrogenases.

  6. Up-regulation of phosphoinositide metabolism in tobacco cells constitutively expressing the human type I inositol polyphosphate 5-phosphatase

    NASA Technical Reports Server (NTRS)

    Perera, Imara Y.; Love, John; Heilmann, Ingo; Thompson, William F.; Boss, Wendy F.; Brown, C. S. (Principal Investigator)

    2002-01-01

    To evaluate the impact of suppressing inositol 1,4,5-trisphosphate (InsP(3)) in plants, tobacco (Nicotiana tabacum) cells were transformed with the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme which specifically hydrolyzes InsP(3). The transgenic cell lines showed a 12- to 25-fold increase in InsP 5-ptase activity in vitro and a 60% to 80% reduction in basal InsP(3) compared with wild-type cells. Stimulation with Mas-7, a synthetic analog of the wasp venom peptide mastoparan, resulted in an approximately 2-fold increase in InsP(3) in both wild-type and transgenic cells. However, even with stimulation, InsP(3) levels in the transgenic cells did not reach wild-type basal values, suggesting that InsP(3) signaling is compromised. Analysis of whole-cell lipids indicated that phosphatidylinositol 4,5-bisphosphate (PtdInsP(2)), the lipid precursor of InsP(3), was greatly reduced in the transgenic cells. In vitro assays of enzymes involved in PtdInsP(2) metabolism showed that the activity of the PtdInsP(2)-hydrolyzing enzyme phospholipase C was not significantly altered in the transgenic cells. In contrast, the activity of the plasma membrane PtdInsP 5 kinase was increased by approximately 3-fold in the transgenic cells. In vivo labeling studies revealed a greater incorporation of (32)P into PtdInsP(2) in the transgenic cells compared with the wild type, indicating that the rate of PtdInsP(2) synthesis was increased. These studies show that the constitutive expression of the human type I InsP 5-ptase in tobacco cells leads to an up-regulation of the phosphoinositide pathway and highlight the importance of PtdInsP(2) synthesis as a regulatory step in this system.

  7. Insulin-mimicking bioactivities of acylated inositol glycans in several mouse models of diabetes with or without obesity.

    PubMed

    Suzuki, Susumu; Suzuki, Chitose; Hinokio, Yoshinori; Ishigaki, Yasushi; Katagiri, Hideki; Kanzaki, Makoto; Azev, Viatcheslav N; Chakraborty, Nilanjana; d'Alarcao, Marc

    2014-01-01

    Insulin-mimetic species of low molecular weight are speculated to mediate some intracellular insulin actions. These inositol glycans, which are generated upon insulin stimulation from glycosylphosphatidylinositols, might control the activity of a multitude of insulin effector enzymes. Acylated inositol glycans (AIGs) are generated by cleavage of protein-free GPI precursors through the action of GPI-specific phospholipase C (GPI-PLC) and D (GPI-PLD). We synthesized AIGs (IG-1, IG-2, IG-13, IG-14, and IG-15) and then evaluated their insulin-mimicking bioactivities. IG-1 significantly stimulated glycogen synthesis and lipogenesis in 3T3-L1 adipocytes and rat isolated adipocytes dose-dependently. IG-2 significantly stimulated lipogenesis in rat isolated adipocytes dose-dependently. IG-15 also enhanced glycogen synthesis and lipogenesis in 3T3-L1 adipocytes. The administration of IG-1 decreased plasma glucose, increased glycogen content in liver and skeletal muscles and improved glucose tolerance in C57B6N mice with normal diets. The administration of IG-1 decreased plasma glucose in STZ-diabetic C57B6N mice. The treatment of IG-1 decreased plasma glucose, increased glycogen content in liver and skeletal muscles and improved glucose tolerance in C57B6N mice with high fat-diets and db/db mice. The long-term treatment of IG-1 decreased plasma glucose and reduced food intake and body weight in C57B6N mice with high fat-diets and ob/ob mice. Thus, IG-1 has insulin-mimicking bioactivities and improves glucose tolerance in mice models of diabetes with or without obesity.

  8. Computational Analysis Reveals a Successive Adaptation of Multiple Inositol Polyphosphate Phosphatase 1 in Higher Organisms Through Evolution

    PubMed Central

    Kilaparty, Surya P; Singh, Awantika; Baltosser, William H; Ali, Nawab

    2014-01-01

    Multiple inositol polyphosphate phosphatase 1 (Minpp1) in higher organisms dephosphorylates InsP6, the most abundant inositol phosphate. It also dephosphorylates less phosphorylated InsP5 and InsP4 and more phosphorylated InsP7 or InsP8. Minpp1 is classified as a member of the histidine acid phosphatase super family of proteins with functional resemblance to phytases found in lower organisms. This study took a bioinformatics approach to explore the extent of evolutionary diversification in Minpp1 structure and function in order to understand its physiological relevance in higher organisms. The human Minpp1 amino acid (AA) sequence was BLAST searched against available national protein databases. Phylogenetic analysis revealed that Minpp1 was widely distributed from lower to higher organisms. Further, we have identified that there exist four isoforms of Minpp1. Multiple computational tools were used to identify key functional motifs and their conservation among various species. Analyses showed that certain motifs predominant in higher organisms were absent in lower organisms. Variation in AA sequences within motifs was also analyzed. We found that there is diversification of key motifs and thus their functions present in Minpp1 from lower organisms to higher organisms. Another interesting result of this analysis was the presence of a glucose-1-phosphate interaction site in Minpp1; the functional significance of which has yet to be determined experimentally. The overall findings of our study point to an evolutionary adaptability of Minpp1 functions from lower to higher life forms. PMID:25574123

  9. Differentially expressed myo-inositol monophosphatase gene (CaIMP) in chickpea (Cicer arietinum L.) encodes a lithium-sensitive phosphatase enzyme with broad substrate specificity and improves seed germination and seedling growth under abiotic stresses.

    PubMed

    Saxena, Saurabh C; Salvi, Prafull; Kaur, Harmeet; Verma, Pooja; Petla, Bhanu Prakash; Rao, Venkateswara; Kamble, Nitin; Majee, Manoj

    2013-12-01

    myo-Inositol monophosphatase (IMP) is an essential enzyme in the myo-inositol metabolic pathway where it primarily dephosphorylates myo-inositol 1-phosphate to maintain the cellular inositol pool which is important for many metabolic and signalling pathways in plants. The stress-induced increased accumulation of inositol has been reported in a few plants including chickpea; however, the role and regulation of IMP is not well defined in response to stress. In this work, it has been shown that IMP activity is distributed in all organs in chickpea and was noticeably enhanced during environmental stresses. Subsequently, using degenerate oligonucleotides and RACE strategy, a full-length IMP cDNA (CaIMP) was cloned and sequenced. Biochemical study revealed that CaIMP encodes a lithium-sensitive phosphatase enzyme with broad substrate specificity, although maximum activity was observed with the myo-inositol 1-phosphate and l-galactose 1-phosphate substrates. Transcript analysis revealed that CaIMP is differentially expressed and regulated in different organs, stresses and phytohormones. Complementation analysis in Arabidopsis further confirmed the role of CaIMP in l-galactose 1-phosphate and myo-inositol 1-phosphate hydrolysis and its participation in myo-inositol and ascorbate biosynthesis. Moreover, Arabidopsis transgenic plants over-expressing CaIMP exhibited improved tolerance to stress during seed germination and seedling growth, while the VTC4/IMP loss-of-function mutants exhibited sensitivity to stress. Collectively, CaIMP links various metabolic pathways and plays an important role in improving seed germination and seedling growth, particularly under stressful environments.

  10. Differentially expressed myo-inositol monophosphatase gene (CaIMP) in chickpea (Cicer arietinum L.) encodes a lithium-sensitive phosphatase enzyme with broad substrate specificity and improves seed germination and seedling growth under abiotic stresses.

    PubMed

    Saxena, Saurabh C; Salvi, Prafull; Kaur, Harmeet; Verma, Pooja; Petla, Bhanu Prakash; Rao, Venkateswara; Kamble, Nitin; Majee, Manoj

    2013-12-01

    myo-Inositol monophosphatase (IMP) is an essential enzyme in the myo-inositol metabolic pathway where it primarily dephosphorylates myo-inositol 1-phosphate to maintain the cellular inositol pool which is important for many metabolic and signalling pathways in plants. The stress-induced increased accumulation of inositol has been reported in a few plants including chickpea; however, the role and regulation of IMP is not well defined in response to stress. In this work, it has been shown that IMP activity is distributed in all organs in chickpea and was noticeably enhanced during environmental stresses. Subsequently, using degenerate oligonucleotides and RACE strategy, a full-length IMP cDNA (CaIMP) was cloned and sequenced. Biochemical study revealed that CaIMP encodes a lithium-sensitive phosphatase enzyme with broad substrate specificity, although maximum activity was observed with the myo-inositol 1-phosphate and l-galactose 1-phosphate substrates. Transcript analysis revealed that CaIMP is differentially expressed and regulated in different organs, stresses and phytohormones. Complementation analysis in Arabidopsis further confirmed the role of CaIMP in l-galactose 1-phosphate and myo-inositol 1-phosphate hydrolysis and its participation in myo-inositol and ascorbate biosynthesis. Moreover, Arabidopsis transgenic plants over-expressing CaIMP exhibited improved tolerance to stress during seed germination and seedling growth, while the VTC4/IMP loss-of-function mutants exhibited sensitivity to stress. Collectively, CaIMP links various metabolic pathways and plays an important role in improving seed germination and seedling growth, particularly under stressful environments. PMID:24123252

  11. G(q/11) is involved in insulin-stimulated inositol phosphoglycan putative mediator generation in rat liver membranes: co-localization of G(q/11) with the insulin receptor in membrane vesicles.

    PubMed

    Sleight, S; Wilson, B A; Heimark, D B; Larner, J

    2002-07-12

    Insulin signaling to generate inositol phosphoglycans (IPGs) was demonstrated to occur via the participation of the heterotrimeric G-proteins G(q/11). IPGs were measured as two specific inositol markers, myo-inositol and chiro-inositol after strong acid hydrolysis. Insulin and Pasteurella multocida toxin (PMT) generated both myo-inositol and chiro-inositol IPGs in a dose-dependent manner. PMT has been shown to activate G(q) specifically. Insulin action was abrogated by pre-treatment with anti G(q/11) antibody. Western blotting demonstrated the enrichment of both insulin receptor beta subunit and G(q/11) in the liver membrane vesicles. Vesicles also contained clathrin, caveolin PLC beta 1 and PLC Delta. Immunogold staining revealed the co-localization of both insulin receptor beta subunit and G(q/11) in an approximate stochiometric ratio of 1:3. No vesicles were detected with either component alone. The present and considerable published data provide strong evidence for insulin signaling both via a tyrosine kinase cascade mechanism and via heterotrimeric G-protein interactions. PMID:12150987

  12. Determination of mannitol sorbitol and myo-inositol in olive tree roots and rhizospheric soil by gas chromatography and effect of severe drought conditions on their profiles.

    PubMed

    Mechri, Beligh; Tekaya, Meriem; Cheheb, Hechmi; Hammami, Mohamed

    2015-01-01

    This study reports a method for the analysis of mannitol, sorbitol and myo-inositol in olive tree roots and rhizospheric soil with gas chromatography. The analytical method consists of extraction with a mixture of dichloromethane:methanol (2:1, v/v) for soil samples and a mixture of ethanol:water (80:20) for root samples, silylation using pyridine, hexamethyldisilazane (HMDS) and trimethylchlorosilane (TMCS). The recovery of mannitol sorbitol and myo-inositol (for extraction and analysis in dichloromethane:methanol and ethanol:water) was acceptable and ranged from 100.3 to 114.7%. The time of analysis was <24 min. Among identified polyols extracted from rhizosphere and roots of olive plants, mannitol was the major compound. A marked increase in mannitol content occurred in rhizosphere and roots of water-stressed plants, suggesting a much broader role of mannitol in stress response based on its ability to act as a compatible solute.

  13. Inositol 1,4,5-trisphosphate and diacylglycerol mimic bradykinin effects on mouse neuroblastoma x rat glioma hybrid cells.

    PubMed Central

    Brown, D A; Higashida, H

    1988-01-01

    1. The role of inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG) as possible mediators of the membrane current responses of NG108-15 neuroblastoma x glioma hybrid cells to bradykinin (BK, Brown & Higashida, 1988b) has been tested using intracellular ionophoresis of InsP3 and external application of phorbol dibutyrate (PDBu) and 1-oleoyl-2-acetylglycerol (OAG). 2. Intracellular ionophoresis of InsP3 into cells clamped at -30 to -50 mV produced (i) a transient outward current, (ii) a transient outward current followed by an inward current, or (iii) an inward current. All currents were accompanied by an increased input conductance. 3. The transient outward current reversed at between -80 and -90 mV. The reversal potential was shifted to more positive potentials on raising extracellular [K+], suggesting that it resulted from an increased K+ conductance. 4. The outward current was inhibited by apamin (0.4 microM) or d-tubocurarine (0.2-0.5 mM); these drugs also inhibit the outward current produced by BK or by intracellular Ca2+ injections (Brown & Higashida, 1988 a, b). The outward current was also slowly reduced in 0 mM [Ca2+] or 0.5 mM [Cd2+] plus 2 mM [Co2+] solution. 5. Ionophoretic injection of inositol 1,3,4-trisphosphate and inositol 1,3,4,5-tetrakisphosphate, guanosine trisphosphate or inorganic phosphate did not evoke an outward current but produced only an inward current with an increased conductance, reversing at between -10 and -20 mV. 6. Bath application of PDBu (10 nM-1 microM) or OAG (1-10 microM) produced an inward current with a fall in input conductance. The inward current was voltage dependent and was accompanied by an inhibition of the time-dependent current relaxations associated with activation or deactivation of the voltage-dependent K+ current, IM. 7. PDBu did not clearly reduce the Ca2+ current or the Ca2+-dependent K+ current recorded in these cells. During superfusion with PDBu, the outward current produced by intracellular

  14. Enzyme activities of Arabidopsis inositol polyphosphate kinases AtIPK2α and AtIPK2β are involved in pollen development, pollen tube guidance and embryogenesis.

    PubMed

    Zhan, Huadong; Zhong, Yujiao; Yang, Zhongnan; Xia, Huijun

    2015-06-01

    Inositol polyphosphate kinase (IPK2) is a key component of inositol polyphosphate signaling. There are two highly homologous inositol polyphosphate kinases (AtIPK2α and AtIPK2β) in Arabidopsis. Previous studies that overexpressed or reduced the expression of AtIPK2α and AtIPK2β revealed their roles in auxiliary shoot branching, abiotic stress responses and root growth. Here, we report that AtIPK2α and AtIPK2β act redundantly during pollen development, pollen tube guidance and embryogenesis. Single knock-out mutants of atipk2α and atipk2β were indistinguishable from the wild type, whereas the atipk2α atipk2β double mutant could not be obtained. Detailed genetic and cytological investigations showed that the mutation of AtIPK2α and AtIPK2β resulted in severely reduced transmission of male gametophyte as a result of abnormal pollen development and defective pollen tube guidance. In addition, the early embryo development of the atipk2α atipk2β double mutant was also aborted. Expressing either catalytically inactive or substrate specificity-altered variants of AtIPK2β could not rescue the male gametophyte and embryogenesis defects of the atipk2α atipk2β double mutant, implying that the kinase activity of AtIPK2 is required for pollen development, pollen tube guidance and embryogenesis. Taken together, our results provide genetic evidence for the requirement of inositol polyphosphate signaling in plant sexual reproduction.

  15. Metabolic Studies on Intermediates in the myo-Inositol Oxidation Pathway in Lilium longiflorum Pollen: III. Polysaccharidic Origin of Labeled Glucose.

    PubMed

    Rosenfield, C L; Loewus, F A

    1978-01-01

    On the basis of solubility, hydrolysis by glucoamylase (EC 3.2.1.3), and monomeric composition, starch appears to be the major glucose-containing, hot water-soluble polysaccharide that is labeled when germinated lily (Lilium longiflorum Thunb., cv. Ace) pollen is grown in the presence of myo-[2-(3)H]inositol, d-[R5,S5-(3)H]xylose, or l-[1-(14)C]arabinose.

  16. Systematic analysis of time-dependent neural effects of soluble amyloid β oligomers in culture and in vivo: prevention by scyllo-inositol

    PubMed Central

    Jin, Ming; Selkoe, Dennis J.

    2015-01-01

    Alzheimer’s disease (AD) is currently being addressed by intensive investment in pre-clinical and clinical research on the amyloid hypothesis, but concern remains about the validity of the concept that soluble Aβ oligomers are principally responsible for initiating AD phenotypes. Here, we apply well-defined Aβ oligomers isolated from AD brains or made synthetically to document a systematic accrual of first subtle and then more profound changes in certain synaptic proteins in both primary neuronal cultures and behaving adult mice. Among the first (within hours) synaptic changes are selective decreases in surface levels of certain (e.g., GluA1) but not other (e.g., GluN2B) glutamate receptors and subtle microglial activation. After 4 days, numerous additional synaptic proteins are altered. Moreover, Aβ oligomers induce hyperphosphorylation of tau and subsequent neuritic dystrophy. All changes are prevented by scyllo-inositol in a dose-and stereoisomer-specific manner. Mechanistically, scyllo-inositol interferes quantitatively with the binding of Aβ oligomers to plasma membranes. These comprehensive analyses in culture and in vivo provide direct evidence that diffusible oligomers of human Aβ (without plaques) induce multiple phenotypic changes in healthy neurons, indicating their role as principal endogenous cytotoxins in AD. Our data recommend a re-examination of scyllo-inositol as an anti-oligomer therapeutic in humans with early AD. PMID:26054438

  17. Inositol's and other nutraceuticals' synergistic actions counteract insulin resistance in polycystic ovarian syndrome and metabolic syndrome: state-of-the-art and future perspectives.

    PubMed

    Paul, Cristiana; Laganà, Antonio Simone; Maniglio, Paolo; Triolo, Onofrio; Brady, David M

    2016-06-01

    The incidence of metabolic syndrome (MetS), type II diabetes (T2D) and polycystic ovarian syndrome (PCOS) has been progressively increasing. Insulin resistance (InsR) seems to play a key role in a majority of phenotypes of these conditions, altering metabolic homeostasis, within muscle, liver, adipose and other tissues. Hyperinsulinemia is often associated with InsR and causes hormonal imbalances especially within ovaries and adrenals. Inositol is a polyalcohol, naturally occurring as nine stereoisomers, including D-chiro-inositol (DCI) and myo-inositol (MI), which have prominent roles in the metabolism of glucose and free fatty acids. MI and DCI have been classified as insulin-sensitizers and seem to adequately counteract several InsR-related metabolic alterations with a safe nutraceutical profile. Based on our analysis of selected studies that investigated MI and/or DCI, we conclude that supplementation with MI and/or DCI complement each other in their metabolic actions and act in synergy with other insulin sensitizing drugs and/or nutraceuticals. Nevertheless, considering the possible severe bias due to different methodologies across published studies, we conclude that there is a need for further studies on larger cohorts and with greater statistical power. These should further clarify outcomes and suitable therapeutic dosages of MI and DCI, possibly based on each patient's clinical status. PMID:26927948

  18. Molecular and biochemical identification of inositol 1,3,4,5,6-pentakisphosphate 2-kinase encoding mRNA variants in castor bean (Ricinus communis L.) seeds.

    PubMed

    Yu, Jaeju; Saiardi, Adolfo; Greenwood, John S; Bewley, J Derek

    2014-05-01

    During seed development, phytic acid (PA) associated with mineral cations is stored as phytin and mobilized following germination in support of seedling growth. Two parallel biosynthetic pathways for PA have been proposed; yet the pathway is still poorly understood in terms of its regulation and the enzymes involved. Here, the castor bean (Ricinus communis L.) gene for inositol 1,3,4,5,6-pentakisphosphate 2-kinase (RcIPK1) has been identified. This encodes the enzyme implicated in catalyzing the final reaction in PA biosynthesis, and its expression is enhanced in isolated germinated embryos by application of phosphate and myo-inositol (Ins). Even though only one copy of the RcIPK1 gene is present in the genome, numerous RNA variants are present, most likely due to alternative splicing. These are translated into six closely related protein isoforms according to in silico analysis. Functional analyses using yeast ipk1Δ revealed that only three of the mRNA variants can rescue a temperature-sensitive growth phenotype of this strain. High-performance liquid chromatography (HPLC) analysis of the synthesized inositol phosphates demonstrated that the ability to complement the missing yeast IPK1 enzyme is associated with the production of enzyme activity. The three active isoforms possess unique conserved motifs important for IPK1 catalytic activity.

  19. Inositol's and other nutraceuticals' synergistic actions counteract insulin resistance in polycystic ovarian syndrome and metabolic syndrome: state-of-the-art and future perspectives.

    PubMed

    Paul, Cristiana; Laganà, Antonio Simone; Maniglio, Paolo; Triolo, Onofrio; Brady, David M

    2016-06-01

    The incidence of metabolic syndrome (MetS), type II diabetes (T2D) and polycystic ovarian syndrome (PCOS) has been progressively increasing. Insulin resistance (InsR) seems to play a key role in a majority of phenotypes of these conditions, altering metabolic homeostasis, within muscle, liver, adipose and other tissues. Hyperinsulinemia is often associated with InsR and causes hormonal imbalances especially within ovaries and adrenals. Inositol is a polyalcohol, naturally occurring as nine stereoisomers, including D-chiro-inositol (DCI) and myo-inositol (MI), which have prominent roles in the metabolism of glucose and free fatty acids. MI and DCI have been classified as insulin-sensitizers and seem to adequately counteract several InsR-related metabolic alterations with a safe nutraceutical profile. Based on our analysis of selected studies that investigated MI and/or DCI, we conclude that supplementation with MI and/or DCI complement each other in their metabolic actions and act in synergy with other insulin sensitizing drugs and/or nutraceuticals. Nevertheless, considering the possible severe bias due to different methodologies across published studies, we conclude that there is a need for further studies on larger cohorts and with greater statistical power. These should further clarify outcomes and suitable therapeutic dosages of MI and DCI, possibly based on each patient's clinical status.

  20. Ectopic expression of myo-inositol 3-phosphate synthase induces a wide range of metabolic changes and confers salt tolerance in rice.

    PubMed

    Kusuda, Hiroki; Koga, Wataru; Kusano, Miyako; Oikawa, Akira; Saito, Kazuki; Hirai, Masami Yokota; Yoshida, Kaoru T

    2015-03-01

    Salt stress is an important factor that limits crop production worldwide. The salt tolerance of plants is a complex biological process mediated by changes in gene expression and metabolite composition. The enzyme myo-inositol 3-phosphate synthase (MIPS; EC 5.5.1.4) catalyzes the first step of myo-inositol biosynthesis, and overexpression of the MIPS gene enhances salt stress tolerance in several plant species. In this study, we performed metabolite profiling of both MIPS-overexpressing and wild-type rice. The enhanced salt stress tolerance of MIPS-overexpressing plants was clear based on growth and the metabolites under salt stress. We found that constitutive overexpression of the rice MIPS gene resulted in a wide range of metabolic changes. This study demonstrates for the first time that overexpression of the MIPS gene increases various metabolites responsible for protecting plants from abiotic stress. Activation of both basal metabolism, such as glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle, and inositol metabolism is induced in MIPS-overexpressing plants. We discuss the relationship between the metabolic changes and the improved salt tolerance observed in transgenic rice.

  1. Systematic analysis of time-dependent neural effects of soluble amyloid β oligomers in culture and in vivo: Prevention by scyllo-inositol.

    PubMed

    Jin, Ming; Selkoe, Dennis J

    2015-10-01

    Alzheimer's disease (AD) is currently being addressed by intensive investment in pre-clinical and clinical research on the amyloid hypothesis, but concern remains about the validity of the concept that soluble Aβ oligomers are principally responsible for initiating AD phenotypes. Here, we apply well-defined Aβ oligomers isolated from AD brains or made synthetically to document a systematic accrual of first subtle and then more profound changes in certain synaptic proteins in both primary neuronal cultures and behaving adult mice. Among the first (within hours) synaptic changes are selective decreases in surface levels of certain (e.g., GluA1) but not other (e.g., GluN2B) glutamate receptors and subtle microglial activation. After 4 days, numerous additional synaptic proteins are altered. Moreover, Aβ oligomers induce hyperphosphorylation of tau and subsequent neuritic dystrophy. All changes are prevented by scyllo-inositol in a dose- and stereoisomer-specific manner. Mechanistically, scyllo-inositol interferes quantitatively with the binding of Aβ oligomers to plasma membranes. These comprehensive analyses in culture and in vivo provide direct evidence that diffusible oligomers of human Aβ (without plaques) induce multiple phenotypic changes in healthy neurons, indicating their role as principal endogenous cytotoxins in AD. Our data recommend a re-examination of scyllo-inositol as an anti-oligomer therapeutic in humans with early AD.

  2. Lithium carbonate teratogenic effects in chick cardiomyocyte micromass system and mouse embryonic stem cell derived cardiomyocyte--possible protective role of myo-inositol.

    PubMed

    Qureshi, W M Shaikh; Latif, M L; Parker, T L; Pratten, M K

    2014-07-01

    The drug lithium carbonate (Li2CO3) use during pregnancy increases the possibility of cardiovascular anomalies. The earlier studies confirm its phosphatidylinositol cycle (PI) inhibition and Wnt pathways mimicking properties, which might contribute to its teratogenic effects. In this study the toxic effects of Li2CO3 in chick embryonic cardiomyocyte micromass system (MM) and embryonic stem cell derived cardiomyocyte (ESDC) were evaluated, with possible protective role of myo-inositol. In MM system the Li2CO3 did not alter the toxicity estimation endpoints, whereas in ESDC system the cardiomyocytes contractile activity stopped at 1500 μM and above with significant increase in total cellular protein contents. In ESDC system when myo-inositol was added along with Li2CO3 to continue PI cycle, the contractile activity was recovered with decreased protein content. The lithium toxic effects depend on the role of PI cycle at particular stage of cardiogenesis, while relation between myo-inositol and reduced cellular protein contents remains unknown.

  3. Toxoplasma gondii microneme secretion involves intracellular Ca(2+) release from inositol 1,4,5-triphosphate (IP(3))/ryanodine-sensitive stores.

    PubMed

    Lovett, Jennie L; Marchesini, Norma; M