Science.gov

Sample records for inositol phosphate production

  1. Phosphate, inositol and polyphosphates.

    PubMed

    Livermore, Thomas M; Azevedo, Cristina; Kolozsvari, Bernadett; Wilson, Miranda S C; Saiardi, Adolfo

    2016-02-01

    Eukaryotic cells have ubiquitously utilized the myo-inositol backbone to generate a diverse array of signalling molecules. This is achieved by arranging phosphate groups around the six-carbon inositol ring. There is virtually no biological process that does not take advantage of the uniquely variable architecture of phosphorylated inositol. In inositol biology, phosphates are able to form three distinct covalent bonds: phosphoester, phosphodiester and phosphoanhydride bonds, with each providing different properties. The phosphoester bond links phosphate groups to the inositol ring, the variable arrangement of which forms the basis of the signalling capacity of the inositol phosphates. Phosphate groups can also form the structural bridge between myo-inositol and diacylglycerol through the phosphodiester bond. The resulting lipid-bound inositol phosphates, or phosphoinositides, further expand the signalling potential of this family of molecules. Finally, inositol is also notable for its ability to host more phosphates than it has carbons. These unusual organic molecules are commonly referred to as the inositol pyrophosphates (PP-IPs), due to the presence of high-energy phosphoanhydride bonds (pyro- or diphospho-). PP-IPs themselves constitute a varied family of molecules with one or more pyrophosphate moiety/ies located around the inositol. Considering the relationship between phosphate and inositol, it is no surprise that members of the inositol phosphate family also regulate cellular phosphate homoeostasis. Notably, the PP-IPs play a fundamental role in controlling the metabolism of the ancient polymeric form of phosphate, inorganic polyphosphate (polyP). Here we explore the intimate links between phosphate, inositol phosphates and polyP, speculating on the evolution of these relationships. © 2016 Authors; published by Portland Press Limited.

  2. 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

  3. Production of partially phosphorylated myo-inositol phosphates using phytases immobilised on magnetic nanoparticles.

    PubMed

    Greiner, Ralf; Konietzny, Ursula; Blackburn, Daniel Menezes; Jorquera, Milko A

    2013-08-01

    Phytases of different origin were covalently bound onto Fe3O4 magnetic nanoparticles (12 nm). Binding efficiencies of all three phytases were well above 70% relative to the number of aldehyde groups available on the surface of the magnetic nanoparticles. Temperature stability for all three phytases was enhanced as a consequence of immobilisation, whereas pH dependence of enzyme activity was not affected. Maximum catalytic activity of the immobilised phytases was found at 60°C (rye), 65°C (Aspergillus niger) and 70°C (Escherichia albertii). The immobilised enzymes exhibited the same excellent substrate specificities and unique myo-inositol phosphate phosphatase activities as their soluble counterparts. However, the catalytic turnover number dropped drastically for the immobilised phytases. The amount of the desired partially phosphorylated myo-inositol phosphate isomer could be easily controlled by the contact time of substrate solution and immobilised enzymes. The immobilised phytases showed a high operational stability by retaining almost full activity even after fifty uses.

  4. Effects of inositol phosphates on mineral utilization

    SciTech Connect

    Tao, S.H.; Fox, M.R.S.; Phillippy, B.Q.; Fry, B.E. Jr.; Johnson, M.L.; Johnston, M.R.

    1986-03-05

    The present study was designed to compare the effects of inositol tri-, tetra-, and pentaphosphate (IP3, IP4, and IP5) with those of phytic acid (IP6) on growth, development and mineral utilization of young quail. Day-old Japanese quail were fed a purified casein-gelatin control diet containing 20 ppm Zn with 0 or 8.33 mmoles/kg of each inositol phosphate, corresponding to 0.55% of IP6, for a week. As compared with controls, IP6 caused reduced body weight, poor feathering, severe perosis, decreased tibia Zn and ash, and decreased pancreas Zn and liver Mn. IP5 produced all the same adverse effects and tissue mineral changes as those by phytic acid, whereas birds fed IP3 or IP4 were normal. Moreover, IP3 and IP4 caused an increased tibia weight and ash. None of the above effects was produced by feeding inositol or inorganic phosphate. In a second experiment, the inositol phosphates were fed at either 8.33 or 16.66 mmoles/kg diet. Doubling inositol phosphate levels resulted in similar effects as those observed previously. Additionally, IP4 decreased pancreas Zn and IP3 increased tibia Zn. These results indicate that unlike IP6 and IP5, inositol phosphates with 4 or fewer phosphate groups, which can arise from hydrolysis of phytic acid during food processing, have very minor adverse effects but may be beneficial for bone mineralization.

  5. Inositol phosphates induce DAPI fluorescence shift.

    PubMed

    Kolozsvari, Bernadett; Parisi, Federica; Saiardi, Adolfo

    2014-06-15

    The polymer inorganic polyP (polyphosphate) and inositol phosphates, such as IP6 (inositol hexakisphosphate; also known as phytic acid), share many biophysical features. These similarities must be attributed to the phosphate groups present in these molecules. Given the ability of polyP to modify the excitation-emission spectra of DAPI we decided to investigate whether inositol phosphates possess the same property. We discovered that DAPI-IP6 complexes emit at approximately 550 nm when excited with light of wavelength 410-420 nm. IP5 (inositol pentakisphosphate) is also able to induce a similar shift in DAPI fluorescence. Conversely, IP3 (inositol trisphosphate) and IP4 (inositol tetrakisphosphate) are unable to shift DAPI fluorescence. We have employed this newly discovered feature of DAPI to study the enzymatic activity of the inositol polyphosphate multikinase and to monitor phytase phosphatase reactions. Finally, we used DAPI-IP6 fluorescence to determine the amount of IP6 in plant seeds. Using an IP6 standard curve this straight-forward analysis revealed that among the samples tested, borlotti beans possess the highest level of IP6 (9.4 mg/g of dry mass), whereas the Indian urad bean the lowest (3.2 mg/g of dry mass). The newly identified fluorescence properties of the DAPI-IP5 and DAPI-IP6 complexes allow the levels and enzymatic conversion of these two important messengers to be rapidly and reliably monitored.

  6. Synthesis of inositol phosphate ligands of plant hormone-receptor complexes: pathways of inositol hexakisphosphate turnover.

    PubMed

    Hanke, David E; Parmar, Paroo N; Caddick, Samuel E K; Green, Porntip; Brearley, Charles A

    2012-06-15

    Reduction of phytate is a major goal of plant breeding programs to improve the nutritional quality of crops. Remarkably, except for the storage organs of crops such as barley, maize and soybean, we know little of the stereoisomeric composition of inositol phosphates in plant tissues. To investigate the metabolic origins of higher inositol phosphates in photosynthetic tissues, we have radiolabelled leaf tissue of Solanum tuberosum with myo-[2-3H]inositol, undertaken a detailed analysis of inositol phosphate stereoisomerism and permeabilized mesophyll protoplasts in media containing inositol phosphates. We describe the inositol phosphate composition of leaf tissue and identify pathways of inositol phosphate metabolism that we reveal to be common to other kingdoms. Our results identify the metabolic origins of a number of higher inositol phosphates including ones that are precursors of cofactors, or cofactors of plant hormone-receptor complexes. The present study affords alternative explanations of the effects of disruption of inositol phosphate metabolism reported in other species, and identifies different inositol phosphates from that described in photosynthetic tissue of the monocot Spirodela polyrhiza. We define the pathways of inositol hexakisphosphate turnover and shed light on the occurrence of a number of inositol phosphates identified in animals, for which metabolic origins have not been defined.

  7. Inositol pyrophosphates: why so many phosphates?

    PubMed Central

    Shears, Stephen B.

    2014-01-01

    The inositol pyrophosphates (PP-InsPs) are a specialized group of “energetic” signaling molecules found in yeasts, plants and animals. PP-InsPs boast the most crowded three dimensional phosphate arrays found in Nature; multiple phosphates and diphosphates are crammed around the six-carbon, inositol ring. Yet, phosphate esters are also a major energy currency in cells. So the synthesis of PP-InsPs, and the maintenance of their levels in the face of a high rate of ongoing turnover, all requires significant bioenergetic input. What are the particular properties of PP-InsPs that repay this investment of cellular energy? Potential answers to that question are discussed here, against the backdrop of a recent hypothesis that signaling by PP-InsPs is evolutionarily ancient. The latter idea is extended herein, with the proposal that the primordial origins of PP-InsPs is reflected in the apparent lack of isomeric specificity of certain of their actions. Nevertheless, there are other aspects of signaling by these polyphosphates that are more selective for a particular PP-InsP isomer. Consideration of the nature of both specific and non-specific effects of PP-InsPs can help rationalize why such molecules possess so many phosphates. PMID:25453220

  8. 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

  9. Structural Studies and Protein Engineering of Inositol Phosphate Multikinase*

    PubMed Central

    Endo-Streeter, Stuart; Tsui, Man-Kin Marco; Odom, Audrey R.; Block, Jeremy; York, John D.

    2012-01-01

    Inositol phosphates (IPs) regulate vital processes in eukaryotes, and their production downstream of phospholipase C activation is controlled through a network of evolutionarily conserved kinases and phosphatases. Inositol phosphate multikinase (IPMK, also called Ipk2 and Arg82) accounts for phosphorylation of IP3 to IP5, as well as production of several other IP molecules. Here, we report the structure of Arabidopsis thaliana IPMKα at 2.9 Å and find it is similar to the yeast homolog Ipk2, despite 17% sequence identity, as well as the active site architecture of human IP3 3-kinase. Structural comparison and substrate modeling were used to identify a putative basis for IPMK selectivity. To test this model, we re-engineered binding site residues predicted to have restricted substrate specificity. Using steady-state kinetics and in vivo metabolic labeling studies in modified yeast strains, we observed that K117W and K117W:K121W mutants exhibited nearly normal 6-kinase function but harbored significantly reduced 3-kinase activity. These mutants complemented conditional nutritional growth defects observed in ipmk null yeast and, remarkably, suppressed lethality observed in ipmk null flies. Our data are consistent with the hypothesis that IPMK 6-kinase activity and production of Ins(1,4,5,6)P4 are critical for cellular signaling. Overall, our studies provide new insights into the structure and function of IPMK and utilize a synthetic biological approach to redesign inositol phosphate signaling pathways. PMID:22896696

  10. Inositol phosphate signaling and gibberellic acid

    PubMed Central

    Fleet, Christine M; Ercetin, Mustafa E

    2009-01-01

    To respond to physical signals and endogenous hormones, plants use specific signal transduction pathways. We and others have previously shown that second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] is used in abscisic acid (ABA) signaling, and that some mutants with altered Ins(1,4,5)P3 have altered responses to ABA. Specifically, mutants defective in the myo-inositol polyphosphate 5-phosphatases (5PTases) 1 and 2 genes that hydrolyze 5-phosphates from Ins(1,4,5)P3 and other PtdInsP and InsP substrates, have elevated Ins (1,4,5)P3, and are ABA-hypersensitive. Given the antagonistic relationship between ABA and gibberellic acid (GA), we tested the response of these same mutants to a GA synthesis inhibitor, paclobutrazol (PAC). We report here that 5ptase1, 5ptase2 and 5ptase11 mutants are hypersensitive to PAC, suggesting a relationship between elevated Ins(1,4,5)P3 and decreased GA signal transduction. These data provide insight into signaling cross-talk between ABA and GA pathways. PMID:19704714

  11. Effect of tricyclic antidepressants on transmitter-stimulated inositol phosphate production in rat brain cortex in vitro

    SciTech Connect

    Nomura, S.; Enna, S.J.

    1986-03-01

    Tricyclic antidepressants (TCAs) have anticholinergic and ..cap alpha..-adrenergic blocking properties. The present study was undertaken to examine the effects of amitriptyline, imipramine, and desipramine on inositol phosphate accumulation, a brain second messenger system associated with cholinergic and adrenergic receptors. Whereas the TCAs were 28 to 400-fold weaker than atropine as inhibitors of /sup 3/H-QNB binding to brain cholinergic receptors, they were 600 to 2000-fold less active than atropine as inhibitors of carbachol-stimulated IP accumulation in brain. In contrast, the relative potencies of the TCAs and prazosin to inhibit norepinephrine-stimulated IP accumulation and /sup 3/H-prazosin binding appeared to be similar in the two assays. The results suggest pharmacological differences between the cholinergic receptors labeled in the ONB binding assay and those mediating the IP response, whereas the ..cap alpha../sub 1/-adrenergic receptors appear to be similar in the two systems. Since atropine is considered a nonselective muscarinic antagonist, it is possible that the TCAs may differentiate between cholinergic receptor subtypes, which may be an important component of their clinical response.

  12. Phosphoinositide and Inositol Phosphate Analysis in Lymphocyte Activation

    PubMed Central

    Sauer, Karsten; Huang, Yina Hsing; Lin, Hongying; Sandberg, Mark; Mayr, Georg W.

    2015-01-01

    Lymphocyte antigen receptor engagement profoundly changes the cellular content of phosphoinositide lipids and soluble inositol phosphates. Among these, the phosphoinositides phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) play key signaling roles by acting as pleckstrin homology (PH) domain ligands that recruit signaling proteins to the plasma membrane. Moreover, PIP2 acts as a precursor for the second messenger molecules diacylglycerol and soluble inositol 1,4,5-trisphosphate (IP3), essential mediators of PKC, Ras/Erk, and Ca2+ signaling in lymphocytes. IP3 phosphorylation by IP3 3-kinases generates inositol 1,3,4,5-tetrakisphosphate (IP4), an essential soluble regulator of PH domain binding to PIP3 in developing T cells. Besides PIP2, PIP3, IP3, and IP4, lymphocytes produce multiple other phosphoinositides and soluble inositol phosphates that could have important physiological functions. To aid their analysis, detailed protocols that allow one to simultaneously measure the levels of multiple different phosphoinositide or inositol phosphate isomers in lymphocytes are provided here. They are based on thin layer, conventional and high-performance liquid chromatographic separation methods followed by radiolabeling or non-radioactive metal-dye detection. Finally, less broadly applicable nonchromatographic methods for detection of specific phosphoinositide or inositol phosphate isomers are discussed. Support protocols describe how to obtain pure unstimulated CD4+CD8+ thymocyte populations for analyses of inositol phosphate turnover during positive and negative selection, key steps in T cell development. PMID:19918943

  13. Inositol phosphates in barley (Hordeum vulgare L.) aleurone tissue are stereochemically similar to the products of breakdown of InsP6 in vitro by wheat-bran phytase.

    PubMed

    Brearley, C A; Hanke, D E

    1996-08-15

    Partisphere SAX HPLC analysis of endogenous inositol phosphates in [3H]inositol-labelled barley aleurone tissue revealed a range of isomers, including D- and/or L-Ins3P, D- and/or L-Ins(1,4)P2, D- and/or L-Ins(1,2)P2, a third unidentified InsP2, Ins(1,2,3)P3, D- and/or L-Ins(1,2,6)P3, D-and/or L-Ins(1,2,3,4)P4, D- and/or L-Ins(1,2,5,6)P4, Ins(1,3,4,5,6)P5, D- and/or L-Ins(1,2,3,4,5)P5, Ins(1,2,3,4,6)P5, InsP6 and a molecule with the chromatographic properties of an inositol pyrophosphate. The striking match between the identities of the stereoisomers, and in some cases enantiomers, detected in vivo and those stereoisomers produced in vitro by the action of wheat-bran phytase on InsP6 [Cosgrove (1980) Inositol Phosphates: Their Chemistry, Bio-chemistry and Physiology. Elsevier, Amsterdam] strongly suggests that most of the inositol phosphates identified are products of the breakdown of InsP4 by endogenous phytase(s) with stereospecificity similar to that of the wheat-bran enzyme(s).

  14. 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.

  15. 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.

  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. Genetic variants in the inositol phosphate metabolism pathway and risk of different types of cancer.

    PubMed

    Tan, Juan; Yu, Chen-Yang; Wang, Zhen-Hua; Chen, Hao-Yan; Guan, Jian; Chen, Ying-Xuan; Fang, Jing-Yuan

    2015-02-16

    Members of the inositol phosphate metabolism pathway regulate cell proliferation, migration and phosphatidylinositol-3-kinase (PI3K)/Akt signaling, and are frequently dysregulated in cancer. Whether germline genetic variants in inositol phosphate metabolism pathway are associated with cancer risk remains to be clarified. We examined the association between inositol phosphate metabolism pathway genes and risk of eight types of cancer using data from genome-wide association studies. Logistic regression models were applied to evaluate SNP-level associations. Gene- and pathway-based associations were tested using the permutation-based adaptive rank-truncated product method. The overall inositol phosphate metabolism pathway was significantly associated with risk of lung cancer (P = 2.00 × 10(-4)), esophageal squamous cell carcinoma (P = 5.70 × 10(-3)), gastric cancer (P = 3.03 × 10(-2)) and renal cell carcinoma (P = 1.26 × 10(-2)), but not with pancreatic cancer (P = 1.40 × 10(-1)), breast cancer (P = 3.03 × 10(-1)), prostate cancer (P = 4.51 × 10(-1)), and bladder cancer (P = 6.30 × 10(-1)). Our results provide a link between inherited variation in the overall inositol phosphate metabolism pathway and several individual genes and cancer. Further studies will be needed to validate these positive findings, and to explore its mechanisms.

  18. The “Other” Inositols and Their Phosphates: Synthesis, Biology and Medicine (with Recent Advances in myo-Inositol Chemistry)

    PubMed Central

    Thomas, Mark P; Mills, Stephen J; Potter, Barry V L

    2016-01-01

    Cell signalling via inositol phosphates, eg the second messenger myo-inositol 1,4,5-trisphosphate, and phosphoinositides comprises a huge field of biology. Of nine 1,2,3,4,5,6-cyclohexanehexol isomers, myo-inositol is pre-eminent, with “other” inositols (cis-, epi-, allo-, muco-, neo-, l-chiro-, d-chiro- and scyllo-) and derivatives rarer or thought not to exist in nature. However, recently, neo- and d-chiro-inositol hexakisphosphates were revealed in both terrestrial and aquatic ecosystems, highlighting the paucity of knowledge of the origins and potential biological functions of such stereoisomers, a prevalent group of environmental organic phosphates, and their parent inositols. Some “other” inositols are medically relevant, e.g. scyllo-inositol (neurodegenerative diseases), and d-chiro-inositol (diabetes). It is timely to consider exploration of roles and applications of “other” isomers and their derivatives, likely by exploiting techniques now well developed for the myo-series. PMID:26694856

  19. Leishmanial phosphatase hydrolyzes phosphoproteins and inositol phosphates

    SciTech Connect

    Saha, A.K.; Das, S.; Glew, R.H.

    1986-05-01

    An extensively purified preparation of the predominant, tartrate-resistant acid phosphatase (ACP) from the external surface of Leishmania donovani promastigotes form catalyzes the dephosphorylation of several phosphoproteins; these include: pyruvate kinase, phosphorylase kinase and histones. However, the protein phosphatase activity of ACP is very low compared with that of other protein phosphates known to be involved in regulating various metabolic pathways. /sup 32/P-labelled inositoltriphosphate (IP3), a well-established second messenger derived from phosphatidylinositol-4,5-diphosphate (PIP2), was a substrate for the leishmanial acid phosphatase; incubation of the IP3 preparation with 13.2 milliunits (1 unit equals 1 ..mu..mol 4-methylumbelliferyl phosphate (MUP) cleaved per min at pH 5.5) of ACP at pH 5.5 for 4 hr resulted in hydrolysis of 75% of the radiolabelled substrate resulting in a mixture of inositoldiphosphate and inositolmonophosphate. In addition PIP2 was hydrolyzed rapidly by ACP at pH 5.5 (V/sub max/, 71 units/mg protein; k/sub m/, 4.16 ..mu..M). In contrast, to MUP which is hydrolzyed most rapidly at pH 5.5, PIP2 hydrolysis was optimal at pH 6.8. These observations raise the possibility that ACP could play a role in the host-phagocyte interaction by degrading the precursor of the second messenger, PIP2 or the second messenger itself, IP3.

  20. Intracellular delivery of phosphoinositides and inositol phosphates using polyamine carriers

    PubMed Central

    Ozaki, Shoichiro; DeWald, Daryll B.; Shope, Joseph C.; Chen, Jian; Prestwich, Glenn D.

    2000-01-01

    Phosphoinositide signaling regulates events in endocytosis and exocytosis, vesicular trafficking of proteins, transduction of extracellular signals, remodeling of the actin cytoskeleton, regulation of calcium flux, and apoptosis. Obtaining mechanistic insights in living cells is impeded by the membrane impermeability of these anionic lipids. We describe a carrier system for intracellular delivery of phosphoinositide polyphosphates (PIPns) and fluorescently labeled PIPns into living cells, such that intracellular localization can be directly observed. Preincubation of PIPns or inositol phosphates with carrier polyamines produced complexes that entered mammalian, plant, yeast, bacterial, and protozoal cells in seconds to minutes via a nonendocytic mechanism. Time-dependent transit of both PIPns and the carrier to specific cytosolic and nuclear compartments was readily visualized by fluorescence microscopy. Platelet-derived growth factor treatment of NIH 3T3 fibroblasts containing carrier-delivered phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]-7-nitrobenz-2-oxa-1,3-diazole resulted in the redistribution of the fluorescent signal, suggesting that fluorescent PtdIns(4,5)P2 was a substrate for phospholipase C. We also observed a calcium flux in NIH 3T3 cells when complexes of carrier and PtdIns(4,5)P2 or inositol 1,4,5-trisphosphate were added extracellularly. This simple intracellular delivery system allows for the efficient translocation of biologically active PIPns, inositol phosphates, and their fluorescent derivatives into living cells in a physiologically relevant context. PMID:11005844

  1. Separation of phytic acid and other related inositol phosphates by high-performance ion chromatography and its applications.

    PubMed

    Chen, Qing-Chuan; Li, Betty W

    2003-11-07

    A high-performance anion-exchange chromatographic method was developed for the separation of phytic acid and other inositol phosphates (myo-inositol bis-, tris-, tetrakis-, and pentakisphosphates) with gradient elution and ultraviolet absorbance detection after post-column derivatization. With the acidic eluents, the combination of anion-exchange and ion suppression retention mechanisms led to the separation of 35 inositol phosphates (excluding enantiomers) into 27 peaks for the first time, and the retention behaviors of all myo-inositol bis- to hexakisphosphate isomers were studied. The whole separation procedure was completed within 65 min. Based on the investigations of nonenzymatic hydrolysis of phytic acid under different conditions by using this method, an in-house reference standard solution was produced, which can be used for method development. In addition, by applying this method to in vitro kinetic studies, at least one new enzymatic hydrolysis pathway of phytic acid was found, and one rule of enzymatic dephosphorylation of inositol phosphates (position effect) was proposed and another one (neighboring effect) was confirmed. The principle of the proposed identification approach for several inositol phosphate isomers based on hydrolysis products study will be applicable to other natural products analysis, for which standards are very expensive or not available.

  2. Anion exchange chromatographic separation of inositol phosphates and their quantification by gas chromatography.

    PubMed

    Heathers, G P; Juehne, T; Rubin, L J; Corr, P B; Evers, A S

    1989-01-01

    The direct measurement of mass of inositol trisphosphate from biologic samples is described. Separation of inositol monophosphate, bisphosphate, trisphosphate, and inositol tetrakisphosphate was achieved using anion exchange chromatography with a sodium sulfate gradient. In addition, separation of the isomers of each inositol phosphate was performed using HPLC procedures. The individual inositol phosphate fractions were subsequently dephosphorylated and desalted. The myo-inositol from each fraction was then derivatized to the hexatrimethylsilyl derivative and the myo-inositol derivatives were quantified by a novel gas chromatographic analysis using the hexatrimethylsilyl derivative of chiro-inositol as an internal concentration reference. This method is a reproducible and relatively rapid procedure for the direct quantification of inositol phosphate mass which overcomes many of the problems associated with the use of radiolabeled precursors. The method is a significant improvement over existing procedures for the quantitative determination of the mass of inositol phosphate by virtue of improved recovery, sensitivity, and technical simplicity. The applicability of this method is illustrated by the quantitative determination of inositol trisphosphate in response to norepinephrine stimulation of adult canine myocytes and cerebral cortical brain slices and by measurement of the isomers of inositol trisphosphate in isolated myocytes.

  3. Expression of the Saccharomyces cerevisiae inositol-1-phosphate synthase (INO1) gene is regulated by factors that affect phospholipid synthesis.

    PubMed Central

    Hirsch, J P; Henry, S A

    1986-01-01

    The INO1 gene of Saccharomyces cerevisiae encodes the regulated enzyme inositol-1-phosphate synthase, which catalyzes the first committed step in the synthesis of inositol-containing phospholipids. The expression of this gene was analyzed under conditions known to regulate phospholipid synthesis. RNA blot hybridization with a genomic clone for INO1 detected two RNA species of 1.8 and 0.6 kb. The abundance of the 1.8-kb RNA was greatly decreased when the cells were grown in the presence of the phospholipid precursor inositol, as was the enzyme activity of the synthase. Complementation analysis showed that this transcript encoded the INO1 gene product. The level of INO1 RNA was repressed 12-fold when the cells were grown in medium containing inositol, and it was repressed 33-fold when the cells were grown in the presence of inositol and choline together. The INO1 transcript was present at a very low level in cells containing mutations (ino2 and ino4) in regulatory genes unlinked to INO1 that result in inositol auxotrophy. The transcript was constitutively overproduced in cells containing a mutation (opi1) that causes constitutive expression of inositol-1-phosphate synthase and results in excretion of inositol. The expression of INO1 RNA was also examined in cells containing a mutation (cho2) affecting the synthesis of phosphatidylcholine. In contrast to what was observed in wild-type cells, growth of cho2 cells in medium containing inositol did not result in a significant decrease in INO1 RNA abundance. Inositol and choline together were required for repression of the INO1 transcript in these cells, providing evidence for a regulatory link between the synthesis of inositol- and choline-containing lipids. The level of the 0.6-kb RNA was affected, although to a lesser degree, by many of the same factors that influence INO1 expression. Images PMID:3025587

  4. Calcium-activated hydrolysis of phosphatidyl-myo-inositol 4-phosphate and phosphatidyl-myo-inositol 4,5-bisphosphate in guinea-pig synaptosomes

    PubMed Central

    Griffin, Harry D.; Hawthorne, John N.

    1978-01-01

    1. Addition of the bivalent ionophore A23187 to synaptosomes isolated from guinea-pig brain cortex and labelled with [32P]phosphate in vitro or in vivo caused a marked loss of radioactivity from phosphatidyl-myo-inositol 4-phosphate (diphosphoinositide) and phosphatidyl-myo-inositol 4,5-bisphosphate (triphosphoinositide) and stimulated labelling of phosphatidate. No change occurred in the labelling of other phospholipids. 2. In conditions that minimized changes in internal Mg2+ concentrations, the effect of ionophore A23187 on labelling of synaptosomal di- and tri-phosphoinositide was dependent on Ca2+ and was apparent at Ca2+ concentrations in the medium as low as 10−5m. 3. An increase in internal Mg2+ concentration stimulated incorporation of [32P]phosphate into di- and tri-phosphoinositide, whereas lowering internal Mg2+ decreased labelling. 4. Increased labelling of phosphatidate was independent of medium Mg2+ concentration and apparently only partly dependent on medium Ca2+ concentration. 5. The loss of label from di- and tri-phosphoinositide caused by ionophore A23187 was accompanied by losses in the amounts of both lipids. 6. Addition of excess of EGTA to synaptosomes treated with ionophore A23187 in the presence of Ca2+ caused a rapid resynthesis of di- and tri-phosphoinositide and a further stimulation of phosphatidate labelling. 7. Addition of ionophore A23187 to synaptosomes labelled in vivo with [3H]inositol caused a significant loss of label from di- and tri-phosphoinositide, but not from phosphatidylinositol. There was a considerable rise in labelling of inositol diphosphate, a small increase in that of inositol phosphate, but no significant production of inositol triphosphate. 8. 32P-labelled di- and tri-phosphoinositides appeared to be located in the synaptosomal plasma membrane. 9. The results indicate that increased Ca2+ influx into synaptosomes markedly activates triphosphoinositide phosphatase and diphosphoinositide phosphodiesterase, but has

  5. 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

  6. 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.

  7. 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

  8. Recombinant expression of a functional myo-inositol-1-phosphate synthase (MIPS) in Mycobacterium smegmatis.

    PubMed

    Huang, Xinyi; Hernick, Marcy

    2015-10-01

    Myo-inositol-1-phosphate synthase (MIPS, E.C. 5.5.1.4) catalyzes the first step in inositol production-the conversion of glucose-6-phosphate (Glc-6P) to myo-inositol-1-phosphate. While the three dimensional structure of MIPS from Mycobacterium tuberculosis has been solved, biochemical studies examining the in vitro activity have not been reported to date. Herein we report the in vitro activity of mycobacterial MIPS expressed in E. coli and Mycobacterium smegmatis. Recombinant expression in E. coli yields a soluble protein capable of binding the NAD(+) cofactor; however, it has no significant activity with the Glc-6P substrate. In contrast, recombinant expression in M. smegmatis mc(2)4517 yields a functionally active protein. Examination of structural data suggests that MtMIPS expressed in E. coli adopts a fold that is missing a key helix containing two critical (conserved) Lys side chains, which likely explains the inability of the E. coli expressed protein to bind and turnover the Glc-6P substrate. Recombinant expression in M. smegmatis may yield a protein that adopts a fold in which this key helix is formed enabling proper positioning of important side chains, thereby allowing for Glc-6P substrate binding and turnover. Detailed mechanistic studies may be feasible following optimization of the recombinant MIPS expression protocol in M. smegmatis.

  9. High-performance reversed-phase ion-pair chromatographic study of myo-inositol phosphates. Separation of myo-inositol phosphates, some common nucleotides and sugar phosphates.

    PubMed

    Patthy, M; Balla, T; Arányi, P

    1990-12-07

    A detailed study of all the major chromatographic variables affecting the retention behaviour and separation of myo-inositol phosphates in reversed-phase ion-pair chromatographic systems was carried out. The parameters studied included the eluent concentration of the pairing ion, the eluent concentration of the organic modifier and the buffer salt, the pH of the eluent, the minimum column plate count necessary for the separation of the inositol trisphosphate isomers and isocratic and gradient modes of separation. The retention behaviour of some common nucleotides and sugar phosphates was also investigated as these phosphates present chromatographic interference problems in biochemical studies based on the cellular incorporation of [32P]Pi. The separation methods developed appear to be superior to established anion-exchange separation techniques in terms of separation speed and "mildness" of the chromatographic conditions.

  10. High-performance thin-layer chromatography method for inositol phosphate analysis.

    PubMed

    Hatzack, F; Rasmussen, S K

    1999-12-24

    A simple and inexpensive high-performance thin-layer chromatography (HPTLC) method for the analysis of inositol mono- to hexakisphosphates on cellulose precoated plates is described. Plates were developed in 1-propanol-25% ammonia solution-water (5:4:1) and substance quantities as low as 100-200 pmol were detected by molybdate staining. Chromatographic mobilities of nucleotides and phosphorylated carbohydrates were also characterized. Charcoal treatment was employed to separate nucleotides from inositol phosphates with similar R(F) values prior to HPTLC analysis. Practical application of the HPTLC system is demonstrated by analysis of grain extracts from wild type and low-phytate mutant barley as well as phytate degradation products resulting from barley phytase activity.

  11. 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

  12. 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.

  13. Phytate (Inositol Hexakisphosphate) in Soil and Phosphate Acquisition from Inositol Phosphates by Higher Plants. A Review

    PubMed Central

    Gerke, Jörg

    2015-01-01

    Phosphate (P) fixation to the soil solid phase is considered to be important for P availability and is often attributed to the strong binding of orthophosphate anion species. However, the fixation and subsequent immobilization of inositolhexa and pentaphosphate isomers (phytate) in soil is often much stronger than that of the orthosphate anion species. The result is that phytate is a main organic P form in soil and the dominating form of identifiable organic P. The reasons for the accumulation are not fully clear. Two hypothesis can be found in the literature in the last 20 years, the low activity of phytase (phosphatases) in soil, which makes phytate P unavailable to the plant roots, and, on the other hand, the strong binding of phytate to the soil solid phase with its consequent stabilization and accumulation in soil. The hypothesis that low phytase activity is responsible for phytate accumulation led to the development of genetically modified plant genotypes with a higher expression of phytase activity at the root surface and research on the effect of a higher phytate activity on P acquisition. Obviously, this hypothesis has a basic assumption, that the phytate mobility in soil is not the limiting step for P acquisition of higher plants from soil phytate. This assumption is, however, not justified considering the results on the sorption, immobilization and fixation of phytate to the soil solid phase reported in the last two decades. Phytate is strongly bound, and the P sorption maximum and probably the sorption strength of phytate P to the soil solid phase is much higher, compared to that of orthophosphate P. Mobilization of phytate seems to be a promising step to make it available to the plant roots. The excretion of organic acid anions, citrate and to a lesser extend oxalate, seems to be an important way to make phytate P available to the plants. Phytase activity at the root surface seems not be the limiting step in P acquisition from phytate. Phytate is not

  14. 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.

  15. 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)

  16. Levoprotiline ((-)-oxaprotiline) effects on inositol phosphate generation in human peripheral lymphocytes.

    PubMed

    Schubert, T; Müller, W E

    1991-01-01

    The effects of the atypical antidepressant levoprotiline (LPT) on inositol phosphate metabolism were investigated in N-formyl-methionyl-leucylphenylalanine (fMLP) activated human lymphocytes. In the presence of LPT, stimulation of phosphoinositide hydrolysis by fMLP lead to an increased accumulation of inositol bisphosphates, an effect which could be detected within the range of therapuetic plasma concentrations and which is exerted by lithium in a similar way. Furthermore, incubation of lymphocytes with LPT and subsequent stimulation with fMLP lead to a pronounced decrease in the level of free intracellular [3H]inositol. Both LPT effects, the increased accumulation of inositol bisphosphates and the reduction of free intracellular [3H]inositol, were found to be more pronounced for LPT than for its enantiomer (+)-oxaprotiline. The results are discussed in view of a possible biochemical mechanism which may contribute to the antidepressive activity of LPT.

  17. Metabolic and structural evidence for the existence of a third species of polyphosphoinositide in cells: D-phosphatidyl-myo-inositol 3-phosphate.

    PubMed Central

    Stephens, L; Hawkins, P T; Downes, C P

    1989-01-01

    When human 1321 N1 astrocytoma cells were labelled to steady state with [3H]inositol and briefly with [32P]orthophosphate, a compound which contained both radiotracers and which co-migrated with phosphatidylinositol-myo-inositol 4-phosphate during t.l.c. could be extracted in acidic chloroform/methanol. Treatment with methylamine under conditions which lead to deacylation of conventional glycerophospholipids yielded a water-soluble moiety which was labelled with both radioisotopes and was eluted from an anion-exchange h.p.l.c. column with a retention time similar to, but distinct from, that of glycerophosphoinositol 4-phosphate. Experiments using sodium periodate and selective phosphatase enzymes to degrade this compound systematically generated a series of products which suggested the structure of the parent phospholipid was phosphatidyl-myo-inositol 3-phosphate (PtdIns3P). PtdIns3P is metabolically closely related to the pool(s) of inositol phospholipid(s) that serves as substrate(s) for an agonist-sensitive phosphoinositidase C, as the levels of PtdIns3P fell significantly when 1321 N1 cells were stimulated with carbachol. The relative rate of turnover of the inositol moiety of PtdIns3P is similar to that of both of the major polyphosphoinositides and significantly higher than that of total cellular phosphatidyl-myo-inositol. This suggests that all three polyphosphoinositides are synthesized from a common, rapidly metabolized, pool of phosphatidyl-myo-inositol. PMID:2541684

  18. Simulations of inositol phosphate metabolism and its interaction with InsP(3)-mediated calcium release.

    PubMed Central

    Mishra, Jyoti; Bhalla, Upinder S

    2002-01-01

    Inositol phosphates function as second messengers for a variety of extracellular signals. Ins(1,4,5)P(3) generated by phospholipase C-mediated hydrolysis of phosphatidylinositol bisphosphate, triggers numerous cellular processes by regulating calcium release from internal stores. The Ins(1,4,5)P(3) signal is coupled to a complex metabolic cascade involving a series of phosphatases and kinases. These enzymes generate a range of inositol phosphate derivatives, many of which have signaling roles of their own. We have integrated published biochemical data to build a mass action model for InsP(3) metabolism. The model includes most inositol phosphates that are currently known to interact with each other. We have used this model to study the effects of a G-protein coupled receptor stimulus that activates phospholipase C on the inositol phosphates. We have also monitored how the metabolic cascade interacts with Ins(1,4,5)P(3)-mediated calcium release. We find temporal dynamics of most inositol phosphates to be strongly influenced by the elaborate networking. We also show that Ins(1,3,4,5)P(4) plays a key role in InsP(3) dynamics and allows for paired pulse facilitation of calcium release. Calcium oscillations produce oscillatory responses in parts of the metabolic network and are in turn temporally modulated by the metabolism of InsP(3). PMID:12202356

  19. Design and Synthesis of an Inositol Phosphate Analog Based on Computational Docking Studies.

    PubMed

    Peng, Zhenghong; Maxwell, David; Sun, Duoli; Ying, Yunming; Schuber, Paul T; Bhanu Prasad, Basvoju A; Gelovani, Juri; Yung, Wai-Kwan Alfred; Bornmann, William G

    2014-01-28

    A virtual library of 54 inositol analog mimics of In(1,4,5)P3 has been docked, scored, and ranked within the binding site of human inositol 1,4,5-trisphosphate 3-kinase A (IP3-3KA). Chemical synthesis of the best scoring structure that also met distance criteria for 3'-OH to -P in Phosphate has been attempted along with the synthesis of (1S,2R,3S,4S)-3-fluoro-2,4-dihydroxycyclohexanecarboxylic acid as an inositol analog, useful for non-invasive visualization and quantitation of IP3-3KA enzymatic activity.

  20. 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.

  1. HPLC with inductively coupled plasma optical emission spectrometric detection for the analysis of inositol phosphates.

    PubMed

    Amaro, Rosa; Escalona, Andrés; Murillo, Miguel

    2004-10-01

    The use of inductively coupled plasma optimal emission spectroscopy as a detector for the high-performance liquid chromatographic analysis of inositol phosphates is studied. It is found that separation of different inositol phosphates with a mobile phase consisting of tetraethylammonium (0.14%, w/v), methanol (5%, v/v), and formic acid (0.18%, w/v) may be obtained on a PRP-1 column with an analysis time of 18 min. In addition, high specificity and sensitivity of the detection system used permits detection of the inositol phosphates from bi- to hexaphosphate free from interference of other chromatographic peaks, which could be from the sample or mobile phase. Additionally, it is possible to use less sample because of the high sensitivity of the detection system.

  2. A tyrosine kinase signaling pathway, regulated by calcium entry and dissociated from tyrosine phosphorylation of phospholipase Cgamma-1, is involved in inositol phosphate production by activated G protein-coupled receptors in myometrium.

    PubMed

    Palmier, B; Vacher, M; Harbon, S; Leiber, D

    1999-05-01

    Our experiments were conducted to evaluate, in rat myometrium, the potential contribution of a protein tyrosine kinase (PTK) pathway in the hydrolysis of phosphatidylinositol-4,5-bisphosphate mediated by bombesin, endothelin-1 (ET-1), and carbachol. The production of inositol phosphates (InsP) by agonists and AlF4- was partly inhibited (35-40%) by genistein and tyrphostins, two PTK inhibitors. Genistein attenuated uterine contractions elicited by the stimulation of muscarinic and bombesin receptors, whereas pervanadate, a protein tyrosine phosphatase inhibitor, potentiated receptor-mediated contraction. Tyrosine-phosphorylated proteins were detected in detergent extracts from agonist- and pervanadate-stimulated myometrium. The amount of InsP produced in response to pervanadate was related to the tyrosine phosphorylation status of phospholipase C-gamma1. In contrast, with ET-1 and bombesin, phosphorylated phospholipase C-gamma1 made a minor contribution. Additional findings were rather consistent with a role for Ca2+. In fura-2-loaded cells, genistein partly decreased both the transient and sustained intracellular Ca2+ concentration phases induced by bombesin. The removal of extracellular Ca2+ or the addition of nifedipine inhibited (35%) InsP production due to bombesin and ET-1. The inhibitory effects of genistein and tyrphostins were abolished in Ca2+-depleted medium, were not additive with that of nifedipine, and (as for nifedipine) were counteracted by the Ca2+ channel agonist Bay K 8644. The data are consistent with a PTK-mediated process in the activation of the voltage-gated Ca2+ influx that is involved in the production of InsP by stimulated G protein-coupled receptors.

  3. Degradation of phytate in the gut of pigs--pathway of gastro-intestinal inositol phosphate hydrolysis and enzymes involved.

    PubMed

    Schlemmer, U; Jany, K D; Berk, A; Schulz, E; Rechkemmer, G

    2001-01-01

    The present study gives an overview on the whole mechanism of phytate degradation in the gut and the enzymes involved. Based on the similarity of the human and pigs gut, the study was carried out in pigs as model for humans. To differentiate between intrinsic feed phytases and endogenous phytases hydrolysing phytate in the gut, two diets, one high (control diet) and the other one very low in intrinsic feed phytases (phytase inactivated diet) were applied. In the chyme of stomach, small intestine and colon inositol phosphate isomers and activities of phytases and alkaline phosphatases were determined. In parallel total tract phytate degradation and apparent phosphorus digestibility were assessed. In the stomach chyme of pigs fed the control diet, comparable high phytase activity and strong phytate degradation were observed. The predominant phytate hydrolysis products were inositol phosphates, typically formed by plant phytases. For the phytase inactivated diet, comparable very low phytase activity and almost no phytate degradation in the stomach were determined. In the small intestine and colon, high activity of alkaline phosphatases and low activity of phytases were observed, irrespective of the diet fed. In the colon, stronger phytate degradation for the phytase inactivated diet than for the control diet was detected. Phytate degradation throughout the whole gut was nearly complete and very similar for both diets while the apparent availability of total phosphorus was significantly higher for the pigs fed the control diet than the phytase inactivated diet. The pathway of inositol phosphate hydrolysis in the gut has been elucidated.

  4. 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

    2015-06-09

    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.

  5. Synthesis of myo-inositol 1,2,3-tris- and 1,2,3,5-tetrakis(dihydrogen phosphate)s as a tool for the inhibition of iron-gall-ink corrosion.

    PubMed

    Sala, Martin; Kolar, Jana; Strlic, Matija; Kocevar, Marijan

    2006-05-22

    Two myo-inositol phosphates, myo-inositol 1,2,3-tris(dihydrogen phosphate) and myo-inositol 1,2,3,5-tetrakis(dihydrogen phosphate), have been synthesised in several steps from myo-inositol (in Chem. Abstr.: d-myo-inositol) in the form of their sodium salts. They were shown to prevent iron-gall-ink decay in cellulose items at the same level as phytic acid dodecasodium salt.

  6. A potassium-18-crown-6 salt of a cyclic myo-inositol phosphate.

    PubMed

    Kumara Swamy, K C; Kumaraswamy, S

    2001-10-01

    The six-membered phosphorinane ring in (1,4,7,10,13,16-hexaoxacyclooctadecane)potassium 2-O-benzoyl-1,3,5-O-methylidyne-myo-inositol 4,6-cyclophosphate trihydrate, [K(C(12)H(24)O(6))](C(14)H(12)O(9)P).3H(2)O, has a boat rather than a chair conformation. The K(+) ion is eight-coordinate and is connected to one of the phosphate O atoms, one of the O atoms of the myo-inositol residue and the six O atoms of the crown ether.

  7. Dephosphorylation of myo-inositol phosphates in the in vitro intestinal Caco-2 cell model.

    PubMed

    Briviba, Karlis; Schollenberger, Margit; Rodehutscord, Markus; Greiner, Ralf

    2017-05-30

    Plant and microbial phytases present in raw materials can cause a dephosphorylation of phytate (myo-inositol hexakisphosphate) (InsP6)) during food processing resulting in a broad range of different myo-inositol phosphates such as pentakisphosphate (InsP5) and tetrakisphosphate (InsP4) in foods. Here, we investigated whether the human intestinal epithelium is able to dephosphorylate myo-inositol phosphates (InsP6, InsP5-, InsP4-, InsP3-isomers) using an in vitro model with differentiated human Caco-2 cells cultured on semipermeable inserts. Incubation of InsP6 and an InsP5-isomer with cells for 3 h showed no dephosphorylation of both InsPs. Treatment of cells with a mixture of different InsP4-isomers, however, caused a formation of about 3.5% of an InsP3-isomer (Ins(1,5,6)P3) and treatment with a mixture of different InsP3-isomers caused about 20% formation of InsP2-isomers, respectively. Thus, human intestinal cells can contribute to the dephosphorylation of myo-inositol phosphates of partly dephosphorylated forms such as InsP3 and InsP4.

  8. 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.

  9. RNAi mediated down regulation of myo-inositol-3-phosphate synthase to generate low phytate rice.

    PubMed

    Ali, Nusrat; Paul, Soumitra; Gayen, Dipak; Sarkar, Sailendra Nath; Datta, Swapan K; Datta, Karabi

    2013-05-15

    Phytic acid (InsP6) is considered as the major source of phosphorus and inositol phosphates in cereal grains. Reduction of phytic acid level in cereal grains is desirable in view of its antinutrient properties to maximize mineral bioavailability and minimize the load of phosphorus waste management. We report here RNAi mediated seed-specific silencing of myo-inositol-3-phosphate synthase (MIPS) gene catalyzing the first step of phytic acid biosynthesis in rice. Moreover, we also studied the possible implications of MIPS silencing on myo-inositol and related metabolism, since, first step of phytic acid biosynthesis is also the rate limiting step of myo-inositol synthesis, catalyzed by MIPS. The resulting transgenic rice plants (T3) showed a 4.59 fold down regulation in MIPS gene expression, which corresponds to a significant decrease in phytate levels and a simultaneous increment in the amount of inorganic phosphate in the seeds. A diminution in the myo-inositol content of transgenic plants was also observed due to disruption of the first step of phytic acid biosynthetic pathway, which further reduced the level of ascorbate and altered abscisic acid (ABA) sensitivity of the transgenic plants. In addition, our results shows that in the transgenic plants, the lower phytate levels has led to an increment of divalent cations, of which a 1.6 fold increase in the iron concentration in milled rice seeds was noteworthy. This increase could be attributed to reduced chelation of divalent metal (iron) cations, which may correlate to higher iron bioavailability in the endosperm of rice grains. The present study evidently suggests that seed-specific silencing of MIPS in transgenic rice plants can yield substantial reduction in levels of phytic acid along with an increase in inorganic phosphate content. However, it was also demonstrated that the low phytate seeds had an undesirable diminution in levels of myo-inositol and ascorbate, which probably led to sensitiveness of seeds to

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

    PubMed

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

    2015-05-05

    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.

  11. Synthesis of myo-inositol 1,3,4,5,6-pentakisphosphate from inositol phosphates generated by receptor activation.

    PubMed Central

    Stephens, L R; Hawkins, P T; Barker, C J; Downes, C P

    1988-01-01

    myo-[3H]Inositol 1,3,4,5,6-pentakisphosphate can be made from myo-[3H]inositol 1,4,5-trisphosphate in a rat brain homogenate or soluble fraction. Although D-myo-inositol 3,4,5,6-tetrakisphosphate can be phosphorylated by a soluble rat brain enzyme to give myo-inositol 1,3,4,5,6-pentakisphosphate, it is not an intermediate in the pathway from myo-inositol 1,4,5-trisphosphate. The intermediates in the above pathway are myo-inositol 1,3,4,5-tetrakisphosphate, myo-inositol 1,3,4-trisphosphate and myo-inositol 1,3,4,6-tetrakisphosphate [Shears, Parry, Tang, Irvine, Michell & Kirk (1987) Biochem. J. 246, 139-147; Balla, Guillemette, Baukal & Catt (1987) J. Biol. Chem. 262, 9952-9955], and it is catalysed by soluble kinase activities of similar anion-exchange mobility and Mr value. Compounds with chromatographic and chemical properties consistent with the structures myo-inositol 1,3,4,5-tetrakisphosphate, myo-inositol 1,3,4,6-tetrakisphosphate and myo-inositol 3,4,5,6-tetrakisphosphate are present in avian erythrocytes, human 1321 N1 astrocytoma cells and primary-cultured murine bone-marrow-derived macrophages. The amounts of these inositol tetrakisphosphates rise upon muscarinic cholinergic stimulation of the astrocytoma cells or stimulation of macrophages with platelet-activating factor. PMID:2845930

  12. Inositol phosphates from barley low-phytate grain mutants analysed by metal-dye detection HPLC and NMR.

    PubMed

    Hatzack, F; Hübel, F; Zhang, W; Hansen, P E; Rasmussen, S K

    2001-03-01

    Inositol phosphates from barley low-phytate grain mutants and their parent variety were analysed by metal-dye detection HPLC and NMR. Compound assignment was carried out by comparison of retention times using a chemical hydrolysate of phytate [Ins(1,2,3,4,5,6)P(6)] as a reference. Co-inciding retention times indicated the presence of phytate, D/L-Ins(1,2,3,4,5)P(5), Ins(1,2,3,4,6)P(5), D/L-(1,2,4,5,6)P(5), D/L-(1,2,3,4)P(4), D/L-Ins(1,2,5,6)P(4) and D/L-Ins(1,4,5,6)P(4) in PLP1B mutants as well as the parent variety. In grain extracts from mutant lines PLP1A, PLP2A and PLP3A unusual accumulations of D/L-Ins(1,3,4,5)P(4) were observed whereas phytate and the above-mentioned inositol phosphates were present in relatively small amounts. Assignment of D/L-Ins(1,3,4,5)P(4) was corroborated by precise co-chromatography with a commercial Ins(1,3,4,5)P(4) standard and by NMR spectroscopy. Analysis of inositol phosphates during grain development revealed accumulation of phytate and D/L-Ins(1,3,4,5)P(4), which suggested the tetrakisphosphate compound to be an intermediate of phytate synthesis. This assumption was strengthened further by phytate degradation assays showing that D/L-Ins(1,3,4,5)P(4) did not belong to the spectrum of degradation products generated by endogenous phytase activity. Metabolic scenarios leading to accumulation of D/L-Ins(1,3,4,5)P(4) in barley low-phytate mutants are discussed.

  13. myo-inositol phosphate isomers generated by the action of a phytase from a malaysian waste-water bacterium.

    PubMed

    Greiner, Ralf; Farouk, Abd-Elaziem; Carlsson, Nils-Gunnar; Konietzny, Ursula

    2007-12-01

    Using a combination of High-Performance Ion Chromatography analysis and kinetic studies, the pathway of myo-inositol hexakisphosphate dephosphorylation by a phytase from a Malaysian waste-water bacterium was established. The data demonstrate that the phytase preferably dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-I(1,2,3,4,5)P(5), D-I(2,3,4,5)P(4), D-I(2,3,4)P(3), D-I(2,3)P(2) to finally I(2)P. It was estimated that more than 90% of phytate hydrolysis occurs via D-I(1,2,3,4,5)P(5). Thus, the phytase from the Malaysian waste-water bacterium has to be considered a 6-phytase (E.C. 3.1.3.26). A second pathway of minor importance could be proposed which is in accordance with the results obtained from analysis of the dephosphorylation products formed by the action of the phytase under investigation on myo-inositol hexakisphosphate. It proceeds via D/L-I(1,2,4,5,6)P(5), D/L-I(1,2,4,5)P(4), D/L-I(1,2,4)P(3), D/L-I(2,4)P(2) to finally I(2)P.

  14. Inositol phosphate pathway controls transcription of telomeric expression sites in trypanosomes.

    PubMed

    Cestari, Igor; Stuart, Ken

    2015-05-26

    African trypanosomes evade clearance by host antibodies by periodically changing their variant surface glycoprotein (VSG) coat. They transcribe only one VSG gene at a time from 1 of about 20 telomeric expression sites (ESs). They undergo antigenic variation by switching transcription between telomeric ESs or by recombination of the VSG gene expressed. We show that the inositol phosphate (IP) pathway controls transcription of telomeric ESs and VSG antigenic switching in Trypanosoma brucei. Conditional knockdown of phosphatidylinositol 5-kinase (TbPIP5K) or phosphatidylinositol 5-phosphatase (TbPIP5Pase) or overexpression of phospholipase C (TbPLC) derepresses numerous silent ESs in T. brucei bloodstream forms. The derepression is specific to telomeric ESs, and it coincides with an increase in the number of colocalizing telomeric and RNA polymerase I foci in the nucleus. Monoallelic VSG transcription resumes after reexpression of TbPIP5K; however, most of the resultant cells switched the VSG gene expressed. TbPIP5K, TbPLC, their substrates, and products localize to the plasma membrane, whereas TbPIP5Pase localizes to the nucleus proximal to telomeres. TbPIP5Pase associates with repressor/activator protein 1 (TbRAP1), and their telomeric silencing function is altered by TbPIP5K knockdown. These results show that specific steps in the IP pathway control ES transcription and antigenic switching in T. brucei by epigenetic regulation of telomere silencing.

  15. Inositol phosphate pathway controls transcription of telomeric expression sites in trypanosomes

    PubMed Central

    Cestari, Igor; Stuart, Ken

    2015-01-01

    African trypanosomes evade clearance by host antibodies by periodically changing their variant surface glycoprotein (VSG) coat. They transcribe only one VSG gene at a time from 1 of about 20 telomeric expression sites (ESs). They undergo antigenic variation by switching transcription between telomeric ESs or by recombination of the VSG gene expressed. We show that the inositol phosphate (IP) pathway controls transcription of telomeric ESs and VSG antigenic switching in Trypanosoma brucei. Conditional knockdown of phosphatidylinositol 5-kinase (TbPIP5K) or phosphatidylinositol 5-phosphatase (TbPIP5Pase) or overexpression of phospholipase C (TbPLC) derepresses numerous silent ESs in T. brucei bloodstream forms. The derepression is specific to telomeric ESs, and it coincides with an increase in the number of colocalizing telomeric and RNA polymerase I foci in the nucleus. Monoallelic VSG transcription resumes after reexpression of TbPIP5K; however, most of the resultant cells switched the VSG gene expressed. TbPIP5K, TbPLC, their substrates, and products localize to the plasma membrane, whereas TbPIP5Pase localizes to the nucleus proximal to telomeres. TbPIP5Pase associates with repressor/activator protein 1 (TbRAP1), and their telomeric silencing function is altered by TbPIP5K knockdown. These results show that specific steps in the IP pathway control ES transcription and antigenic switching in T. brucei by epigenetic regulation of telomere silencing. PMID:25964327

  16. Regulatory Mutations of Inositol Biosynthesis in Yeast: Isolation of Inositol-Excreting Mutants

    PubMed Central

    Greenberg, Miriam L.; Reiner, Barry; Henry, Susan A.

    1982-01-01

    The enzyme inositol-1-phosphate synthase (I-1-P synthase), product of the INO1 locus, catalyzes the synthesis of inositol-1-phosphate from the substrate glucose-6-phosphate. The activity of this enzyme is dramatically repressed in the presence of inositol. By selecting for mutants which overproduce and excrete inositol, we have identified mutants constitutive for inositol-1-phosphate synthase as well as a mutation in phospholipid biosynthesis. Genetic analysis of the mutants indicates that at least three loci (designated OPI1, OPI2 and OPI4) direct inositol-mediated repression of I-1-P synthase. Mutants of these loci synthesize I-1-P synthase constitutively. Three loci are unlinked to each other and to INO1, the structural gene for the enzyme. A mutant of a fourth locus, OPI3, does not synthesize I-1-P synthase constitutively, despite its inositol excretion phenotype. This mutant is preliminarily identified as having a defect in phospholipid synthesis. PMID:7047296

  17. Inositol phosphates and phosphoinositides activate insulin-degrading enzyme, while phosphoinositides also mediate binding to endosomes.

    PubMed

    Song, Eun Suk; Jang, HyeIn; Guo, Hou-Fu; Juliano, Maria A; Juliano, Luiz; Morris, Andrew J; Galperin, Emilia; Rodgers, David W; Hersh, Louis B

    2017-04-04

    Insulin-degrading enzyme (IDE) hydrolyzes bioactive peptides, including insulin, amylin, and the amyloid β peptides. Polyanions activate IDE toward some substrates, yet an endogenous polyanion activator has not yet been identified. Here we report that inositol phosphates (InsPs) and phosphatdidylinositol phosphates (PtdInsPs) serve as activators of IDE. InsPs and PtdInsPs interact with the polyanion-binding site located on an inner chamber wall of the enzyme. InsPs activate IDE by up to ∼95-fold, affecting primarily Vmax The extent of activation and binding affinity correlate with the number of phosphate groups on the inositol ring, with phosphate positional effects observed. IDE binds PtdInsPs from solution, immobilized on membranes, or presented in liposomes. Interaction with PtdInsPs, likely PtdIns(3)P, plays a role in localizing IDE to endosomes, where the enzyme reportedly encounters physiological substrates. Thus, InsPs and PtdInsPs can serve as endogenous modulators of IDE activity, as well as regulators of its intracellular spatial distribution.

  18. A chromogenic substrate for phosphatidylinositol-specific phospholipase C: 4-nitrophenyl myo-inositol-1-phosphate.

    PubMed

    Shashidhar, M S; Volwerk, J J; Griffith, O H; Keana, J F

    1991-12-01

    A chromogenic water-soluble substrate for phosphatidylinositol-specific phospholipase C was synthesized starting from myo-inositol employing isopropylidene and 4-methoxytetrahydropyranyl protecting groups. In this analogue of phosphatidylinositol, 4-nitrophenol replaces the diacylglycerol moiety, resulting in synthetic, racemic 4-nitrophenyl myo-inositol-1-phosphate. Using this synthetic substrate a rapid, convenient and sensitive spectrophotometric assay for the phosphatidylinositol-specific phospholipase C from Bacillus cereus was developed. Initial rates of the cleavage of the nitrophenol substrate were linear with time and the amount of enzyme used. At pH 7.0, specific activities for the B. cereus enzyme were 77 and 150 mumol substrate cleaved min-1 (mg protein)-1 at substrate concentrations of 1 and 2 mM, respectively. Under these conditions, less than 50 ng quantities of enzyme were easily detected. The chromogenic substrate was stable during long term storage (6 months) as a solid at -20 degrees C.

  19. A role for rat inositol polyphosphate kinases rIPK2 and rIPK1 in inositol pentakisphosphate and inositol hexakisphosphate production in rat-1 cells.

    PubMed

    Fujii, Makoto; York, John D

    2005-01-14

    Over 30 inositol polyphosphates are known to exist in mammalian cells; however, the majority of them have uncharacterized functions. In this study we investigated the molecular basis of synthesis of highly phosphorylated inositol polyphosphates (such as inositol tetrakisphosphate, inositol pentakisphosphate (IP5), and inositol hexakisphosphate (IP6)) in rat cells. We report that heterologous expression of rat inositol polyphosphate kinases rIPK2, a dual specificity inositol trisphosphate/inositol tetrakisphosphate kinase, and rIPK1, an IP5 2-kinase, were sufficient to recapitulate IP6 synthesis from inositol 1,4,5-trisphosphate in mutant yeast cells. Overexpression of rIPK2 in Rat-1 cells increased inositol 1,3,4,5,6-pentakisphosphate (I(1,3,4,5,6)P5) levels about 2-3-fold compared with control. Likewise in Rat-1 cells, overexpression of rIPK1 was capable of completely converting I(1,3,4,5,6)P5 to IP6. Simultaneous overexpression of both rIPK2 and rIPK1 in Rat-1 cells increased both IP5 and IP6 levels. To reduce IPK2 activity in Rat-1 cells, we introduced vector-based short interference RNA against rIPK2. Cells harboring the short interference RNA had a 90% reduction of mRNA levels and a 75% decrease of I(1,3,4,5,6)P5. These data confirm the involvement of IPK2 and IPK1 in the conversion of inositol 1,4,5-trisphosphate to IP6 in rat cells. Furthermore these data suggest that rIPK2 and rIPK1 act as key determining steps in production of IP5 and IP6, respectively. The ability to modulate the intracellular inositol polyphosphate levels by altering IPK2 and IPK1 expression in rat cells will provide powerful tools to study the roles of I(1,3,4,5,6)P5 and IP6 in cell signaling.

  20. Effect of traditional, microwave and industrial cooking on inositol phosphate content in beans, chickpeas and lentils.

    PubMed

    Máñez, G; Alegría, A; Farré, R; Frígola, A

    2002-11-01

    An high-performance liquid chromatography method for determining inositol phosphate fractions was adapted to legumes. The validity of the method was assessed by estimating the following analytical parameters: linearity (linear response between 125 and 5000 microg inositol hexaphosphate (IP(6))/ml); instrumental precision and method precision (relative standard deviation, %) were 1.9% (IP(6)) for instrumental, and 2.5% (IP(6)) and 8.2% (IP(5)) for method precision. An accuracy was estimated by percentage recovery (72 +/- 3%). The application of this method to raw, conventional, microwave-cooked and ready-to-eat beans, chickpeas and lentils gave IP(6) contents ranging from 0.63 g/100 g dry matter in ready-to-eat lentils to 1.87 g/100 g dry matter in raw beans. The IP(6) content was reduced by all the cooking procedures, while the relative percentage of inositol pentaphosphate increased in all the legumes studied, and reached the maximum of 31% (expressed in relation to dry matter) in ready-to-eat beans.

  1. 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

    2005-10-05

    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.

  2. Seed phosphorus and inositol phosphate phenotype of barley low phytic acid genotypes.

    PubMed

    Dorsch, John A; Cook, Allen; Young, Kevin A; Anderson, Joseph M; Bauman, Andrew T; Volkmann, Carla J; Murthy, Pushpalatha P N; Raboy, Victor

    2003-03-01

    myo-Inositol-1,2,3,4,5,6-hexakisphosphate (Ins P(6) or "phytic acid") typically represents approximately 75% of the total phosphorus and >80% of soluble myo-inositol (Ins) phosphates in seeds. The seed phosphorus and Ins phosphate phenotypes of four non-lethal barley (Hordeum vulgare L.) low phytic acid mutations are described. In seeds homozygous for M 635 and M 955 reductions in Ins P(6), approximately 75 and >90% respectively, are accompanied by reductions in other Ins phosphates and molar-equivalent increases in Pi. This phenotype suggests a block in supply of substrate Ins. In seeds homozygous for barley low phytic acid 1-1 (lpa1-1), a 45% decrease in Ins P(6) is mostly matched by an increase in Pi but also accompanied by small increases in Ins(1,2,3,4,6)P(5). In seeds homozygous for barley lpa2-1, reductions in seed Ins P(6) are accompanied by increases in both Pi and in several Ins phosphates, a phenotype that suggests a lesion in Ins phosphate metabolism, rather than Ins supply. The increased Ins phosphates in barley lpa2-1 seed are: Ins(1,2,3,4,6)P(5); Ins(1,2,4,6)P(4) and/or its enantiomer Ins(2,3,4,6)P(4); Ins(1,2,3,4)P(4) and/or its enantiomer Ins(1,2,3,6)P(4); Ins(1,2,6)P(3) and/or its enantiomer Ins(2,3,4)P(3); Ins(1,5,6)P(3) and/or its enantiomer Ins(3,4,5)P(3) (the methods used here cannot distinguish between enantiomers). This primarily "5-OH" series of Ins phosphates differs from the "1-/3-OH" series observed at elevated levels in seed of the maize lpa2 genotype, but previous chromosomal mapping data indicated that the maize and barley lpa2 loci might be orthologs of a single ancestral gene. Therefore one hypothesis that might explain the differing lpa2 phenotypes is that their common ancestral gene encodes a multi-functional, Ins phosphate kinase with both "1-/-3-" and "5-kinase" activities. A putative pyrophosphate-containing Ins phosphate, possibly an Ins P(7), was also observed in the mature seed of all barley genotypes except lpa2-1. Barley M

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

    PubMed

    Rounds, M A; Nielsen, S S

    1993-10-29

    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.

  4. 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.

  5. 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.

  6. The pathway for the production of inositol hexakisphosphate in human cells.

    PubMed

    Verbsky, John W; Chang, Shao-Chun; Wilson, Monita P; Mochizuki, Yasuhiro; Majerus, Philip W

    2005-01-21

    The yeast and Drosophila pathways leading to the production of inositol hexakisphosphate (InsP(6)) have been elucidated recently. The in vivo pathway in humans has been assumed to be similar. Here we show that overexpression of Ins(1,3,4)P(3) 5/6-kinase in human cell lines results in an increase of inositol tetrakisphosphate (InsP(4)) isomers, inositol pentakisphosphate (InsP(5)) and InsP(6), whereas its depletion by RNA interference decreases the amounts of these inositol phosphates. Expression of Ins(1,3,4,6)P(4) 5-kinase does not increase the amount of InsP(5) and InsP(6), although its depletion does block InsP(5) and InsP(6) production, showing that it is necessary for production of InsP(5) and InsP(6). Expression of Ins(1,3,4,5,6)P(5) 2-kinase increases the amount of InsP(6) by depleting the InsP(5) in the cell, and depletion of 2-kinase decreases the amount of InsP(6) and causes an increase in InsP(5). These results are consistent with a pathway that produces InsP(6) through the sequential action of Ins(1,3,4)P(3) 5/6-kinase, Ins(1,3,4,6)P(4) 5-kinase, and Ins(1,3,4,5,6)P5 2-kinase to convert Ins(1,3,4)P(3) to InsP(6). Furthermore, the evidence implicates 5/6-kinase as the rate-limiting enzyme in this pathway.

  7. Structural Studies of Medicago truncatula Histidinol Phosphate Phosphatase from Inositol Monophosphatase Superfamily Reveal Details of Penultimate Step of Histidine Biosynthesis in Plants.

    PubMed

    Ruszkowski, Milosz; Dauter, Zbigniew

    2016-05-06

    The penultimate enzyme in the histidine biosynthetic pathway catalyzes dephosphorylation of l-histidinol 1-phosphate (HOLP) into l-histidinol. The recently discovered in Arabidopsis thaliana plant-type histidinol phosphate phosphatase (HPP) shares no homology with the two other HPP superfamilies known previously in prokaryotes and resembles myo-inositol monophosphatases (IMPases). In this work, identification of an HPP enzyme from a model legume, Medicago truncatula (MtHPP) was based on the highest sequence identity to A. thaliana enzyme. Biochemical assays confirmed that MtHPP was able to cleave inorganic phosphate from HOLP but not from d-myo-inositol-1-phosphate, the main substrate of IMPases. Dimers of MtHPP, determined by size exclusion chromatography, in the presence of CO2 or formaldehyde form mutual, methylene-bridged cross-links between Lys(158) and Cys(245) residues. Four high resolution crystal structures, namely complexes with HOLP (substrate), l-histidinol (product), and PO4 (3-) (by-product) as well as the structure showing the cross-linking between two MtHPP molecules, provide detailed structural information on the enzyme. Based on the crystal structures, the enzymatic reaction mechanism of IMPases is accustomed to fit the data for MtHPP. The enzymatic reaction, which requires Mg(2+) cations, is catalyzed mainly by amino acid residues from the N-terminal domain. The C-terminal domain, sharing little identity with IMPases, is responsible for the substrate specificity (i.e. allows the enzyme to distinguish between HOLP and d-myo-inositol-1-phosphate). Structural features, mainly the presence of a conserved Asp(246), allow MtHPP to bind HOLP specifically.

  8. myo-Inositol phosphate isomers generated by the action of a phytate-degrading enzyme from Klebsiella terrigena on phytate.

    PubMed

    Greiner, Ralf; Carlsson, Nils-Gunnar

    2006-08-01

    For the first time a dual pathway for dephosphorylation of myo-inositol hexakisphosphate by a histidine acid phytase was established. The phytate-degrading enzyme of Klebsiella terrigena degrades myo-inositol hexakisphosphate by stepwise dephosphorylation, preferably via D-Ins(1,2,4,5,6)P5, D-Ins(1,2,5,6)P4, D-Ins(1,2,6)P3, D-Ins(1,2)P2 and alternatively via D-Ins(1,2,4,5,6)P5, Ins(2,4,5,6)P4, D-Ins(2,4,5)P3, D-Ins(2,4)P2 to finally Ins(2)P. It was estimated that more than 98% of phytate hydrolysis occurs via D-Ins(1,2,4,5,6)P5. Therefore, the phytate-degrading enzyme from K. terrigena has to be considered a 3-phytase (EC 3.1.3.8). A second dual pathway of minor importance could be proposed that is in accordance with the results obtained by analysis of the dephosphorylation products formed by the action of the phytate-degrading enzyme of K. terrigena on myo-inositol hexakisphosphate. It proceeds preferably via D-Ins(1,2,3,5,6)P5, D-Ins(1,2,3,6)P4, Ins(1,2,3)P3, D-Ins(2,3)P2 and alternatively via D-Ins(1,2,3,5,6)P5, D-Ins(2,3,5,6)P4, D-Ins(2,3,5)P3, D-Ins(2,3)P2 to finally Ins(2)P. D-Ins(2,3,5,6)P4, D-Ins(2,3,5)P3, and D-Ins(2,4)P2 were reported for the first time as intermediates of enzymatic phytate dephosphorylation. A role of the phytate-degrading enzyme from K. terrigena in phytate breakdown could not be ruled out. Because of its cytoplasmatic localization and the suggestions for substrate recognition, D-Ins(1,3,4,5,6)P5 might be the natural substrate of this enzyme and, therefore, may play a role in microbial pathogenesis or cellular myo-inositol phosphate metabolism.

  9. myo-Inositol 1-Phosphate Synthase Inhibition and Control of Uridine Diphosphate-d-glucuronic Acid Biosynthesis in Plants 12

    PubMed Central

    Loewus, Mary W.; Loewus, Frank

    1974-01-01

    Of the eight intermediates associated with the two pathways of UDP-d-glucuronic acid biosynthesis found in plants, only d-glucuronic acid inhibited myo-inositol 1-phosphate synthase (EC 5.5.1.4), formerly referred to as d-glucose 6-phosphate cycloaldolase. Inhibition was competitive. An attempt to demonstrate over-all reversibility of the synthase indicated that it was less than 5% reversible, if at all. PMID:16658890

  10. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Neurotransmitter agonists inhibit inositol phosphate formation in the brain of bupropione-treated rats

    SciTech Connect

    Butler, P.D.; Hungund, B.; Suckow, R.; Barkai, A.I.

    1986-03-05

    Bupropione is a chemically unique antidepressant whose mechanism of action is not known. In this study they have evaluated the effect of chronic treatment with bupropione on the receptor-mediated release of inositol phosphates (IP) from brain slices in rats. Animals were implanted with Alzet osmotic pumps that delivered bupropione at a constant rate (40mg/kg/day) for 2 weeks. Cross-chopped slices of cerebral cortex from control and drug-treated rats were prelabelled with myo-/sup 3/H-inositol in HEPES buffer containing 11 mM LiCl. Accumulation of IP was measured in the presence and absence of the following agonists: Carbamylcholine (100..mu..m); norepinephrine (5..mu..M) and serotonin (10..mu..M). All agonists stimulated release of IP from slices of control animals but appeared to inhibit IP release in bupropione-treated rats. These results indicate that a phospholipase C inhibitor may appear following the activation of this enzyme by the agonist, and that the agonist-induced formation of the apparent inhibitor may be markedly enhanced after treatment with bupropione.

  12. Inhibitory effects of phytic acid and other inositol phosphates on zinc and calcium absorption in suckling rats.

    PubMed

    Lönnerdal, B; Sandberg, A S; Sandström, B; Kunz, C

    1989-02-01

    While it is known that phytic acid, inositol hexaphosphate, has a negative effect on zinc and calcium absorption, the effects of inositol which is phosphorylated to a lesser extent are less known. We have prepared inositol triphosphate (IP-3), tetraphosphate (IP-4), pentaphosphate (IP-5) and hexaphosphate (IP-6) by hydrolysis of sodium phytate and separation by ion-exchange chromatography and have studied their effect on zinc and calcium absorption. Using a suckling rat pup model, we found that liver uptake of 65Zn after 6 h was 5% of the total dose from solutions of IP-6, 19% from IP-5, 28% from IP-4, 29% from IP-3 and 31% from ZnCl2 (control). Non-absorbed calcium was 17%, 1.4%, 0.5%, 0.5% and 0.5% of the given dose of 45Ca, respectively. Thus, at a high degree of phosphorylation (IP-6, IP-5), zinc and calcium uptake was inhibited, while no effect was observed for the other phosphates. Consequently, total "phytate" analysis, which includes inositol phosphates with varying degrees of phosphorylation, can give misleading information with regard to mineral availability. In addition, even limited dephosphorylation of inositol hexaphosphate can have a positive effect on mineral absorption.

  13. 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

  14. A novel Entamoeba histolytica inositol phosphate kinase catalyzes the formation of 5PP-Ins(1,2,3,4,6)P(5).

    PubMed

    Löser, Benjamin; Nalaskowski, Marcus M; Fanick, Werner; Lin, Hongying; Tannich, Egbert; Mayr, Georg W

    2012-01-01

    The parasitic protozoan Entamoeba histolytica is able to invade human tissues by secreting proteolytic enzymes. This secretion is regulated by inositol phosphate-mediated Ca(2+) release from internal stores. To further investigate the inositol phosphate metabolism of Entamoeba histolytica four putative inositol phosphate kinase genes (ehipk1-4) were identified and their expression analyzed by real-time quantitative PCR using RNA of trophozoites. Furthermore inositol phosphate kinase EhIPK1 was recombinantly expressed, purified and enzymatically characterized. Its main activity is the conversion of InsP(6) to 5PP-Ins(1,2,3,4,6)P(5), one of the main inositol phosphates found in Entamoeba histolytica. Remarkably, EhIPK1 possesses several additional enzymatic activities, e.g. the phosphorylation of the Ca(2+)-releasing second messenger Ins(1,4,5)P(3).We were able to identify several compounds with inhibitory potential against EhIPK1. Because of the important role of inositol phosphates in the invasion of human tissues by Entamoeba histolytica, inositol phosphate metabolizing enzymes are interesting targets for novel therapeutic approaches.

  15. Inositol Hexakis Phosphate is the Seasonal Phosphorus Reservoir in the Deciduous Woody Plant Populus alba L.

    PubMed

    Kurita, Yuko; Baba, Kei'ichi; Ohnishi, Miwa; Matsubara, Ryosuke; Kosuge, Keiko; Anegawa, Aya; Shichijo, Chizuko; Ishizaki, Kimitsune; Kaneko, Yasuko; Hayashi, Masahiko; Suzaki, Toshinobu; Fukaki, Hidehiro; Mimura, Tetsuro

    2017-09-01

    Seasonal recycling of nutrients is an important strategy for deciduous perennials. Deciduous perennials maintain and expand their nutrient pools by the autumn nutrient remobilization and the subsequent winter storage throughout their long life. Phosphorus (P), one of the most important elements in living organisms, is remobilized from senescing leaves during autumn in deciduous trees. However, it remains unknown how phosphate is stored over winter. Here we show that in poplar trees (Populus alba L.), organic phosphates are accumulated in twigs from late summer to winter, and that IP6 (myo-inositol-1,2,3,4,5,6-hexakis phosphate: phytic acid) is the primary storage form. IP6 was found in high concentrations in twigs during winter and quickly decreased in early spring. In parenchyma cells of winter twigs, P was associated with electron-dense structures, similar to globoids found in seeds of higher plants. Various other deciduous trees were also found to accumulate IP6 in twigs during winter. We conclude that IP6 is the primary storage form of P in poplar trees during winter, and that it may be a common strategy for seasonal P storage in deciduous woody plants. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  16. 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

  17. 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.

  18. Myo-inositol products in polycystic ovary syndrome (PCOS) treatment: quality, labeling accuracy, and cost comparison.

    PubMed

    Papaleo, E; Molgora, M; Quaranta, L; Pellegrino, M; De Michele, F

    2011-02-01

    To evaluate the labeling accuracy of four myo-inositol products, designed for polycystic ovary syndrome (PCOS) treatment, available on the italian market and to perform a cost comparison based on myo-inositol content in milligrams for products analyzed. Four (4) myo-inositol products (3 sachet and 1 tablet formulations) were dissolved using water, and each sample was analyzed for myo-inositol content using a high-performance liquid chromatography (HPLC) method with index refraction detector. The amount of myo-inositol per purchased product was then divided into its purchase price in order to make cost comparisons between the products based on a 2 and 4 g/day dose. A significant difference in the myo-inositol content, compared with the labeling was found for the products. Only 1 product contained more than 95% of the myo-inositol content claimed on the label, and there was a product with less than 75% of the labeling amount. Based on a 2-g myo-inositol per day dose, the cost of a 30-day supply ranged from Euro 20,77 and Euro 71,86, after correction by actual amount of myo-inositol. There is a lack of conformity between declared and actual amount of myo-inositol among the products tested and the majority of the products contained less than 95% of labeled amounts. There should be a better control in the manufacturing process in order to ensure more quality and accuracy. Nowadays consumers cannot trust myo-inositol product labels to represent the product's content accurately or that product pricing is a reflection of myo-inositol content.

  19. H(+)-dependent inorganic phosphate uptake in Trypanosoma brucei is influenced by myo-inositol transporter.

    PubMed

    Russo-Abrahão, Thais; Koeller, Carolina Macedo; Steinmann, Michael E; Silva-Rito, Stephanie; Marins-Lucena, Thaissa; Alves-Bezerra, Michele; Lima-Giarola, Naira Ligia; de-Paula, Iron Francisco; Gonzalez-Salgado, Amaia; Sigel, Erwin; Bütikofer, Peter; Gondim, Katia Calp; Heise, Norton; Meyer-Fernandes, José Roberto

    2017-04-01

    Trypanosoma brucei is an extracellular protozoan parasite that causes human African trypanosomiasis or "sleeping sickness". During the different phases of its life cycle, T. brucei depends on exogenous inorganic phosphate (Pi), but little is known about the transport of Pi in this organism. In the present study, we showed that the transport of (32)Pi across the plasma membrane follows Michaelis-Menten kinetics and is modulated by pH variation, with higher activity at acidic pH. Bloodstream forms presented lower Pi transport in comparison to procyclic forms, that displayed an apparent K0.5 = 0.093 ± 0.008 mM. Additionally, FCCP (H(+)-ionophore), valinomycin (K(+)-ionophore) and SCH28080 (H(+), K(+)-ATPase inhibitor) inhibited the Pi transport. Gene Tb11.02.3020, previously described to encode the parasite H(+):myo-inositol transporter (TbHMIT), was hypothesized to be potentially involved in the H(+):Pi cotransport because of its similarity with the Pho84 transporter described in S. cerevisiae and other trypanosomatids. Indeed, the RNAi mediated knockdown remarkably reduced TbHMIT gene expression, compromised cell growth and decreased Pi transport by half. In addition, Pi transport was inhibited when parasites were incubated in the presence of concentrations of myo-inositol that are above 300 μM. However, when expressed in Xenopus laevis oocytes, two-electrode voltage clamp experiments provided direct electrophysiological evidence that the protein encoded by TbHMIT is definitely a myo-inositol transporter that may be only marginally affected by the presence of Pi. These results confirmed the presence of a Pi carrier in T. brucei, similar to the H(+)-dependent inorganic phosphate system described in S. cerevisiae and other trypanosomatids. This transport system contributes to the acquisition of Pi and may be involved in the growth and survival of procyclic forms. In summary, this work presents the first description of a Pi transport system in T. brucei.

  20. 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.

  1. The Maize Low-Phytic Acid Mutant lpa2 Is Caused by Mutation in an Inositol Phosphate Kinase Gene

    PubMed Central

    Shi, Jinrui; Wang, Hongyu; Wu, Yunsheng; Hazebroek, Jan; Meeley, Robert B.; Ertl, David S.

    2003-01-01

    Reduced phytic acid content in seeds is a desired goal for genetic improvement in several crops. Low-phytic acid mutants have been used in genetic breeding, but it is not known what genes are responsible for the low-phytic acid phenotype. Using a reverse genetics approach, we found that the maize (Zea mays) low-phytic acid lpa2 mutant is caused by mutation in an inositol phosphate kinase gene. The maize inositol phosphate kinase (ZmIpk) gene was identified through sequence comparison with human and Arabidopsis Ins(1,3,4)P3 5/6-kinase genes. The purified recombinant ZmIpk protein has kinase activity on several inositol polyphosphates, including Ins(1,3,4)P3, Ins(3,5,6)P3, Ins(3,4,5,6)P4, and Ins(1,2,5,6)P4. The ZmIpk mRNA is expressed in the embryo, the organ where phytic acid accumulates in maize seeds. The ZmIpk Mutator insertion mutants were identified from a Mutator F2 family. In the ZmIpk Mu insertion mutants, seed phytic acid content is reduced approximately 30%, and inorganic phosphate is increased about 3-fold. The mutants also accumulate myo-inositol and inositol phosphates as in the lpa2 mutant. Allelic tests showed that the ZmIpk Mu insertion mutants are allelic to the lpa2. Southern-blot analysis, cloning, and sequencing of the ZmIpk gene from lpa2 revealed that the lpa2-1 allele is caused by the genomic sequence rearrangement in the ZmIpk locus and the lpa2-2 allele has a nucleotide mutation that generated a stop codon in the N-terminal region of the ZmIpk open reading frame. These results provide evidence that ZmIpk is one of the kinases responsible for phytic acid biosynthesis in developing maize seeds. PMID:12586875

  2. A protein tyrosine phosphatase-like inositol polyphosphatase from Selenomonas ruminantium subsp. lactilytica has specificity for the 5-phosphate of myo-inositol hexakisphosphate.

    PubMed

    Puhl, Aaron A; Greiner, Ralf; Selinger, L Brent

    2008-01-01

    Although it is becoming well known that myo-inositol polyphosphates and the enzymes involved in their metabolism play a critical role in eukaryotic systems, little is understood of their significance in prokaryotic systems. A novel protein tyrosine phosphatase (PTP)-like inositol polyphosphatase (IPPase) gene has been cloned from Selenomonas ruminantium subsp. lactilytica (phyAsrl). The deduced amino acid sequence of PhyAsrl is most similar to a PTP-like IPPase from the anaerobic bacterium S. ruminantium (35% identity), but also shows similarity (19-30% identity) to various other putative prokaryotic PTPs. Recombinant PhyAsrl could dephosphorylate myo-inositol hexakisphosphate (Ins P(6)) in vitro, and maximal activity was displayed at an ionic strength of 200 mM, a pH of 4.5, and a temperature of 55 degrees C. In order to elucidate its substrate specificity and pathway of Ins P(6) dephosphorylation, a combination of kinetic and high-performance ion-pair chromatography studies were conducted. The data indicated that PhyAsrl has a general specificity for polyphosphorylated myo-inositol substrates, but can also dephosphorylate molecules containing high energy pyrophosphate bonds in vitro. PhyAsrl is unique from other microbial IPPases in that it preferentially cleaves the 5-phosphate position of Ins P(6). Furthermore, it can produce Ins(2)P via a highly unique and ordered pathway of sequential dephosphorylation: Ins P(6), Ins(1,2,3,4,6)P(5), D-Ins(1,2,3,6)P(4), Ins(1,2,3)P(3), and D/L-Ins(1,2)P(2). Finally, reverse transcription PCR was used to determine that phyAsrl is constitutively expressed, and together with bioinformatic analysis, was used to gain an understanding of its physiological significance.

  3. Norepinephrine and veratrine stimulated formation of inositol phosphates in rat brain slices

    SciTech Connect

    Maier, K.U.; Rutledge, C.O.

    1986-03-05

    Stimulation of phosphoinositide (PIn) hydrolysis by depolarization with veratrine was compared to that produced by stimulation of alpha/sub 1/ adrenoceptors by norepinephrine (NE). The PIns in rat cerebral cortex were labelled with /sup 3/H-myoinositol and the effects of the drugs on the formation of the three /sup 3/H-inositol phosphates (IP, IP2, IP3) were determined. The amounts of IP and IP2 formed by a maximal concentration of veratrine were about 50% of that formed by a maximal concentration of NE while the amount of IP3 formed after stimulation by veratrine was only about 10% of that produced by NE. The increase in IP was linear with time (30 min) for both NE and veratrine. IP2 and IP3 stimulation by veratrine reached a maximum at 5 min whereas that produced by NE continued to increase for 30 min. Blockade of voltage dependent calcium channels with manganese produced nearly complete antagonism of the veratrine response while only partially antagonizing the NE response. NE-induced IP2 formation was less sensitive to manganese than IP or IP3. These data suggest that veratrine causes hydrolysis of either a different pool of PIn or that the hydrolysis occurs by a different mechanism compared to NE. The data also suggest that IP2 may be produced directly from phosphatidylinositol 4-phosphate rather than solely as a metabolite of IP3.

  4. Interactions between HIV-1 Gag molecules in solution: an inositol phosphate-mediated switch.

    PubMed

    Datta, Siddhartha A K; Zhao, Zhuojun; Clark, Patrick K; Tarasov, Sergey; Alexandratos, Jerry N; Campbell, Stephen J; Kvaratskhelia, Mamuka; Lebowitz, Jacob; Rein, Alan

    2007-01-19

    Retrovirus particle assembly is mediated by the Gag protein. Gag is a multi-domain protein containing discrete domains connected by flexible linkers. When recombinant HIV-1 Gag protein (lacking myristate at its N terminus and the p6 domain at its C terminus) is mixed with nucleic acid, it assembles into virus-like particles (VLPs) in a fully defined system in vitro. However, this assembly is defective in that the radius of curvature of the VLPs is far smaller than that of authentic immature virions. This defect can be corrected to varying degrees by addition of inositol phosphates to the assembly reaction. We have now explored the binding of inositol hexakisphosphate (IP6) to Gag and its effects upon the interactions between Gag protein molecules in solution. Our data indicate that basic regions at both ends of the protein contribute to IP6 binding. Gag is in monomer-dimer equilibrium in solution, and mutation of the previously described dimer interface within its capsid domain drastically reduces Gag dimerization. In contrast, when IP6 is added, Gag is in monomer-trimer rather than monomer-dimer equilibrium. The Gag protein with a mutation at the dimer interface also remains almost exclusively monomeric in IP6; thus the "dimer interface" is essential for the trimeric interaction in IP6. We discuss possible explanations for these results, including a change in conformation within the capsid domain induced by the binding of IP6 to other domains within the protein. The participation of both ends of Gag in IP6 interaction suggests that Gag is folded over in solution, with its ends near each other in three-dimensional space; direct support for this conclusion is provided in a companion manuscript. As Gag is an extended rod in immature virions, this apparent proximity of the ends in solution implies that it undergoes a major conformational change during particle assembly.

  5. 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)

  6. The interrelationships of the inositol phosphates formed in vasopressin-stimulated WRK-1 rat mammary tumour cells.

    PubMed Central

    Barker, C J; Wong, N S; Maccallum, S M; Hunt, P A; Michell, R H; Kirk, C J

    1992-01-01

    1. Temporal changes in the levels of many inositol phosphates, whose structural characterization is presented in the preceding paper [Wong, Barker, Morris, Craxton, Kirk & Michell (1991) Biochem. J. 286, 459-468], have been monitored in vasopressin-stimulated WRK-1 cells. 2. Upon stimulation, Ins(1,4,5)P3 accumulated within 1 s, consistent with its role as a rapidly acting second messenger produced by receptor activation of phosphoinositidase C. Ins(1,4)P2 and Ins(1,3,4,5)P4, both of which are immediate products of Ins(1,4,5)P3 metabolism, also accumulated quickly. Ins4P, Ins(1,3,4)P3, Ins(3,4)P2, Ins(1,3)P2, Ins1P and Ins3P, which are intermediates in the metabolism of Ins(1,4)P2 and Ins(1,3,4,5)P4 to inositol, accumulated after seconds or within a few minutes, and in a temporal sequence consistent with their known metabolic interrelationships. 3. The stimulated accumulation of Ins(1,3,4,6)P4 was delayed, as expected if it is formed by phosphorylation of Ins(1,3,4)P3. 4. Ins(3,4,5,6)P4 accumulated 2-3-fold in a few minutes, and mainly before Ins(1,3,4,6)P4. 5. Using a [3H]-/[14C]-inositol double-labelling protocol, we obtained evidence that all of the compounds that accumulated upon stimulation, except Ins(3,4,5,6)P4, originated from lipid-derived Ins(1,4,5)P3, but that the newly formed Ins(3,4,5,6)P4 came from a different source. 6. There were no consistent changes in the levels of Ins(1,3,4,5,6)P5 and InsP6 during stimulation. 7. Alongside the gradual accumulation of Ins(1:2-cyclic,4,5)P3 during stimulation [Wong, Barker, Shears, Kirk & Michell (1988) Biochem. J. 252, 1-5], there was an accumulation of Ins(1:2-cyclic,4)P2 and Ins(1:2-cyclic)P, probably as either minor side products of phosphoinositidase C action or metabolites of Ins(1:2-cyclic,4,5)P3. 8. When Li+ was present during stimulation, it redirected the dephosphorylation pathways downstream of Ins(1,4,5)P3 in the manner expected from its inhibition of inositol monophosphatase and Ins(1,4)P2/Ins(1,3,4)P

  7. Developing an Inositol-Phosphate-Actuated Nanochannel System by Mimicking Biological Calcium Ion Channels.

    PubMed

    Lu, Qi; Tang, Qiuhan; Chen, Zhonghui; Zhao, Shilong; Qing, Guangyan; Sun, Taolei

    2017-09-13

    In eukaryotic cells, ion channels, which ubiquitously present as polypeptides or proteins, usually regulate the ion transport across biological membranes by conformational switching of the channel proteins in response to the binding of diverse signaling molecules (e.g., inositol phosphate, abbreviated to InsP). To mimic the gating behaviors of natural Ca(2+) channels manipulated by InsPs, a smart poly[(N-isopropylacrylamide-co-4-(3-acryloylthioureido) benzoic acid)0.2] (denoted as PNI-co-ATBA0.2) was integrated onto a porous anodic alumina (PAA) membrane, building an InsP-actuated nanochannel system. Driven by the intensive hydrogen bonding complexation of ATBA monomer with InsP, the copolymer chains displayed a remarkable and reversible conformational transition from a contracted state to a swollen one, accompanied with significant changes in surface morphology, wettability, and viscoelasticity. Benefiting from these features, dynamic gating behaviors of the nanochannels located on the copolymer-modified PAA membrane could be precisely manipulated by InsPs, reflected as a satisfactory linear relationship between real-time variation in transmembrane ionic current and the InsP concentration over a wide range from 1 nmol L(-1) to 10 μmol L(-1), as well as a clear discrimination among InsP2, InsP3, and InsP6. This study indicates the great potential of biomolecule-responsive polymers in the fabrication of biomimetic ion nanochannels and other nanoscale biodevices.

  8. Quantification of inositol phosphates in almond meal and almond brown skins by HPLC/ESI/MS.

    PubMed

    Duong, Quynh H; Clark, Kevin D; Lapsley, Karen G; Pegg, Ronald B

    2017-08-15

    The extraction and measurement of all six forms of inositol phosphates (InsPs) in almond meal and brown skins were improved from existing methods by pH adjustment, supplementation of EDTA, and rapid analysis via anion-exchange high-performance liquid chromatography coupled with electrospray ionization mass spectrometry. The quantity of InsPs in six major almond cultivars ranged from 8 to 12μmol/g in the meal and 5 to 14μmol/g in the brown skins. InsP6 was the dominant form, but lower forms still accounted for ∼20% of the total InsPs molar concentration in a majority of the samples. InsPs contributed 32-55% of the organic phosphorus content and 20-38% of the total phosphorus content in the meal. In brown skins, these ranges were 44-77% and 30-52%, respectively. The successful application of this analytical method with almonds demonstrates its potential use for re-examination of the reported phytic acid contents in many other tree nuts, legumes, grains, and complex foods. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Comparison of Saccharomyces cerevisiae F-BAR Domain Structures Reveals a Conserved Inositol Phosphate Binding Site

    DOE PAGES

    Moravcevic, Katarina; Alvarado, Diego; Schmitz, Karl R.; ...

    2015-01-22

    F-BAR domains control membrane interactions in endocytosis, cytokinesis, and cell signaling. Although they are generally thought to bind curved membranes containing negatively charged phospholipids, numerous functional studies argue that differences in lipid-binding selectivities of F-BAR domains are functionally important. Here in this paper, we compare membrane-binding properties of the Saccharomyces cerevisiae F-BAR domains in vitro and in vivo. Whereas some F-BAR domains (such as Bzz1p and Hof1p F-BARs) bind equally well to all phospholipids, the F-BAR domain from the RhoGAP Rgd1p preferentially binds phosphoinositides. We determined X-ray crystal structures of F-BAR domains from Hof1p and Rgd1p, the latter bound tomore » an inositol phosphate. The structures explain phospholipid-binding selectivity differences and reveal an F-BAR phosphoinositide binding site that is fully conserved in a mammalian RhoGAP called Gmip and is partly retained in certain other F-BAR domains. In conclusion, our findings reveal previously unappreciated determinants of F-BAR domain lipid-binding specificity and provide a basis for its prediction from sequence.« less

  10. Ion chromatographic determination of inositol in infant formulae and clinical products for enteral feeding.

    PubMed

    Tagliaferri, E G; Bonetti, G; Blake, C J

    2000-05-26

    An ion chromatographic method is described for the determination of inositol in infant formula and products for enteral feeding. A two-step procedure for hydrolysis and extraction of total inositol has been developed, involving alkaline hydrolysis with 3 M potassium hydroxide and enzymatic dephosphorylation. Substances having a long chromatographic retention time were removed with an ion-exchange resin. Inositol was separated on a high-resolution ion-exchange column and detected by pulsed amperometric detection. Phytic acid interferes only slightly in the analysis. This method can be used for determination of total inositol in infant formulae, and enteral feeding products. The analytical method gave an average recovery of 94% from infant formula samples spiked with inositol and a recovery of 86+/-3% from products spiked with lecithin.

  11. Roles of Phosphate Recognition in Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase (IPK1) Substrate Binding and Activation*

    PubMed Central

    Gosein, Varin; Miller, Gregory J.

    2013-01-01

    Inositol phosphate kinases (IPKs) sequentially phosphorylate inositol phosphates (IPs) to yield a group of small signaling molecules involved in diverse cellular processes. IPK1 (inositol 1,3,4,5,6-pentakisphosphate 2-kinase) phosphorylates inositol 1,3,4,5,6-pentakisphosphate to inositol 1,2,3,4,5,6-hexakisphosphate; however, the mechanism of IP recognition employed by IPK1 is currently unresolved. We demonstrated previously that IPK1 possesses an unstable N-terminal lobe in the absence of IP, which led us to propose that the phosphate profile of the IP was linked to stabilization of IPK1. Here, we describe a systematic study to determine the roles of the 1-, 3-, 5-, and 6-phosphate groups of inositol 1,3,4,5,6-pentakisphosphate in IP binding and IPK1 activation. The 5- and 6-phosphate groups were the most important for IP binding to IPK1, and the 1- and 3-phosphate groups were more important for IPK1 activation than the others. Moreover, we demonstrate that there are three critical residues (Arg-130, Lys-170, and Lys-411) necessary for IPK1 activity. Arg-130 is the only substrate-binding N-terminal lobe residue that can render IPK1 inactive; its 1-phosphate is critical for full IPK1 activity and for stabilization of the active conformation of IPK1. Taken together, our results support the model for recognition of the IP substrate by IPK1 in which (i) the 4-, 5-, and 6-phosphates are initially recognized by the C-terminal lobe, and subsequently, (ii) the interaction between the 1-phosphate and Arg-130 stabilizes the N-terminal lobe and activates IPK1. This model of IP recognition, believed to be unique among IPKs, could be exploited for selective inhibition of IPK1 in future studies that investigate the role of higher IPs. PMID:23884422

  12. Roles of phosphate recognition in inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) substrate binding and activation.

    PubMed

    Gosein, Varin; Miller, Gregory J

    2013-09-13

    Inositol phosphate kinases (IPKs) sequentially phosphorylate inositol phosphates (IPs) to yield a group of small signaling molecules involved in diverse cellular processes. IPK1 (inositol 1,3,4,5,6-pentakisphosphate 2-kinase) phosphorylates inositol 1,3,4,5,6-pentakisphosphate to inositol 1,2,3,4,5,6-hexakisphosphate; however, the mechanism of IP recognition employed by IPK1 is currently unresolved. We demonstrated previously that IPK1 possesses an unstable N-terminal lobe in the absence of IP, which led us to propose that the phosphate profile of the IP was linked to stabilization of IPK1. Here, we describe a systematic study to determine the roles of the 1-, 3-, 5-, and 6-phosphate groups of inositol 1,3,4,5,6-pentakisphosphate in IP binding and IPK1 activation. The 5- and 6-phosphate groups were the most important for IP binding to IPK1, and the 1- and 3-phosphate groups were more important for IPK1 activation than the others. Moreover, we demonstrate that there are three critical residues (Arg-130, Lys-170, and Lys-411) necessary for IPK1 activity. Arg-130 is the only substrate-binding N-terminal lobe residue that can render IPK1 inactive; its 1-phosphate is critical for full IPK1 activity and for stabilization of the active conformation of IPK1. Taken together, our results support the model for recognition of the IP substrate by IPK1 in which (i) the 4-, 5-, and 6-phosphates are initially recognized by the C-terminal lobe, and subsequently, (ii) the interaction between the 1-phosphate and Arg-130 stabilizes the N-terminal lobe and activates IPK1. This model of IP recognition, believed to be unique among IPKs, could be exploited for selective inhibition of IPK1 in future studies that investigate the role of higher IPs.

  13. 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.

  14. Comparison of calcium ionophore and receptor-activated inositol phosphate formation in primary glial cell cultures.

    PubMed

    Wigginton, S A; Minneman, K P

    1991-11-13

    The possible role of Ca2+ influx in alpha 1-adrenoceptor-stimulated [3H]inositol phosphate [( 3H]InsP) formation was examined in primary cultures of glial cells from 1-day-old rat brain. The Ca2+ ionophore A23187 caused a concentration- and time-dependent increase in [3H]InsP formation similar in magnitude to that caused by norepinephrine (NE). Responses to A23187 and NE were both completely dependent on extracellular Ca2+, with a similar concentration dependence. However, cadmium was more potent in blocking the response to A23187 than to NE. Lanthanum (1 mM) blocked the response to NE, although cobalt (5 mM) did not. The [3H]InsP response to A23187 was not additive with the response to NE or to the muscarinic agonist carbachol, although responses to NE and carbachol were addictive Both A23187 and ionomycin inhibited the additive stimulation caused by a combination of NE and carbachol, and this inhibition was potentiated by cadmium. Ionomycin stimulated [3H]InsP formation at concentrations lower than those inhibiting receptor-mediated responses, and this stimulation was not additive with responses to NE or carbachol. High-performance liquid chromatography separation showed similar patterns of [3H]InsPs formed in response to both Ca2+ ionophore and receptor agonists. These results raise the possibility that receptor-activated Ca2+ influx may be involved in stimulation of [3H]InsP formation in these cells.

  15. Isolation and Characterization of a myo-inositol-1-phosphate Synthase Gene from Yellow Passion Fruit (Passiflora edulis f. flavicarpa) Expressed During Seed Development and Environmental Stress

    PubMed Central

    Abreu, Emanuel F. M.; Aragão, Francisco J. L.

    2007-01-01

    Background and Aims Myo-inositol-1l-phosphate synthase (MIPS) catalyses the conversion of d-glucose 6-phosphate to 1-l-myo-inositol-1-phosphate, the first and rate-limiting step in the biosynthesis of all inositol-containing compounds. Inositol phospholipids play a vital role in membrane trafficking and signalling pathways, auxin storage and transport, phytic acid biosynthesis, cell wall biosynthesis and production of stress-related molecules. In the present study, an MIPS cDNA from developing Passiflora edulis f. flavicarpa seeds was characterized and an investigation made into its spatial and differential expression, as well as changes in its transcription during exposure of growing plants to cold and heat stresses. Methods The MIPS-encoding gene was isolated by polymerase chain reaction (PCR) methods, and transcript levels were examined using semi-quantitative reverse transcription–PCR (RT–PCR) during seed development and in response to heat and cold stress. In addition, the copy number of the cloned PeMIPS1 gene in the genome of Passiflora edulis, P. eichleriana, P. caerulea, P. nitida and P. coccinea was determined by Southern blot analyses. Key Results A full-length cDNA clone of the PeMIPS1 from P. edulis was isolated and characterized. Southern blot analyses indicated that the genomic DNA might have diverse sequences of MIPS-encoding genes and one copy of the cloned PeMIPS1 gene in the genomes of P. edulis, P. eichleriana, P. caerulea, P. nitida and P. coccinea. RT–PCR expression analyses revealed the presence of PeMIPS1 transcripts in ovules, pollen grains and leaves, and during the seed developmental stages, where it peaked at 9 d after pollination. The PeMIPS1 gene is differentially regulated under cold and heat stress, presenting a light-responsive transcription. Conclusions Experimental data suggest that PeMIPS1 transcription plays an important role in the establishment of developmental programmes and during the response of plants to

  16. Isolation and characterization of a myo-inositol-1-phosphate synthase gene from yellow passion fruit (Passiflora edulis f. flavicarpa) expressed during seed development and environmental stress.

    PubMed

    Abreu, Emanuel F M; Aragão, Francisco J L

    2007-02-01

    Myo-inositol-1l-phosphate synthase (MIPS) catalyses the conversion of d-glucose 6-phosphate to 1-l-myo-inositol-1-phosphate, the first and rate-limiting step in the biosynthesis of all inositol-containing compounds. Inositol phospholipids play a vital role in membrane trafficking and signalling pathways, auxin storage and transport, phytic acid biosynthesis, cell wall biosynthesis and production of stress-related molecules. In the present study, an MIPS cDNA from developing Passiflora edulis f. flavicarpa seeds was characterized and an investigation made into its spatial and differential expression, as well as changes in its transcription during exposure of growing plants to cold and heat stresses. The MIPS-encoding gene was isolated by polymerase chain reaction (PCR) methods, and transcript levels were examined using semi-quantitative reverse transcription-PCR (RT-PCR) during seed development and in response to heat and cold stress. In addition, the copy number of the cloned PeMIPS1 gene in the genome of Passiflora edulis, P. eichleriana, P. caerulea, P. nitida and P. coccinea was determined by Southern blot analyses. A full-length cDNA clone of the PeMIPS1 from P. edulis was isolated and characterized. Southern blot analyses indicated that the genomic DNA might have diverse sequences of MIPS-encoding genes and one copy of the cloned PeMIPS1 gene in the genomes of P. edulis, P. eichleriana, P. caerulea, P. nitida and P. coccinea. RT-PCR expression analyses revealed the presence of PeMIPS1 transcripts in ovules, pollen grains and leaves, and during the seed developmental stages, where it peaked at 9 d after pollination. The PeMIPS1 gene is differentially regulated under cold and heat stress, presenting a light-responsive transcription. Experimental data suggest that PeMIPS1 transcription plays an important role in the establishment of developmental programmes and during the response of plants to environmental changes. The PeMIPS1 is differentially transcribed

  17. 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.

  18. Receptor-recognized alpha 2-macroglobulin-methylamine elevates intracellular calcium, inositol phosphates and cyclic AMP in murine peritoneal macrophages.

    PubMed Central

    Misra, U K; Chu, C T; Rubenstein, D S; Gawdi, G; Pizzo, S V

    1993-01-01

    Human plasma alpha 2-macroglobulin (alpha 2M) is a tetrameric proteinase inhibitor, which undergoes a conformational change upon reaction with either a proteinase or methylamine. As a result, a receptor recognition site is exposed on each subunit of the molecule enabling it to bind to its receptors on macrophages. We have used Fura-2-loaded murine peritoneal macrophages and digital video fluorescence microscopy to examine the effects of receptor binding on second messenger levels. alpha 2M-methylamine caused a rapid 2-4-fold increase in intracellular Ca2+ concentration ([Ca2+]i) within 5 s of binding to receptors. The agonists induced a focal increase in [Ca2+]i that spread out to other areas of the cell. The increase in [Ca2+]i was dependent on the alpha 2M-methylamine concentration and on the extracellular [Ca2+]. Both sinusoidal and transitory oscillations were observed, which varied from cell to cell. Neither alpha 2M nor boiled alpha 2M-methylamine, forms that are not recognized by the receptor, affected [Ca2+]i in peritoneal macrophages under identical conditions of incubation. The alpha 2M-methylamine-induced rise in [Ca2+]i was accompanied by a rapid and transient increase in macrophage inositol phosphates, including inositol tris- and tetrakis-phosphates. Native alpha 2M did not stimulate a rise in inositol phosphates. Finally, binding of alpha 2M-methylamine to macrophages increased cyclic AMP transiently. Thus receptor-recognized alpha-macroglobulins behave as agonists whose receptor binding causes stimulation of signal transduction pathways. Images Figure 2 PMID:7681282

  19. The influence of extrusion process on myo-inositol phosphate content and profile in snacks containing rye bran.

    PubMed

    Gambuś, H; Matusz-Mirlak, A; Duliński, R; Ziobro, R; Golachowski, A

    2012-02-01

    The analysis of phytates in extrudates containing rye bran was done by a colorimetric method with the Wade reagent. The changes in profiles of myo-inositol phosphates indicate that, although no isomers with positive physiological functions (such as I(1,4,5)P3, I(1,2,3)P3 or I(1,3,4,5)P4) were formed, the beneficial impact of extrusion is the reduction of phytates, as compared to the raw material. The obtained snacks containing rye bran received acceptable to desirable sensory scores.

  20. Effect of exogenous phytase on feed inositol phosphate hydrolysis in an in vitro rumen fluid buffer system.

    PubMed

    Brask-Pedersen, D N; Glitsø, L V; Skov, L K; Lund, P; Sehested, J

    2011-02-01

    Three in vitro experiments using a rumen fluid buffer system were performed to investigate the effect of addition of 4 experimental phytases (Phy1, Phy2, Phy3, and Phy4) compared with no addition of phytase on feed inositol phosphate hydrolysis in wheat and rapeseed cake to determine which of the 4 phytases was most suitable under rumen-like conditions. The feedstuffs were incubated with a mixture of physiological buffer, ruminal fluid, and exogenous phytase at pH 6.2, after which the samples were incubated for different periods. Incubations were stopped using HCl, and the samples were analyzed for inositol phosphates via high performance ion chromatography. Addition of phytase (Phy1) resulted in enhanced degradation of myo-inositol hexakisphosphate (InsP(6)) in rapeseed cake, whereas addition of exogenous phytase did not improve the degradation of InsP(6) in wheat. Only rapeseed cake was therefore used subsequently. All 4 phytases increased degradation of InsP(6) in rapeseed cake in the in vitro system, and degradability of InsP(6) increased with higher incubation time and higher phytase dosages, independent of phytase. Addition of 2 units of phytase per gram of substrate of the phytases Phy1, Phy2, Phy3, and Phy4 led to an undegraded InsP(6) content of 56, 49, 70, and 18%, respectively, when incubated with rapeseed cake for 6h, indicating that Phy2 and Phy4 were the most effective phytases. However, Phy2 had a higher specific activity than Phy4, as 60% of the original InsP(6) content was remaining after 3h when 5mg of enzyme protein per gram of substrate of Phy2 was added to rapeseed cake, whereas 150 mg of enzyme protein per gram of substrate of Phy4 was necessary to achieve a similar result. Therefore, Phy2 appeared to be most applicable under rumen-like conditions.

  1. 3-Deoxy-3-substituted-D-myo-inositol imidazolyl ether lipid phosphates and carbonate as inhibitors of the phosphatidylinositol 3-kinase pathway and cancer cell growth.

    PubMed

    Hu, Y; Meuillet, E J; Berggren, M; Powis, G; Kozikowski, A P

    2001-01-22

    3-Modified D-myo-inositol imidazolyl ether lipid phosphates and a carbonate were synthesized and evaluated as inhibitors of P13-K and Akt. These data are presented along with IC50 values for the inhibition of the growth of three cancer cell lines.

  2. Identification and quantification of inositol hexa-kis phosphate (IHP) in environmental samples at neutral pH using electro-spray ionization and raman spectroscopy

    USDA-ARS?s Scientific Manuscript database

    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...

  3. Osmotic stress-induced phosphoinositide and inositol phosphate signalling in plants.

    PubMed

    Munnik, Teun; Vermeer, Joop E M

    2010-04-01

    Polyphosphoinositides (PPIs) became famous for their role in inositol-1,4,5-trisphosphate (InsP3) mediated-Ca(2+) signalling in mammalian cells, generated through signal-activated phospholipase C (PLC) hydrolysis of the minor membrane lipid, phosphatidylinositol-4,5-bisphosphate. For many years, the plant field followed the same paradigm, however, slowly a completely different picture is emerging. Moreover, various novel PPI-signalling compounds have been identified meanwhile, with new functions and targets coming to light. These include lipids phosphorylated at the D3-position of inositol but also water-soluble inositolpolyphosphates (IPPs). For several of them, a relationship to water stress has been reported. This review summarizes the current status of PPIs and IPPs in plants and discusses their potential in osmotic stress signalling and drought.

  4. Modification and application of an in vitro assay to examine inositol phosphate degradation in the digestive tract of poultry.

    PubMed

    Sommerfeld, Vera; Schollenberger, Margit; Hemberle, Luca; Rodehutscord, Markus

    2017-09-01

    An in vitro assay was modified to study the disappearance of inositol hexakisphosphate (InsP6 ) and the formation of lower inositol phosphate (InsP) isomers in the poultry digestive tract, and three experiments investigated the influence of diets with different ingredients and additives. Using the poultry diet as a matrix, the assay simulated the conditions (e.g. pH, temperature, proteolytic enzymes, water content, and retention time) of the crop, stomach, and small intestine, and extraction and analysis of InsP isomers were immediately conducted. The assay produced highly reproducible results with coefficients of variation ≤10% for an InsP isomer concentration ≥0.4 µmol g(-1) DM (n = 3), and it was sensitive to the factors that varied in the three experiments. The described assay is a suitable tool that can be used to screen feed enzymes and to investigate the effects of supplements in the absence of endogenous phytases. The ease of handling and high reproducibility of the assay indicated that the assay is a rapid and feasible method that can be used to examine the degradation pathway of phytate in feed under gastrointestinal conditions. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

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

    PubMed Central

    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. PMID:25482235

  7. 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.

  8. Impacts of dietary calcium, phytate, and phytase on inositol hexakisphosphate degradation and inositol phosphate release in different segments of digestive tract of broilers.

    PubMed

    Li, W; Angel, R; Kim, S-W; Brady, K; Yu, S; Plumstead, P W

    2017-10-01

    A total of 720 straight-run Heritage 56 M × fast feathering Cobb 500F broiler chickens was fed from 11 to 13 d of age to determine the impacts of dietary calcium (Ca), phytate phosphorus (PP), and phytase concentrations on inositol phosphate (IP3-6) profile in different digestive tract (GI) segments. The experiment was a 2 × 2 × 3 randomized block design with 2 Ca (0.7 and 1.0%) and 2 PP (0.23 and 0.34%) concentrations and 3 doses of Buttiauxella sp. phytase (0, 500, and 1,000 FTU/kg). The experiment was replicated in time (block) with 3 replicates per treatment (Trt) of 10 birds per block. Concentrations of IP3-6 in the crop, proventriculus (Prov) plus (+) gizzard (Giz), and distal ileum, as well as the ileal IP6 and P disappearance were determined at 13 d of age. The detrimental impact of Ca on IP6 and P disappearance was observed only in the ileum, where 11% reduction in both IP6 and P disappearance was seen when Ca increased from 0.7 to 1.0% (P < 0.05). Higher IP5 and IP6 concentrations were seen in both the crop and Prov+Giz at 0.34% PP as compared to birds fed to 0.23% PP diets, regardless of Ca or phytase (P < 0.05), whereas IP3 and IP4 concentrations were not affected by PP (P > 0.05). Inclusion of phytase, at both 500 and 1,000 FTU/kg, resulted in lower IP6 and the accumulation of lower IP ester (IP3-5) concentrations in all GI segments (P < 0.05). Improved IP6 and P disappearance was seen as a result of phytase inclusion, despite the degree of improvement affected by PP (P < 0.05). On average, 5.5 and 6.7 times improvement in IP6 was observed with 500 and 1,000 FTU phytase/kg inclusion, respectively, resulting in 41 and 64% greater P digestibility, respectively. In conclusion, phytase can effectively degrade IP6 to lower esters and increase P utilization. However, the efficacy of phytase can be affected by diet Ca and PP concentrations. © The Author 2017. Published by Oxford University Press on behalf of Poultry Science Association.

  9. 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

  10. Inframolecular acid base studies of the tris and tetrakis myo-inositol phosphates including the 1,2,3-trisphosphate motif

    NASA Astrophysics Data System (ADS)

    Dozol, Hélène; Blum-Held, Corinne; Guédat, Philippe; Maechling, Clarisse; Lanners, Steve; Schlewer, Gilbert; Spiess, Bernard

    2002-12-01

    The intrinsic acid-base properties of the phosphate groups of three myo-inositol derivatives which display the 1,2,3-trisphosphate motif, i.e. (±)- myo-inositol 1,2,3-trisphosphate (Ins(1,2,3)P 3), (±)- myo-inositol 1,2,3,6-tetrakisphosphate (Ins(1,2,3,6)P 4), and (±)- myo-inositol 1,2,3,5-tetrakisphosphate (Ins(1,2,3,5)P 4) are reported. The studies were performed in 0.2 M KCl solution at 37 °C, near physiological ionic strength and temperature. In addition, in order to shed light on the transition metal complexation properties of Ins(1,2,3)P 3, the influence of the Zn 2+ cations on its 31P NMR titration curves was investigated. From the titration curves as well as from the determined protonation microconstants, it appears that for Ins(1,2,3)P 3, the two lateral P1 and P3 phosphates strongly contribute to stabilise a proton on the central P2 phosphate. However, in the fully deprotonated form of Ins(1,2,3)P 3, P1 and P3 repulse each other so that they establish hydrogen bonds with, respectively, their neighbouring OH6 and OH4 hydroxyls. The 1,2,3-trisphosphate motif of Ins(1,2,3,5)P 4 behaves very similarly to that of Ins(1,2,3)P 3 indicating a poor interaction with the distant P5 phosphate. By contrast, moving a phosphate group from position 5 to position 6 on the myo-inositol ring as in Ins(1,2,3,6)P 4, leads to major changes in the basicity and cooperativity of the phosphate groups. Finally, the presence of Zn 2+ cations has a marked influence on the 31P NMR titration curves of Ins(1,2,3)P 3, leading to the conclusion that two equatorial phosphates, assisted by a middle axial one, afford an optimal chelating moiety that is able to occupy all sites of the metal coordination polyhedron which could be the reason for its antioxidant properties.

  11. A novel method for the purification of inositol phosphates from biological samples reveals that no phytate is present in human plasma or urine

    PubMed Central

    Wilson, Miranda S. C.; Bulley, Simon J.; Pisani, Francesca; Irvine, Robin F.; Saiardi, Adolfo

    2015-01-01

    Inositol phosphates are a large and diverse family of signalling molecules. While genetic studies have discovered important functions for them, the biochemistry behind these roles is often not fully characterized. A key obstacle in inositol phosphate research in mammalian cells has been the lack of straightforward techniques for their purification and analysis. Here we describe the ability of titanium dioxide (TiO2) beads to bind inositol phosphates. This discovery allowed the development of a new purification protocol that, coupled with gel analysis, permitted easy identification and quantification of InsP6 (phytate), its pyrophosphate derivatives InsP7 and InsP8, and the nucleotides ATP and GTP from cell or tissue extracts. Using this approach, InsP6, InsP7 and InsP8 were visualized in Dictyostelium extracts and a variety of mammalian cell lines and tissues, and the effects of metabolic perturbation on these were explored. TiO2 bead purification also enabled us to quantify InsP6 in human plasma and urine, which led to two distinct but related observations. Firstly, there is an active InsP6 phosphatase in human plasma, and secondly, InsP6 is undetectable in either fluid. These observations seriously question reports that InsP6 is present in human biofluids and the advisability of using InsP6 as a dietary supplement. PMID:25808508

  12. 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.

  13. Increasing inositol (1,4,5)-trisphosphate metabolism affects drought tolerance, carbohydrate metabolism and phosphate-sensitive biomass increases in tomato.

    PubMed

    Khodakovskaya, Mariya; Sword, Courtney; Wu, Qian; Perera, Imara Y; Boss, Wendy F; Brown, Christopher S; Winter Sederoff, Heike

    2010-02-01

    Inositol-(1,4,5)-trisphosphate (InsP(3)) is a second messenger in plants that increases in response to many stimuli. The metabolic consequences of this signalling pathway are not known. We reduced the basal level of InsP(3) in tomato (Solanum lycopersicum cv. Micro-Tom) by expressing the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase) gene. Transgenic lines producing InsP 5-ptase protein had between 15% and 30% of the basal InsP(3) level of control plants. This increased hydrolysis of InsP(3) caused dramatic increases in drought tolerance, vegetative biomass and lycopene and hexose concentrations in the fruits. Transcript profiling of root, leaf and fruit tissues identified a small group of genes, including a cell-wall invertase inhibitor gene, that were differentially regulated in all tissues of the InsP 5-ptase expressing plants. Significant differences were found in the amounts of carbohydrates and organic phosphate in these plants. Plants with increased hydrolysis of InsP(3) in the cytosol also showed increased net CO(2)-fixation and sucrose export into sink tissue and storage of hexoses in the source leaves. The increase in biomass was dependent on the supply of inorganic phosphate in the nutrient medium. Uptake and storage of phosphate was increased in the transgene expressing lines. This suggests that in tomato, increased flux through the inositol phosphate pathway uncoupled phosphate sensing from phosphate metabolism. Altering the second messenger, InsP(3), revealed multiple coordinated changes in development and metabolism in tomato that have potential for crop improvement.

  14. 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. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  15. Early hypergravity exposure effects calbindin-D28k and inositol-3-phosphate expression in Purkinje cells.

    PubMed

    Bouët, Valentine; Dijk, Freark; Ijkema-Paassen, Jos; Wubbels, René J; van der Want, Johannes J; Gramsbergen, Albert

    In this study the effects of hypergravity were analyzed on cerebellar Purkinje cells during early development in rats. The cerebellum is a key structure in the control and the adaptation of posture and anti-gravity activities. This holds particularly when external conditions are modified. Three groups of rats were conceived, born and reared in hypergravity (2g). At postnatal day 5 (P5), P10 or P15, they were exposed to normal gravity and at P40, the cerebella were investigated on the expression of calbindin-D28k and inositol-3-phosphate (IP3) in Purkinje cells. Control animals were bred in the same conditions but at 1g. Immunoreactivity of Purkinje cells was studied in lobules III and IX of the vermis. Lobule IX of the vermis is one of the targets of primary otolithic vestibular projections, and lobule III served as a control, being much less related with vestibular inputs. The results show that hypergravity induces a decrease in calbindin and IP3 labeling in 20% of Purkinje cells of lobule IX without any change in lobule III. Animals transferred from 2g to 1g at P5 or P10 showed the most pronounced effects and much less at P15. This study demonstrates that early development of the cerebellum is highly sensitive to changes in gravity. Ages until P10 are critical for the development of vestibulo-cerebellar connections, and in particularly the calcium signaling in Purkinje cells.

  16. Synthesis of radiolabeled chiral probes for binding and receptor studies: Radiolabeled juvenoids and inositol phosphates

    SciTech Connect

    Boehm, M.F.

    1988-01-01

    This study is composed of two parts. Part I describes the synthesis of seven high specific activity radioligands for the characterization of macromolecular receptors for juvenile hormone analog-type labeled insect growth regulators. These radioligands include (1) ({sup 125}I)-radioiodinated iodovinyl methoprenol and iodovinyl methoprene (2000 Ci/mmol), (2) ({sup 3}H)-labeled (7S)-methoprene and (7S)-hydroprene (>60 Ci/mmol), potent dodecadienoate insect growth regulators, (3) ({sup 3}H)-labeled fenoxycarb (Maag) and S-31183 Sumitomo, phenoxyphenyl ether IGRs, and (4) ({sup 3}H)-methoprene diazoketone, a photoaffinity label for characterizing receptor sites. The attempted synthesis of high specific activity tritium labeled JH III is also described. Biological studies utilizing these radioligands show separate nuclear receptor proteins for JH homologs and juvenoids. Part II describes the preparation of enantiomerically enriched radiolabeled myo-inositol-1,3,4-trisphosphate (myo-Ins(1,3,4)P{sub 3}) and fluorinated analogs of myo-Ins(1,3,4)P{sub 3} for examining receptors for myo-Ins(1,3,4)P{sub 3}. Three compounds have been synthesized. These include 2-fluoro- and 2,2-difluoro-2-deoxy analogs of DL-myo-Ins(1,3,4)P{sub 3}, D- and L-myo-Ins(1,3,4)P{sub 3} at >95% enantiomeric excess and, D-and L-({sup 3}H)-myo-Ins(1,3,4)P{sub 3} enantiomers with specific activities of 15 Ci/mmol.

  17. The Structure of the L-myo-inositol-1-phosphate Synthase-NAD[superscript +]-2-deoxy-D-glucitol 6-(E)-Vinylhomophosphonate Complex Demands a Revision of the Enzyme Mechanism

    SciTech Connect

    Jin, Xiangshu; Foley, Kathleen M.; Geiger, James H.

    2010-11-16

    1l-myo-inositol 1-phosphate (MIP) synthase catalyzes the conversion of D-glucose 6-phosphate to 1l-myo-inositol 1-phosphate, the first and rate-limiting step in the biosynthesis of all inositol-containing compounds. It involves an oxidation, enolization, intramolecular aldol cyclization, and reduction. Here we present the structure of MIP synthase in complex with NAD{sup +} and a high-affinity inhibitor, 2-deoxy-D-glucitol 6-(E)-vinylhomophosphonate. This structure reveals interactions between the enzyme active site residues and the inhibitor that are significantly different from that proposed for 2-deoxy-D-glucitol 6-phosphate in the previously published structure of MIP synthase-NAD{sup +}-2-deoxy-D-glucitol 6-phosphate. There are several other conformational changes in NAD{sup +} and the enzyme active site as well. Based on the new structural data, we propose a new and completely different mechanism for MIP synthase.

  18. A new-generation of Bacillus subtilis cell factory for further elevated scyllo-inositol production.

    PubMed

    Tanaka, Kosei; Natsume, Ayane; Ishikawa, Shu; Takenaka, Shinji; Yoshida, Ken-Ichi

    2017-04-21

    A stereoisomer of inositol, scyllo-inositol (SI), has been regarded as a promising therapeutic agent for Alzheimer's disease. However, this compound is relatively rare, whereas another stereoisomer of inositol, myo-inositol (MI) is abundant in nature. Bacillus subtilis 168 has the ability to metabolize inositol stereoisomers, including MI and SI. Previously, we reported a B. subtilis cell factory with modified inositol metabolism that converts MI into SI in the culture medium. The strain was constructed by deleting all genes related to inositol metabolism and overexpressing key enzymes, IolG and IolW. By using this strain, 10 g/l of MI initially included in the medium was completely converted into SI within 48 h of cultivation in a rich medium containing 2% (w/v) Bacto soytone. When the initial concentration of MI was increased to 50 g/l, conversion was limited to 15.1 g/l of SI. Therefore, overexpression systems of IolT and PntAB, the main transporter of MI in B. subtilis and the membrane-integral nicotinamide nucleotide transhydrogenase in Escherichia coli respectively, were additionally introduced into the B. subtilis cell factory, but the conversion efficiency hardly improved. We systematically determined the amount of Bacto soytone necessary for ultimate conversion, which was 4% (w/v). As a result, the conversion of SI reached to 27.6 g/l within 48 h of cultivation. The B. subtilis cell factory was improved to yield a SI production rate of 27.6 g/l/48 h by simultaneous overexpression of IolT and PntAB, and by addition of 4% (w/v) Bacto soytone in the conversion medium. The concentration of SI was increased even in the stationary phase perhaps due to nutrients in the Bacto soytone that contribute to the conversion process. Thus, MI conversion to SI may be further optimized via identification and control of these unknown nutrients.

  19. Hydrogen peroxide and nitric oxide mediated cold- and dehydration-induced myo-inositol phosphate synthase that confers multiple resistances to abiotic stresses.

    PubMed

    Tan, Jiali; Wang, Congying; Xiang, Bin; Han, Ruihong; Guo, Zhenfei

    2013-02-01

    myo-Inositol phosphate synthase (MIPS) is the key enzyme of myo-inositol synthesis, which is a central molecule required for cell metabolism and plant growth as a precursor to a large variety of compounds. A full-length fragment of MfMIPS1 cDNA was cloned from Medicago falcata that is more cold-tolerant than Medicago sativa. While MfMIPS1 transcript was induced in response to cold, dehydration and salt stress, MIPS transcript and myo-inositol were maintained longer and at a higher level in M. falcata than in M. sativa during cold acclimation at 5 °C. MfMIPS1 transcript was induced by hydrogen peroxide (H(2) O(2)) and nitric oxide (NO), but was not responsive to abscisic acid (ABA). Pharmacological experiments revealed that H(2) O(2) and NO are involved in the regulation of MfMIPS1 expression by cold and dehydration, but not by salt. Overexpression of MfMIPS1 in tobacco increased the MIPS activity and levels of myo-inositol, galactinol and raffinose, resulting in enhanced resistance to chilling, drought and salt stresses in transgenic tobacco plants. It is suggested that MfMIPS1 is induced by diverse environmental factors and confers resistance to various abiotic stresses.

  20. Noradrenaline stimulation of the phosphoinositide system: evidence for a novel hydrophobic inositol-containing compound in resistance arterioles.

    PubMed Central

    Ollerenshaw, J. D.; Heagerty, A. M.; Swales, J. D.

    1988-01-01

    1. Five inositol phosphates were extracted from adult rat resistance arterioles and separated by ion-exchange high performance liquid chromatography. 2. By use of this technique, inositol phosphates liberated were identified as inositol 1-phosphate, inositol 1,4-bisphosphate, inositol 1,3,4-trisphosphate, inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate. Stimulation of phosphoinositide hydrolysis with noradrenaline produced increases in inositol phosphate production. 3. Three inositol-containing phospholipids extracted from resistance arterioles were measured as their glycerol esters following deacylation, thereby permitting an analysis of both membrane and cytosolic components of the phosphoinositide signalling system. 4. A substantial agonist-sensitive pool of a previously undescribed inositol but not glycerol-containing lipid extract component was also identified in this tissue. 5. These experiments for the first time allow a precise description of phosphoinositide metabolism in resting and agonist-stimulated resistance arterioles and provide data on a novel compound possibly similar to that recently described in other tissues. PMID:2840158

  1. Cytoplasmic inositol hexakisphosphate production is sufficient for mediating the Gle1-mRNA export pathway.

    PubMed

    Miller, Aimee L; Suntharalingam, Mythili; Johnson, Sylvia L; Audhya, Anjon; Emr, Scott D; Wente, Susan R

    2004-12-03

    Production of inositol hexakisphosphate (IP6) by Ipk1, the inositol-1,3,4,5,6-pentakisphosphate 2-kinase, is required for Gle1-mediated mRNA export in Saccharomyces cerevisiae cells. To examine the network of interactions that require IP6 production, an analysis of fitness defects was conducted in mutants harboring both an ipk1 null allele and a mutant allele in genes encoding nucleoporins or transport factors. Enhanced lethality was observed with a specific subset of mutants, including nup42, nup116, nup159, dbp5, and gle2, all of which had been previously connected to Gle1 function. Complementation of the nup116Deltaipk1Delta and nup42Deltaipk1Delta double mutants did not require the Phe-Gly repeat domains in the respective nucleoporins, suggesting that IP6 was acting subsequent to heterogeneous nuclear ribonucleoprotein targeting to the nuclear pore complex. With Nup42 and Nup159 localized exclusively to the nuclear pore complex cytoplasmic side, we speculated that IP6 may regulate a cytoplasmic step in mRNA export. To test this prediction, the spatial requirements for the production of IP6 were investigated. Restriction of Ipk1 to the cytoplasm did not block IP6 production. Moreover, coincident sequestering of both Ipk1 and Mss4 (an enzyme required for phosphatidylinositol 4,5-bisphosphate production) to the cytoplasm also did not block IP6 production. Given that the kinase required for inositol 1,3,4,5,6-pentakisphosphate production (Ipk2) is localized in the nucleus, these results indicated that soluble inositides were diffusing between the nucleus and the cytoplasm. Additionally, the cytoplasmic production of IP6 by plasma membrane-anchored Ipk1 rescued a gle1-2 ipk1-4 synthetic lethal mutant. Thus, cytoplasmic IP6 production is sufficient for mediating the Gle1-mRNA export pathway.

  2. Activation of PLC by an endogenous cytokine (GBP) in Drosophila S3 cells and its application as a model for studying inositol phosphate signalling through ITPK1.

    PubMed

    Zhou, Yixing; Wu, Shilan; Wang, Huanchen; Hayakawa, Yoichi; Bird, Gary S; Shears, Stephen B

    2012-12-01

    Using immortalized [3H]inositol-labelled S3 cells, we demonstrated in the present study that various elements of the inositol phosphate signalling cascade are recruited by a Drosophila homologue from a cytokine family of so-called GBPs (growth-blocking peptides). HPLC analysis revealed that dGBP (Drosophila GBP) elevated Ins(1,4,5)P3 levels 9-fold. By using fluorescent Ca2+ probes, we determined that dGBP initially mobilized Ca2+ from intracellular pools; the ensuing depletion of intracellular Ca2+ stores by dGBP subsequently activated a Ca2+ entry pathway. The addition of dsRNA (double-stranded RNA) to knock down expression of the Drosophila Ins(1,4,5)P3 receptor almost completely eliminated mobilization of intracellular Ca2+ stores by dGBP. Taken together, the results of the present study describe a classical activation of PLC (phospholipase C) by dGBP. The peptide also promoted increases in the levels of other inositol phosphates with signalling credentials: Ins(1,3,4,5)P4, Ins(1,4,5,6)P4 and Ins(1,3,4,5,6)P5. These results greatly expand the regulatory repertoire of the dGBP family, and also characterize S3 cells as a model for studying the regulation of inositol phosphate metabolism and signalling by endogenous cell-surface receptors. We therefore created a cell-line (S3ITPK1) in which heterologous expression of human ITPK (inositol tetrakisphosphate kinase) was controlled by an inducible metallothionein promoter. We found that dGBP-stimulated S3ITPK1 cells did not synthesize Ins(3,4,5,6)P4, contradicting a hypothesis that the PLC-coupled phosphotransferase activity of ITPK1 [Ins(1,3,4,5,6)P5+Ins(1,3,4)P3→Ins(3,4,5,6)P4+Ins(1,3,4,6)P4] is driven solely by the laws of mass action [Chamberlain, Qian, Stiles, Cho, Jones, Lesley, Grabau, Shears and Spraggon (2007) J. Biol. Chem. 282, 28117-28125]. This conclusion represents a fundamental breach in our understanding of ITPK1 signalling.

  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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  4. Evidence for the coupling of Gq protein to D1-like dopamine sites in rat striatum: possible role in dopamine-mediated inositol phosphate formation.

    PubMed

    Wang, H Y; Undie, A S; Friedman, E

    1995-12-01

    The role of G proteins in mediating the coupling of D1 dopamine receptors to inositol phosphate formation was investigated in rat brain striatum. Pertussis toxin-activated ADP-ribosylation ( > or = 95%) did not affect the ability of the D1 agonist SKF38393 to stimulate the generation of inositol phosphates in striatal slices. Stimulation of striatal membranes with dopamine in the presence of [35S]GTP gamma S or [alpha-32P]GTP increased guanine nucleotide binding to G alpha s, G alpha i, and G alpha q in a concentration-dependent fashion. The activation of G alpha s and G alpha q was mimicked by the D1 agonist SKF38393 and blocked by the D1 antagonist SCH23390. In contrast, the D2/3 dopamine receptor agonist quinpirole stimulated guanine nucleotide binding to G alpha i, and dopamine-stimulated activation of G alpha i was attenuated by the D2 antagonist I-sulpiride. Furthermore, antisera directed against G alpha s or G alpha q but not G alpha i, G alpha o, or G alpha z precipitated specific D1-like binding sites labeled with [3H]SCH23390. The D1-like receptors that coprecipitated with G alpha s-but not with G alpha q can be recognized by a specific D1 dopamine receptor antibody. The data provide evidence to suggest that in addition to coupling to Gs/adenylyl cyclase, D1-like dopamine sites that couple to Gq may mediate dopamine-stimulated formation of inositol phosphates in the rat striatum.

  5. Arabidopsis inositol pentakisphosphate 2-kinase, AtIPK1, is required for growth and modulates phosphate homeostasis at the transcriptional level.

    PubMed

    Kuo, Hui-Fen; Chang, Tzu-Yun; Chiang, Su-Fen; Wang, Wei-Di; Charng, Yee-Yung; Chiou, Tzyy-Jen

    2014-11-01

    Inositol hexakisphosphate (IP6 ) provides a phosphorous reservoir in plant seeds; in addition, along with its biosynthesis intermediates and derivatives, IP6 also plays important roles in diverse developmental and physiological processes. Disruption of the Arabidopsis inositol pentakisphosphate 2-kinase coding gene AtIPK1 was previously shown to reduce IP6 content in vegetative tissues and affect phosphate (Pi) sensing. Here we show that AtIPK1 is required for sustaining plant growth, as null mutants are non-viable. An incomplete loss-of-function mutant, atipk1-1, exhibited disturbed Pi homeostasis and overaccumulated Pi as a consequence of increased Pi uptake activity and root-to-shoot Pi translocation. The atipk1-1 mutants also showed a Pi deficiency-like root system architecture with reduced primary root and enhanced lateral root growth. Transcriptome analysis indicated that a subset of Pi starvation-responsive genes was transcriptionally perturbed in the atipk1-1 mutants and the expression of multiple genes involved in Pi uptake, allocation, and remobilization was increased. Genetic and transcriptional analyses suggest that disturbance of Pi homeostasis caused by atipk1 mutation involved components in addition to PHR1(-like) transcription factors. Notably, the transcriptional increase of a number of Pi starvation-responsive genes in the atipk1-1 mutants is correlated with the reduction of histone variant H2A.Z occupation in chromatin. The myo-inositol-1-phosphate synthase mutants, atmips1 and atmips2 with comparable reduction in vegetative IP6 to that in the atipk1-1 mutants did not overaccumulate Pi, suggesting that Pi homeostasis modulated by AtIPK1 is not solely attributable to IP6 level. This study reveals that AtIPK1 has important roles in growth and Pi homeostasis. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  6. Avian multiple inositol polyphosphate phosphatase is an active phytase that can be engineered to help ameliorate the planet’s “phosphate crisis”

    PubMed Central

    Cho, Jaiesoon; Choi, Kuicheon; Darden, Thomas; Reynolds, Paul R.; Petitte, James N.; Shears, Stephen B.

    2007-01-01

    Contemporary phytase research is primarily concerned with ameliorating the problem of inadequate digestion of inositol hexakisphosphate (phytate; InsP6) in monogastric farm animal feed, so as to reduce the pollution that results from the high phosphate content of the manure. In the current study we pursue a new, safe and cost-effective solution. We demonstrate that the rate of hydrolysis of InsP6 by recombinant avian MINPP (0.7 μmol/mg protein/min) defines it as by far the most active phytase found to date in any animal cell (the corresponding activity of recombinant mammalian MINPP is only 0.006 μmol/mg protein/min). Although avian MINPP has less than 20% sequence identity with microbial phytases, we create a homology model of MINPP in which it is predicted that the structure of the phytase active site is well-conserved. This model is validated by site-directed mutagenesis and by use of a substrate analogue, scyllo-InsP6, which we demonstrate is only a weak MINPP substrate. In a model chicken cell line, we overexpressed a mutant form of MINPP that is secretion-competent. This version of the enzyme was actively secreted without affecting either cell viability or the cellular levels of any inositol phosphates. Our studies offer a genetic strategy for greatly improving dietary InsP6 digestion in poultry. PMID:16759730

  7. Arabidopsis plants constitutively overexpressing a myo-inositol 1-phosphate synthase gene (SaINO1) from the halophyte smooth cordgrass exhibits enhanced level of tolerance to salt stress.

    PubMed

    Joshi, Rohit; Ramanarao, Mangu Venkata; Baisakh, Niranjan

    2013-04-01

    Salinity is one of the most important environmental constraints limiting agricultural productivity. Considering the importance of the accumulation of osmolytes, myo-inositol in particular, in halophytic plant's adaptive response to salinity, an effort was made to overexpress the SaINO1 gene from the grass halophyte Spartina alterniflora encoding myo-inositol 1-phosphate synthase (MIPS) in Arabidopsis thaliana. We demonstrated that SaINO1 is a stress-responsive gene and its constitutive over expression in Arabidopsis provides significantly improved tolerance to salt stress during germination and seedling growth and development. The transgenics retained more chlorophyll and carotenoid by protecting the photosystem II. The low level of stress-induced cellular damage in the transgenics was clearly evident by lower accumulation of proline in comparison to WT. Our results indicated that possible overaccumulation of MIPS enzyme in the cytosol protected the transgenic Arabidopsis plants overexpressing SaINO1 from the toxic effect of Na(+) under salt stress by reducing cellular damage and chlorophyll loss.

  8. 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

  9. Reaching for mechanistic consensus across life kingdoms: structure and insights into catalysis of the myo-inositol-1-phosphate synthase (mIPS) from Archaeoglobus fulgidus.

    PubMed

    Stieglitz, Kimberly A; Yang, Hongying; Roberts, Mary F; Stec, Boguslaw

    2005-01-11

    myo-Inositol-1-phosphate synthase (mIPS) catalyzes the first step in the synthesis of l-myo-inositol-1-phosphate. We have solved and refined the structure of the mIPS from the hyperthermophilic sulfate reducer Archaeoglobus fulgidus at 1.9 A resolution. The enzyme crystallized from poly(ethylene glycol) in the P1 space group with one tetramer in the asymmetric unit and provided a view of the entire biologically active oligomer. Despite significant changes in sequence length and amino acid composition, the general architecture of the archaeal enzyme is similar to that of the eukaryotic mIPS from Saccharomyces cerevisiae and bacterial mIPS from Mycobacterium tuberculosis. The enhanced thermostability of the archaeal enzyme as compared to that from yeast is consistent with deletion of a number of surface loops that results in a significantly smaller protein. In the structure of the A. fulgidus mIPS, the active sites of all four subunits were fully ordered and contained NAD(+) and inorganic phosphate. The structure also contained a single metal ion (identified as K(+)) in two of the four subunits. The analysis of the electrostatic potential maps of the protein suggested the presence of a second metal-ion-binding site in close proximity to the first metal ion and NAD(+). The modeling of the substrate and known inhibitors suggests a critical role for the second metal ion in catalysis and provides insights into the common elements of the catalytic cycle in enzymes from different life kingdoms.

  10. 18:1 n7 fatty acids inhibit growth and decrease inositol phosphate release in HT-29 cells compared to n9 fatty acids.

    PubMed

    Awad, A B; Herrmann, T; Fink, C S; Horvath, P J

    1995-05-04

    Studies have shown that trans fatty acids may play a role in the development of chronic diseases such as heart disease and cancer. The objective of the present project was to examine the effect of supplementation with 18:1 isomers, both positional and geometrical, as compared to 18:0 on the growth, membrane fatty acid composition and the phosphoinositide cycle of HT-29 human colon cancer cells. Cells were supplemented with 30 microM stearic acid (18:0), elaidic acid (18:1, n9, trans), oleic acid (18:1, n9, cis), vaccenic acid (18:1, n7, cis) or trans-vaccenic acid (18:1, n7, trans) as sodium salts complexed to fatty acid-free bovine serum. Cells were grown in these media for 9 days. Cell growth was examined by counting the number of cells and expressed as percentage of control (18:0 supplemented cells). The phosphoinositide (PI) cycle was examined by measuring the inositol phosphate (IP) released from phosphoinositides in the absence (basal) or presence of stimuli (0.1 mM carbachol, 0.1 mM A23187 or 20 mM NaF). The results obtained indicated that cis and trans n7 fatty acids inhibited the growth of HT-29 cells by 11% and 23%, respectively, as compared to 18:0 supplementation. 18:1, n9 had no effect on tumor growth. Supplementation with all forms of 18:1 resulted in an increase in IP and IP2 production as compared to 18:0 supplemented cells without influencing IP3. The presence of the double bond at the 9 position in the supplemented fatty acid increases total IP production by 59% and in the cis form by 37% above the control. The breakdown of phosphoinositides in the absence and presence of several stimuli supports the observed finding on IP. Trans fatty acid supplementation resulted in lower hydrolysis of PI as compared to cis fatty acids. It is concluded that the observed inhibition of tumor growth by the vaccenic acids may be mediated by their effect(s) on the PI cycle which may be associated with their incorporation into membrane lipids.

  11. Accumulation of inositol phosphates and cyclic AMP in guinea-pig cerebral cortical preparations. Effects of norepinephrine, histamine, carbamylcholine and 2-chloroadenosine.

    PubMed

    Hollingsworth, E B; Daly, J W

    1985-11-20

    Norepinephrine and serotonin augment by about 2-fold the accumulation of cyclic [3H]AMP elicited by 2-chloroadenosine in [3H]adenine-labeled guinea-pig cerebral cortical slices. Histamine causes a 3-fold augmentation. The first two agents have no effect on cyclic AMP alone, while histamine has only a small effect alone. The augmentation of the 2-chloroadenosine response appears to be mediated by alpha 1-adrenergic, 5HT2-serotonergic and H2-histaminergic receptors. VIP-elicited accumulations of cyclic AMP are also augmented through stimulation of alpha 1-adrenergic, 5HT2-serotonergic and H1-histaminergic receptors. Activation of these amine receptors also increases the turnover of phosphatidylinositols in [3H]inositol-labeled guinea pig cerebral cortical slices. Norepinephrine causes a 5-fold, serotonin a 1.2-fold, and histamine a 2.5-fold increase in accumulations of [3H]inositol phosphates. 2-Chloroadenosine, vasoactive intestinal peptide, baclofen, and somatostatin have no effect on phosphatidylinositol turnover, nor do the last two agents augment accumulations of cyclic AMP elicited by 2-chloroadenosine. The data suggest a possible relationship between turnover of phosphatidylinositol and the augmentations of the cyclic AMP accumulations elicited by biogenic amines in brain slices.

  12. Profile and bioavailability analysis of myo-inositol phosphates in rye bread supplemented with phytases: a study using an in vitro method and Caco-2 monolayers.

    PubMed

    Duliński, R; Cielecka, E K; Pierzchalska, M; Byczyński, Ł; Żyła, K

    2016-06-01

    Commercial preparations of 6-phytase A alone and in combination with phytase B were used in rye breadmaking. Determination of bioavailability of myo-inositol phosphates from bread was performed by an in vitro digestion method followed by the measurement of an uptake by Caco-2 cells in culture. In bread supplemented with a combination of 6-phytase A and phytase B, a significant reduction in phytate content was observed from 3.62 μmol/g in the control to 0.7 μmol/g. Bioavailability of phytate estimated by an in vitro method simulating digestion in the human alimentary tract was 9% in the bread supplemented with phytase B, 7% (6-phytase A) and 50% in the control bread. In cell culture, the bioaccessibilities of inositol triphosphates from bread baked with the addition of 6-phytase A was higher by 36% as compared to the samples baked with phytase B and by 32% in breads baked with combination of both phytases.

  13. Degradation of myo-inositol hexakisphosphate by a phytate-degrading enzyme from Pantoea agglomerans.

    PubMed

    Greiner, Ralf

    2004-11-01

    High-pressure liquid chromatography (HPLC) analysis established myo-inositol pentakisphosphate as the final product of phytate dephosphorylation by the phytate-degrading enzyme from Pantoea agglomerans. Neither product inhibition by phosphate nor inactivation of the Pantoea enzyme during the incubation period were responsible for the limited phytate hydrolysis as shown by addition of phytate-degrading enzyme and phytate, respectively, after the observed stop of enzymatic phytate degradation. In additon, the Pantoea enzyme did not possess activity toward the purified myo-inositol pentakisphosphate. Using a combination of High-Performance Ion Chromatography (HPIC) analysis and kinetic studies, the nature of the generated myo-inositol pentakisphosphate was established. The data demonstrate that the phytate-degrading enzyme from Pantoea agglomerans dephosphorylates myo-inositol hexakisphosphate in a stereospecific way to finally D-myo-inositol(1,2,4,5,6)pentakisphosphate.

  14. Effect of several germination conditions on total P, phytate P, phytase, and acid phosphatase activities and inositol phosphate esters in rye and barley.

    PubMed

    Centeno, C; Viveros, A; Brenes, A; Canales, R; Lozano, A; de la Cuadra, C

    2001-07-01

    Two assays were conducted to study the evolution of rye and barley phosphatases (phytase and acid phosphatase) and the degradation of its substrates (inositol phosphate esters) during seed germination. In this manner we could obtain a low-phytate, endogenous phosphatase rich ingredient to be used in animal nutrition. In the first assay, the seeds were soaked for 1 and 14 h and germinated for 3 and 5 days with and without the addition of gibberellic acid (GA3). In the second assay, the seeds were soaked for 1 h and germinated for 1, 3, and 5 days with GA3. Phytase (up to 5739 and 3151 U x kg(-1)) and acid phosphatase (up to 18288 and 3151 U x g(-1)) activities, and IP6 (6.09 and 6.01 mg x g(-1)), IP5 (0.48 and 0.48 mg x g(-1)), and IP4 (0.13 and 0.06 mg x g(-1)) were detected in ungerminated rye and barley, respectively. The germination process caused a significant increase of Phy and AcPh activities in rye (up to 112 and 213%) and barley (up to 212 and 634%) and a reduction in the phytate phosphorus content (up to 84 and 58%, respectively). Phytate phosphorus content was affected only by soaking time in the case of rye. Finally, during the course of germination, IP6 and IP5 were rapidly degraded in rye (88 and 79%) and barley (67 and 52%), and IP4 was only a short-living intermediate, which was increased during hydrolysis and degraded to IP3. In conclusion, a marked increase of Phy and AcPh activities in rye and barley with a concomitant decrease in phytate phosphorus content and an increase in the content of lower inositol phosphates were observed during the rye and barley germination.

  15. Ethanol stimulates phospholipid turnover and inositol 1,4,5-trisphosphate production in Chlamydomonas eugametos gametes.

    PubMed

    Musgrave, A; Kuin, H; Jongen, M; de Wildt, P; Schuring, F; Klerk, H; van den Ende, H

    1992-02-01

    Alcohols induce mating-structure activation in Chlamydomonas eugametos gametes. From the effect of ethanol on the (32)P-labelling of polyphosphoinositides, we conclude that the synthesis of these lipids is stimulated. Biologically inactive concentrations of ethanol (<6%) had no effect on synthesis, but 6-8% ethanol stimulated synthesis for upto 60 min. The (32)P incorporated into polyphosphoinositides and phosphatidic acid during ethanol treatment was readily chased out when 1 mM unlabelled Na3PO4 was added. Using a binding assay for inositol 1,4,5-trisphosphate, we show that the production of this phospholipid constituent is dramatically increased after ethanol treatment. This effect, coupled to a rise in intracellular calcium concentration, could explain gamete activation. The significance of these results in explaining other ethanol-induced phenomena in algae is discussed.

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

    PubMed

    Krause, Erin L; Villa-García, Manuel J; Henry, Susan A; Walker, Larry P

    2007-11-07

    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.

  17. The presence of inositol phosphates in gastric pig digesta is affected by time after feeding a nonfermented or fermented liquid wheat- and barley-based diet.

    PubMed

    Blaabjerg, K; Jørgensen, H; Tauson, A-H; Poulsen, H D

    2011-10-01

    The objective was to quantify the retention of digesta and evaluate the degradation of phytate or inositol hexakisphosphate (InsP(6)) and lower inositol phosphates (InsP₅, InsP₄, InsP₃, and InsP₂) in the stomach at different times after feeding pigs a fermented liquid diet with microbial phytase or a nonfermented diet with or without microbial phytase. Six barrows fitted with gastric cannulas were used. The experiment was a 3 × 3 Latin square with 3 pigs fed 3 diets during 3 wk in 2 replicates. Each experimental period lasted for 7 d, comprising 3 d of adaptation and 4 d of total collection of gastric digesta. For each pig, the digesta was collected once daily at 1, 2, 3, or 5 h after feeding the morning meal. A basal wheat- and barley-based diet was steam-pelleted at 90°C. The dietary treatments were a nonfermented basal diet (NF-BD), the NF-BD with microbial phytase (750 phytase units of phytase/kg, as-fed basis; NF-BD + phytase), and the NF-BD + phytase fermented for 17.5 h (F-BD + phytase). Gastric InsP₆-P was not detected at all in pigs fed F-BD + phytase because of complete InsP₆ degradation during fermentation of the feed before feeding. Gastric InsP₆-P decreased over time (P < 0.05) in pigs fed NF-BD and NF-BD + phytase. The decreases were 45, 54, 56, and 61 percentage points greater at 1, 2, 3, and 5 h, respectively, in pigs fed NF-BD + phytase compared with NF-BD. However, substantial amounts of InsP₆ still passed into the small intestine in pigs fed NF-BD + phytase, especially within the first hour (estimated to 17% of InsP₆-P intake). The accumulation of lower inositol phosphates in gastric digesta was very small for all treatments and at all times because of a rapid and almost complete degradation. In conclusion, phytase addition to the nonfermented diet increased the degradation of gastric InsP₆. However, considerable amounts of intact InsP₆ still passed into the small intestine because of a shortage of time for Ins

  18. Stereospecificity of myo-inositol hexakisphosphate dephosphorylation by a phytate-degrading enzyme of baker's yeast.

    PubMed

    Greiner, R; Alminger, M L; Carlsson, N G

    2001-05-01

    During food processing such as baking, phytate is dephosphorylated to produce degradation products, such as myo-inositol pentakis-, tetrakis-, tris-, bis-, and monophosphates. Certain myo-inositol phosphates have been proposed to have positive effects on human health. The position of the phosphate groups on the myo-inositol ring is thereby of great significance for their physiological functions. Using a combination of high-performance ion chromatography analysis and kinetic studies the stereospecificity of myo-inositol hexakisphosphate dephosphorylation by a phytate-degrading enzyme from baker's yeast (Saccharomyces cerevisiae) was established. The data demonstrate that the phytate-degrading enzyme from baker's yeast dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-Ins(1,2,4,5,6)P(5), D-Ins(1,2,5,6)P(4), D-Ins(1,2,6)P(3), D-Ins(1,2)P(2), to finally Ins(2)P (notation 3/4/5/6/1). Knowledge of the absolute stereochemical specificity of the baker's yeast phytase allows use of the enzyme to produce defined myo-inositol phosphates for kinetic and physiological studies.

  19. [Coupled excitation and contraction in skeletal muscles: arguments in favor of a mechanism implicating inositol metabolism].

    PubMed

    Potus, J

    1986-01-01

    When mouse diaphragms are incubated in vitro with [14C] inositol, radioactivity is observed at post synaptic areas in phosphatidyl-inositols and in inositol-phosphates. Synaptic desensitization suppresses the labelled inositol-phosphates. Lindane restores the labelling effect without suppressing the desensitized state.

  20. Interactions between supplemented mineral phosphorus and phytase on phytate hydrolysis and inositol phosphates in the small intestine of broilers1,2.

    PubMed

    Zeller, Ellen; Schollenberger, Margit; Witzig, Maren; Shastak, Yauheni; Kühn, Imke; Hoelzle, Ludwig E; Rodehutscord, Markus

    2015-05-01

    Phytate breakdown in the digestive tract of broilers is affected by supplements of mineral phosphorus (P) and phytase with unknown interactions between the 2 factors. It was the objective to study phytate hydrolysis and the presence of inositol phosphate isomers (InsPs) as affected by supplements of mineral P and phytase in the small intestine of broilers. Fifteen-day old broilers were assigned to 48 pens of 20 broilers each (n = 8 pens/treatment). Two low-P corn-soybean meal-based diets without (BD-; 4.4 g P/kg dry matter) or with monocalcium phosphate (MCP; BD+; 5.2 g P/kg dry matter) were supplied without or with added phytase at 500 or 12,500 FTU/kg. On d 24, digesta from the duodenum/jejunum and lower ileum was pooled per segment on a by-pen basis, freeze-dried, and analyzed for P, InsPs, and the marker TiO2. Another 180 broilers (n = 6 pens/treatment, 10 birds each) were fed the 3 BD+ diets from d 1 to 21 to assess the influence of supplemented phytase on tibia mineralization and strength. Significant interactions between MCP and phytase supplements on myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate) (InsP6) hydrolysis (duodenum/jejunum: P ≤ 0.001; ileum: P = 0.004) and level of specific lower InsPs were detected. Supplementation with 12,500 FTU/kg phytase resulted in 92% InsP6 hydrolysis and strong degradation of InsP5. This treatment resulted in higher P net absorption, affirmed by higher BW gain, tibia strength, and mineralization compared to treatments without or with 500 FTU/kg phytase (P ≤ 0.05). MCP supplementation reduced the degradation of InsP6 and specific lower InsPs in birds fed diets without or with 500 FTU/kg of phytase (P ≤ 0.05), but did not reduce InsP6 hydrolysis or degradation of InsP5 at the high phytase dose. Effects of added MCP on phytase efficacy depend on the dose of supplemented phytase. Differences in the concentrations of lower InsPs indicated that the initial step of InsP6 hydrolysis is not the only

  1. Pheromone regulated production of inositol-(1, 4, 5)-trisphosphate in the mammalian vomeronasal organ.

    PubMed

    Wekesa, K S; Anholt, R R

    1997-08-01

    Social behaviors of most mammals are profoundly affected by chemical signals, pheromones, exchanged between conspecifics. Pheromones interact with dendritic microvilli of bipolar neurons in the vomeronasal organ (VNO). To investigate vomeronasal signal transduction pathways, microvillar membranes from porcine VNO were prepared. Incubation of such membranes from prepubertal females with boar seminal fluid or urine results in an increase in production of inositol-(1, 4, 5)-trisphosphate (IP3). The dose response for IP3 production is biphasic with a GTP-dependent component at low stimulus concentrations and a nonspecific increase in IP3 at higher stimulus concentrations. The GTP-dependent stimulation is mimicked by GTPgammaS and blocked by GDPbetaS. Furthermore, the GTP-dependent component of the stimulation of IP3 production is sex specific and tissue dependent. Studies with monospecific antibodies reveal a G alpha(q/11)-related protein in vomeronasal neurons, concentrated at their microvilli. Our observations indicate that pheromones in boar secretions act on vomeronasal neurons in the female VNO via a receptor mediated, G protein-dependent increase in IP3. These observations set the stage for further investigations on the regulation of stimulus-excitation coupling in vomeronasal neurons. The pheromone-induced IP3 response also provides an assay for future purification of mammalian reproductive pheromones.

  2. Guanine nucleotide-dependent, pertussis toxin-insensitive, stimulation of inositol phosphate formation by carbachol in a membrane preparation from astrocytoma cells

    SciTech Connect

    Hepler, J.R.; Harden, T.K.

    1986-03-05

    Formation of the inositol phosphates (InsP), InsP/sub 3/, InsP/sub 2/, and InsP/sub 1/ was increased in a concentration dependent manner (K/sub 0.5/ approx. 5 ..mu..M) by GTP..sigma..S in washed membranes prepared from /sup 3/H-inositol-prelabelled 1321N1 human astrocytoma cells. Both GTP..gamma..S and GppNHp stimulated InsP formation by 2-3 fold over control; GTP and GDP were much less efficacious and GMP had no effect. Although the muscarinic cholinergic receptor agonist carbachol had no effect in the absence of guanine nucleotide, in the presence of 10 ..mu..M GTP..gamma..S, carbachol stimulated (K/sub 0.5/ approx. 10 ..mu.. M) the formation of InsP above the level achieved with GTP..gamma..S alone. The effect of carbachol was completely blocked by atropine. The order of potency for a series of nucleotides for stimulation of InsP formation in the presence of 500 ..mu..M carbachol was GTP..gamma..S > GppNHp > GTP = GDP. Pertussis toxin, at concentrations that fully ADP-ribosylate and functionally inactivate G/sub i/, had no effect on InsP formation in the presence of GTP..gamma..S or GTP..gamma..S plus carbachol. Histamine and bradykinin also stimulated InsP formation in the presence of GTP..gamma..S in washed membranes from 1321N1 cells. These data are consistent with the idea that a guanine nucleotide regulatory protein that is not G/sub i/ is involved in receptor-mediated stimulation of InsP formation in 1321N1 human astrocytoma cells.

  3. Analysis of weighted co-regulatory networks in maize provides insights into new genes and regulatory mechanisms related to inositol phosphate metabolism.

    PubMed

    Zhang, Shaojun; Yang, Wenzhu; Zhao, Qianqian; Zhou, Xiaojin; Jiang, Ling; Ma, Shuai; Liu, Xiaoqing; Li, Ye; Zhang, Chunyi; Fan, Yunliu; Chen, Rumei

    2016-02-24

    D-myo-inositol phosphates (IPs) are a series of phosphate esters. Myo-inositol hexakisphosphate (phytic acid, IP6) is the most abundant IP and has negative effects on animal and human nutrition. IPs play important roles in plant development, stress responses, and signal transduction. However, the metabolic pathways and possible regulatory mechanisms of IPs in maize are unclear. In this study, the B73 (high in phytic acid) and Qi319 (low in phytic acid) lines were selected for RNA-Seq analysis from 427 inbred lines based on a screening of IP levels. By integrating the metabolite data with the RNA-Seq data at three different kernel developmental stages (12, 21 and 30 days after pollination), co-regulatory networks were constructed to explore IP metabolism and its interactions with other pathways. Differentially expressed gene analyses showed that the expression of MIPS and ITPK was related to differences in IP metabolism in Qi319 and B73. Moreover, WRKY and ethylene-responsive transcription factors (TFs) were common among the differentially expressed TFs, and are likely to be involved in the regulation of IP metabolism. Six co-regulatory networks were constructed, and three were chosen for further analysis. Based on network analyses, we proposed that the GA pathway interacts with the IP pathway through the ubiquitination pathway, and that Ca(2+) signaling functions as a bridge between IPs and other pathways. IP pools were found to be transported by specific ATP-binding cassette (ABC) transporters. Finally, three candidate genes (Mf3, DH2 and CB5) were identified and validated using Arabidopsis lines with mutations in orthologous genes or RNA interference (RNAi)-transgenic maize lines. Some mutant or RNAi lines exhibited seeds with a low-phytic-acid phenotype, indicating perturbation of IP metabolism. Mf3 likely encodes an enzyme involved in IP synthesis, DH2 encodes a transporter responsible for IP transport across organs and CB5 encodes a transporter involved in IP

  4. Adhesion to fibronectin stimulates inositol lipid synthesis and enhances PDGF-induced inositol lipid breakdown

    PubMed Central

    1993-01-01

    The aim of these experiments was to investigate whether inositol lipids might mediate some of the effects of extracellular matrix (ECM) on cellular form and functions. The lipid phosphatidylinositol bisphosphate (PIP2) plays a role in cytoskeletal regulation while its hydrolysis products, diacylglycerol and inositol triphosphate, serve as second messengers. We therefore measured the effect of adhesion to fibronectin (FN) on PIP2 and its hydrolysis products, in the presence and absence of the soluble mitogen PDGF. PDGF induced a threefold increase in release of water-soluble inositol phosphates in C3H 10T1/2 fibroblasts when cells were attached to FN, but had little effect in suspended cells. Suppression of inositol phosphate release in unattached cells was not due to dysfunction of the PDGF receptor or failure to activate phospholipase C-gamma; PDGF induced similar tyrosine phosphorylation of PLC-gamma under both conditions. By contrast, the total mass of phosphatidylinositol bisphosphate (PIP2), the substrate for PLC-gamma, was found to decrease by approximately 80% when cells were detached from their ECM attachments and placed in suspension in the absence of PDGF. PIP2 levels were restored when suspended cells were replated on FN, demonstrating that the effect was reversible. Furthermore, a dramatic increase in synthesis of PIP2 could be measured in cells within 2 min after reattachment to FN in the absence of PDGF. These results show that FN acts directly to stimulate PIP2 synthesis, and that it also enhances PIP2 hydrolysis in response to PDGF. The increase in PIP2 induced by adhesion may mediate some of the known effects of FN on cell shape and cytoskeletal organization, while regulation of inositol lipid hydrolysis may provide a means for integrating hormone- and ECM-dependent signaling pathways. PMID:8387531

  5. Epidermal growth factor (EGF)-stimulated inositol phosphate formation in hepatocytes is abolished by pertussis toxin and phorbol esters

    SciTech Connect

    Johnson, R.M.; Garrison, J.C.

    1987-05-01

    The EGF-stimulated rise in intracellular Ca/sup 2 +/ (Ca/sup 2 +/)/sub i/ and Ca/sup 2 +/-dependent protein phosphorylation events in isolated hepatocytes are blocked by pertussis toxin and phorbol ester pretreatment. The present study characterized the EGF-stimulated formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P/sub 3/) and inositol 1,3,4-trisphosphate (Ins(1,3,4)P/sub 3/) in hepatocytes using HPLC methodology to separate the InsP/sub 3/ isomers. Both 66 nM EGF and 10 nM angiotensin II (ANG II) caused a rapid increase in the Ins(1,4,5)P/sub 3/ isomer although EGF-stimulated formation was smaller. At a concentration of ANG II (0.1 nM) which gave an equivalent rise in (Ca/sup 2 +/)/sub i/ as 66 nM EGF, the kinetics and magnitude of Ins(1,4,5)P/sub 3/ formation were similar. EGF or ANG II-stimulated formation of the Ins(1,3,4)P/sub 3/ isomer was more gradual and increased beyond the level of Ins(1,4,5)P/sub 3/ after 60 sec. The initial EGF and ANG II-stimulated increase in both InsP/sub 3/ isomers was not affected by removing external Ca/sup 2 +/ with a 10-fold excess of EGTA. Pretreatment of rats with pertussis toxin for 72 hrs blocked the ability of EGF to increase Ins(1,4,5)P/sub 3/ but did not affect the increase due to ANG II. Three main pretreatment of cells with 1 ..mu..g/ml phorbol 12-myristate-13-acetate (PMA) also inhibited the EGF-stimulated Ins(1,4,5)P/sub 3/ formation. PMA slightly attenuated Ins(1,4,5)P/sub 3/ formation stimulated by 0.1 nM ANG II but not enough to affect the Ca/sup 2 +/ signal. These data suggest that the signal transduction system used by EGF receptors to increase Ins (1,4,5)P/sub 3/ in hepatocytes is somehow different from that used by ANG II receptors.

  6. Antibacterial polyetheretherketone implants immobilized with silver ions based on chelate-bonding ability of inositol phosphate: processing, material characterization, cytotoxicity, and antibacterial properties.

    PubMed

    Kakinuma, H; Ishii, K; Ishihama, H; Honda, M; Toyama, Y; Matsumoto, M; Aizawa, M

    2015-01-01

    We developed a novel antibacterial implant by forming a hydroxyapatite (HAp) film on polyetheretherketone (PEEK) substrate, and then immobilizing silver ions (Ag(+) ) on the HAp film based on the chelate-bonding ability of inositol phosphate (IP6). First, the PEEK surface was modified by immersion into concentrated sulfuric acid for 10 min. HAp film was formed on the acid-treated PEEK via the soft-solution process using simulated body fluid (SBF), urea, and urease. After HAp coating, specimens were immersed into IP6 solution, and followed by immersion into silver nitrite solution at concentrations of 0, 0.5, 1, 5 or 10 mM. Ag(+) ions were immobilized on the resulting HAp film due to the chelate-bonding ability of IP6. On cell-culture tests under indirect conditions by Transwell, MC3T3-E1 cells on the specimens derived from the 0.5 and 1 mM Ag(+) solutions showed high relative growth when compared with controls. Furthermore, on evaluation of antibacterial activity in halo test, elution of Ag(+) ions from Ag(+) -immobilized HAp film inhibited bacterial growth. Therefore, the above-mentioned results demonstrated that specimens had both biocompatibility and strong antibacterial activity. The present coating therefore provides bone bonding ability to the implant surface and prevents the formation of biofilms in the early postoperative period.

  7. Reconstructed Ancestral Myo-Inositol-3-Phosphate Synthases Indicate That Ancestors of the Thermococcales and Thermotoga Species Were More Thermophilic than Their Descendants

    PubMed Central

    Butzin, Nicholas C.; Lapierre, Pascal; Green, Anna G.; Swithers, Kristen S.; Gogarten, J. Peter; Noll, Kenneth M.

    2013-01-01

    The bacterial genomes of Thermotoga species show evidence of significant interdomain horizontal gene transfer from the Archaea. Members of this genus acquired many genes from the Thermococcales, which grow at higher temperatures than Thermotoga species. In order to study the functional history of an interdomain horizontally acquired gene we used ancestral sequence reconstruction to examine the thermal characteristics of reconstructed ancestral proteins of the Thermotoga lineage and its archaeal donors. Several ancestral sequence reconstruction methods were used to determine the possible sequences of the ancestral Thermotoga and Archaea myo-inositol-3-phosphate synthase (MIPS). These sequences were predicted to be more thermostable than the extant proteins using an established sequence composition method. We verified these computational predictions by measuring the activities and thermostabilities of purified proteins from the Thermotoga and the Thermococcales species, and eight ancestral reconstructed proteins. We found that the ancestral proteins from both the archaeal donor and the Thermotoga most recent common ancestor recipient were more thermostable than their descendants. We show that there is a correlation between the thermostability of MIPS protein and the optimal growth temperature (OGT) of its host, which suggests that the OGT of the ancestors of these species of Archaea and the Thermotoga grew at higher OGTs than their descendants. PMID:24391933

  8. Reconstructed ancestral Myo-inositol-3-phosphate synthases indicate that ancestors of the Thermococcales and Thermotoga species were more thermophilic than their descendants.

    PubMed

    Butzin, Nicholas C; Lapierre, Pascal; Green, Anna G; Swithers, Kristen S; Gogarten, J Peter; Noll, Kenneth M

    2013-01-01

    The bacterial genomes of Thermotoga species show evidence of significant interdomain horizontal gene transfer from the Archaea. Members of this genus acquired many genes from the Thermococcales, which grow at higher temperatures than Thermotoga species. In order to study the functional history of an interdomain horizontally acquired gene we used ancestral sequence reconstruction to examine the thermal characteristics of reconstructed ancestral proteins of the Thermotoga lineage and its archaeal donors. Several ancestral sequence reconstruction methods were used to determine the possible sequences of the ancestral Thermotoga and Archaea myo-inositol-3-phosphate synthase (MIPS). These sequences were predicted to be more thermostable than the extant proteins using an established sequence composition method. We verified these computational predictions by measuring the activities and thermostabilities of purified proteins from the Thermotoga and the Thermococcales species, and eight ancestral reconstructed proteins. We found that the ancestral proteins from both the archaeal donor and the Thermotoga most recent common ancestor recipient were more thermostable than their descendants. We show that there is a correlation between the thermostability of MIPS protein and the optimal growth temperature (OGT) of its host, which suggests that the OGT of the ancestors of these species of Archaea and the Thermotoga grew at higher OGTs than their descendants.

  9. 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.

  10. Strategies for acquiring the phospholipid metabolite inositol in pathogenic bacteria, fungi and protozoa: making it and taking it

    PubMed Central

    Reynolds, Todd B.

    2009-01-01

    myo-Inositol (inositol) is an essential nutrient that is used for building phosphatidylinositol and its derivatives in eukaryotes and even in some eubacteria such as the mycobacteria. As a consequence, fungal, protozoan and mycobacterial pathogens must be able to acquire inositol in order to proliferate and cause infection in their hosts. There are two primary mechanisms for acquiring inositol. One is to synthesize inositol from glucose 6-phosphate using two sequentially acting enzymes: inositol-3-phosphate synthase (Ino1p) converts glucose 6-phosphate to inositol 3-phosphate, and then inositol monophosphatase (IMPase) dephosphorylates inositol 3-phosphate to generate inositol. The other mechanism is to import inositol from the environment via inositol transporters. Inositol is readily abundant in the bloodstream of mammalian hosts, providing a source from which many pathogens could potentially import inositol. However, despite this abundance of inositol in the host, some pathogens such as the bacterium Mycobacterium tuberculosis and the protist parasite Trypanosoma brucei must be able to make inositol de novo in order to cause disease (M. tuberculosis) or even grow (T. brucei). Other pathogens such as the fungus Candida albicans are equally adept at causing disease by importing inositol or by making it de novo. The role of inositol acquisition in the biology and pathogenesis of the parasite Leishmania and the fungus Cryptococcus are being explored as well. The specific strategies used by these pathogens to acquire inositol while in the host are discussed in relation to each pathogen's unique metabolic requirements. PMID:19383710

  11. Comparative genomics of pneumocystis species suggests the absence of genes for myo-inositol synthesis and reliance on inositol transport and metabolism.

    PubMed

    Porollo, Aleksey; Sesterhenn, Thomas M; Collins, Margaret S; Welge, Jeffrey A; Cushion, Melanie T

    2014-11-04

    In the context of deciphering the metabolic strategies of the obligate pathogenic fungi in the genus Pneumocystis, the genomes of three species (P. carinii, P. murina, and P. jirovecii) were compared among themselves and with the free-living, phylogenetically related fission yeast (Schizosaccharomyces pombe). The underrepresentation of amino acid metabolism pathways compared to those in S. pombe, as well as the incomplete steroid biosynthesis pathway, were confirmed for P. carinii and P. jirovecii and extended to P. murina. All three Pneumocystis species showed overrepresentation of the inositol phosphate metabolism pathway compared to that in the fission yeast. In addition to those known in S. pombe, four genes, encoding inositol-polyphosphate multikinase (EC 2.7.1.151), inositol-pentakisphosphate 2-kinase (EC 2.7.1.158), phosphoinositide 5-phosphatase (EC 3.1.3.36), and inositol-1,4-bisphosphate 1-phosphatase (EC 3.1.3.57), were identified in the two rodent Pneumocystis genomes, P. carinii and P. murina. The P. jirovecii genome appeared to contain three of these genes but lacked phosphoinositide 5-phosphatase. Notably, two genes encoding enzymes essential for myo-inositol synthesis, inositol-1-phosphate synthase (INO1) and inositol monophosphatase (INM1), were absent from all three genomes, suggesting that Pneumocystis species are inositol auxotrophs. In keeping with the need to acquire exogenous inositol, two genes with products homologous to fungal inositol transporters, ITR1 and ITR2, were identified in P. carinii and P. murina, while P. jirovecii contained only the ITR1 homolog. The ITR and inositol metabolism genes in P. murina and P. carinii were expressed during fulminant infection as determined by reverse transcriptase real-time PCR of cDNA from infected lung tissue. Supplementation of in vitro culture with inositol yielded significant improvement of the viability of P. carinii for days 7 through 14. Microbes in the genus Pneumocystis are obligate

  12. Comparative Genomics of Pneumocystis Species Suggests the Absence of Genes for myo-Inositol Synthesis and Reliance on Inositol Transport and Metabolism

    PubMed Central

    Sesterhenn, Thomas M.; Collins, Margaret S.; Welge, Jeffrey A.

    2014-01-01

    ABSTRACT In the context of deciphering the metabolic strategies of the obligate pathogenic fungi in the genus Pneumocystis, the genomes of three species (P. carinii, P. murina, and P. jirovecii) were compared among themselves and with the free-living, phylogenetically related fission yeast (Schizosaccharomyces pombe). The underrepresentation of amino acid metabolism pathways compared to those in S. pombe, as well as the incomplete steroid biosynthesis pathway, were confirmed for P. carinii and P. jirovecii and extended to P. murina. All three Pneumocystis species showed overrepresentation of the inositol phosphate metabolism pathway compared to that in the fission yeast. In addition to those known in S. pombe, four genes, encoding inositol-polyphosphate multikinase (EC 2.7.1.151), inositol-pentakisphosphate 2-kinase (EC 2.7.1.158), phosphoinositide 5-phosphatase (EC 3.1.3.36), and inositol-1,4-bisphosphate 1-phosphatase (EC 3.1.3.57), were identified in the two rodent Pneumocystis genomes, P. carinii and P. murina. The P. jirovecii genome appeared to contain three of these genes but lacked phosphoinositide 5-phosphatase. Notably, two genes encoding enzymes essential for myo-inositol synthesis, inositol-1-phosphate synthase (INO1) and inositol monophosphatase (INM1), were absent from all three genomes, suggesting that Pneumocystis species are inositol auxotrophs. In keeping with the need to acquire exogenous inositol, two genes with products homologous to fungal inositol transporters, ITR1 and ITR2, were identified in P. carinii and P. murina, while P. jirovecii contained only the ITR1 homolog. The ITR and inositol metabolism genes in P. murina and P. carinii were expressed during fulminant infection as determined by reverse transcriptase real-time PCR of cDNA from infected lung tissue. Supplementation of in vitro culture with inositol yielded significant improvement of the viability of P. carinii for days 7 through 14. PMID:25370490

  13. New Functions of the Inositol Polyphosphate 5-Phosphatases in Cancer.

    PubMed

    Erneux, Christophe; Ghosh, Somadri; Ramos, Ana Raquel; Edimo, William's Elong

    2016-01-01

    Inositol polyphosphate 5-phosphatases act on inositol phosphates and phosphoinositides as substrates. They are 10 different isoenzymes and several splice variants in the human genome that are involved in a series of human pathologies such as the Lowe syndrome, the Joubert and MORM syndromes, breast cancer, glioblastoma, gastric cancer and several other type of cancers. Inositol 5-phosphatases can be amplified in human cancer cells, whereas the 3- and 4- phosphatase tumor suppressor PTEN and INPP4B, repectively are often repressed or deleted. The inositol 5-phosphatases are critically involved in a complex network of higly regulated phosphoinositides, affecting the lipid content of PI(3, 4, 5)P3, PI(4, 5)P2 and PI(3, 4)P2. This has an impact on the normal behavior of many intracellular target proteins e.g. protein kinase B (PKB/Akt) or actin binding proteins and final biological responses. The production of PI(3, 4P)2 by dephosphorylation of the substrate PI(3, 4, 5)P3 is particularly important as it produces a new signal messenger in the control of cell migration, invasion and endocytosis. New inhibitors/activators of inositol 5- phosphatases have recently been identified for the possible control of their activity in several human pathologies such as inflamation and cancer.

  14. Myo-inositol phosphate synthase expression in the European eel (Anguilla anguilla) and Nile tilapia (Oreochromis niloticus): effect of seawater acclimation.

    PubMed

    Kalujnaia, Svetlana; Hazon, Neil; Cramb, Gordon

    2016-08-01

    A single MIPS gene (Isyna1/Ino1) exists in eel and tilapia genomes with a single myo-d-inositol 3-phosphate synthase (MIPS) transcript identified in all eel tissues, although two MIPS spliced variants [termed MIPS(s) and MIPS(l)] are found in all tilapia tissues. The larger tilapia transcript [MIPS(l)] results from the inclusion of the 87-nucleotide intron between exons 5 and 6 in the genomic sequence. In most tilapia tissues, the MIPS(s) transcript exhibits much higher abundance (generally >10-fold) with the exception of white skeletal muscle and oocytes, in which the MIPS(l) transcript predominates. SW acclimation resulted in large (6- to 32-fold) increases in mRNA expression for both MIPS(s) and MIPS(l) in all tilapia tissues tested, whereas in the eel, changes in expression were limited to a more modest 2.5-fold increase and only in the kidney. Western blots identified a number of species- and tissue-specific immunoreactive MIPS proteins ranging from 40 to 67 kDa molecular weight. SW acclimation failed to affect the abundance of any immunoreactive protein in any tissue tested from the eel. However, a major 67-kDa immunoreactive protein (presumed to be MIPS) found in tilapia tissues exhibited 11- and 54-fold increases in expression in gill and fin samples from SW-acclimated fish. Immunohistochemical investigations revealed specific immunoreactivity in the gill, fin, skin, and intestine taken from only SW-acclimated tilapia. Immunofluorescence indicated that MIPS was expressed within gill chondrocytes and epithelial cells of the primary filaments, basal epithelial cell layers of the skin and fin, the cytosol of columnar intestinal epithelial and mucous cells, as well as unknown entero-endocrine-like cells.

  15. Myo-inositol phosphate synthase expression in the European eel (Anguilla anguilla) and Nile tilapia (Oreochromis niloticus): effect of seawater acclimation

    PubMed Central

    Kalujnaia, Svetlana; Hazon, Neil

    2016-01-01

    A single MIPS gene (Isyna1/Ino1) exists in eel and tilapia genomes with a single myo-d-inositol 3-phosphate synthase (MIPS) transcript identified in all eel tissues, although two MIPS spliced variants [termed MIPS(s) and MIPS(l)] are found in all tilapia tissues. The larger tilapia transcript [MIPS(l)] results from the inclusion of the 87-nucleotide intron between exons 5 and 6 in the genomic sequence. In most tilapia tissues, the MIPS(s) transcript exhibits much higher abundance (generally >10-fold) with the exception of white skeletal muscle and oocytes, in which the MIPS(l) transcript predominates. SW acclimation resulted in large (6- to 32-fold) increases in mRNA expression for both MIPS(s) and MIPS(l) in all tilapia tissues tested, whereas in the eel, changes in expression were limited to a more modest 2.5-fold increase and only in the kidney. Western blots identified a number of species- and tissue-specific immunoreactive MIPS proteins ranging from 40 to 67 kDa molecular weight. SW acclimation failed to affect the abundance of any immunoreactive protein in any tissue tested from the eel. However, a major 67-kDa immunoreactive protein (presumed to be MIPS) found in tilapia tissues exhibited 11- and 54-fold increases in expression in gill and fin samples from SW-acclimated fish. Immunohistochemical investigations revealed specific immunoreactivity in the gill, fin, skin, and intestine taken from only SW-acclimated tilapia. Immunofluorescence indicated that MIPS was expressed within gill chondrocytes and epithelial cells of the primary filaments, basal epithelial cell layers of the skin and fin, the cytosol of columnar intestinal epithelial and mucous cells, as well as unknown entero-endocrine-like cells. PMID:27252471

  16. Potential role of D-myo-inositol-3-phosphate synthase and 14-3-3 genes in the crosstalk between Zea mays and Rhizophagus intraradices under drought stress.

    PubMed

    Li, Tao; Sun, Yuqing; Ruan, Yuan; Xu, Lijiiao; Hu, Yajun; Hao, Zhipeng; Zhang, Xin; Li, Hong; Wang, Youshan; Yang, Liguo; Chen, Baodong

    2016-11-01

    Arbuscular mycorrhizal (AM) symbiosis is known to stimulate plant drought tolerance. However, the mechanisms underlying the synergistic responses of the symbiotic partners to drought stress are largely unknown. A split-root experiment was designed to investigate the molecular interactions between a host plant and an AM fungus (AMF) under drought stress. In the two-compartment cultivation system, an entire or only a half root system of a maize plant was inoculated with an AMF, Rhizophagus intraradices, in the presence of localized or systemic drought treatment. Plant physiological parameters including growth, water status, and phosphorus concentration, and the expression of drought tolerance-related genes in both roots and R. intraradices were recorded. Although mycorrhizal inoculation in either one or both compartments systemically decreased abscisic acid (ABA) content in the whole root system subjected to systemic or local drought stress, we observed local and/or systemic AM effects on root physiological traits and the expression of functional genes in both roots and R. intraradices. Interestingly, the simultaneous increase in the expression of plant genes encoding D-myo-inositol-3-phosphate synthase (IPS) and 14-3-3-like protein GF14 (14-3GF), which were responsible for ABA signal transduction, was found to be involved in the activation of 14-3-3 protein and aquaporins (GintAQPF1 and GintAQPF2) in R. intraradices. These findings suggest that coexpression of IPS and 14-3GF is responsible for the crosstalk between maize and R. intraradices under drought stress, and potentially induces the synergistic actions of the symbiotic partners in enhancing plant drought tolerance.

  17. Different pathways of ( sup 3 H)inositol phosphate formation mediated by. alpha. 1a- and. alpha. 1b-adrenergic receptors

    SciTech Connect

    Wilson, K.M.; Minneman, K.P. )

    1990-10-15

    The types of inositol phosphates (InsPs) formed in response to activation of alpha 1-adrenergic receptor subtypes were determined in collagenase-dispersed renal cells and hepatocytes by high pressure liquid chromatography separation. In hepatocytes, which contain only the alpha 1b subtype, norepinephrine stimulated rapid (10-s) formation of (3H)Ins(1,4,5)P3 and (3H)Ins(1,3,4)P3 and slower (5-min) formation of Ins(1,4)P2 and Ins(1)P. Selective inactivation of alpha 1b receptors by chloroethylclonidine almost completely blocked the effects of norepinephrine in hepatocytes. In renal cells, which contain both alpha 1a and alpha 1b receptors in a 60:40 ratio, norepinephrine did not significantly increase the size of any peaks until 5 min after agonist activation. At this time, only a peak eluting with Ins(1)P and one eluting shortly after Ins(1,4)P2 were significantly elevated. Incubation with norepinephrine for 2 h caused small but significant increases in peaks co-eluting with Ins(1)P and Ins(1,4,5)P3 in renal cells; however, only the increase in Ins(1)P was inhibited by chloroethylclonidine pretreatment. Extraction under neutral conditions suggested that cyclic InsPs may be the primary compounds formed in response to norepinephrine in renal cells. Removal of extracellular Ca2+ caused a 60% reduction in the InsP response to norepinephrine in renal cells but had no effect in hepatocytes. These results suggest that activation of alpha 1a and alpha 1b receptor subtypes results in formation of different InsPs and that the response to alpha 1a activation may require influx of extracellular Ca2+.

  18. Expression analysis of a heat-inducible, Myo-inositol-1-phosphate synthase (MIPS) gene from wheat and the alternatively spliced variants of rice and Arabidopsis.

    PubMed

    Khurana, Neetika; Chauhan, Harsh; Khurana, Paramjit

    2012-01-01

    Molecular dissection and a deeper analysis of the heat stress response mechanism in wheat have been poorly understood so far. This study delves into the molecular basis of action of TaMIPS, a heat stress-inducible enzyme that was identified through PCR-select subtraction technology, which is named here as TaMIPS2. MIPS (L-Myo-inositol-phosphate synthase) is important for the normal growth and development in plants. Expression profiling showed that TaMIPS2 is expressed during different developing seed stages upon heat stress. Also, the transcript levels increase in unfertilized ovaries and significant amounts are present during the recovery period providing evidence that MIPS is crucial for its role in heat stress recovery and flower development. Alternatively spliced forms from rice and Arabidopsis were also identified and their expression analysis revealed that apart from heat stress, some of the spliced variants were also inducible by drought, NaCl, Cold, ABA, BR, SA and mannitol. In silico promoter analysis revealed various cis-elements that could contribute for the differential regulation of MIPS in different plant systems. Phylogenetic analysis indicated that MIPS are highly conserved among monocots and dicots and TaMIPS2 grouped specifically with monocots. Comparative analyses was undertaken by different experimental approaches, i.e., semi-quantitative RT-PCR, quantitative RT-PCR, Genevestigator as a reference expression tool and motif analysis to predict the possible function of TaMIPS2 in regulating the different aspects of plant development under abiotic stress in wheat.

  19. Generation of stable 'low phytic acid' transgenic rice through antisense repression of the 1D-myo-inositol 3-phosphate synthase gene (RINO1) using the 18-kDa oleosin promoter.

    PubMed

    Kuwano, Mio; Mimura, Tetsuro; Takaiwa, Fumio; Yoshida, Kaoru T

    2009-01-01

    Phytic acid acts as the major storage form of phosphorus in plant seeds and is poorly digested by monogastric animals. The degradation of phytic acid in animal diets is necessary to overcome both environmental and nutritional issues. The enzyme 1D-myo-inositol 3-phosphate [Ins(3)P(1)] synthase (EC 5.5.1.4) catalyses the first step of myo-inositol biosynthesis and directs phytic acid biosynthesis in seeds. The rice Ins(3)P(1) synthase gene (RINO1) is highly expressed in developing seed embryos and in the aleurone layer, where phytic acid is synthesized and stored. In rice seeds, 18-kDa oleosin (Ole18) is expressed in a seed-specific manner, and its transcripts are restricted to the embryo and the aleurone layer. Therefore, to effectively suppress phytic acid biosynthesis, antisense RINO1 cDNA was expressed under the control of the Ole18 promoter, directing the same spatial pattern in seeds as RINO1 in transgenic rice plants. The generated transgenic rice plants showed strong 'low phytic acid' (lpa) phenotypes, in which seed phytic acid was reduced by 68% and free available phosphate was concomitantly increased. No negative effects on seed weight, germination or plant growth were observed. The available phosphate levels of the stable transgenic plants surpassed those of currently available rice lpa mutants.

  20. Scale-up potential of ion-pair high-performance liquid chromatography method to produce biologically active inositol phosphates.

    PubMed

    Hamada, Jamel S

    2002-01-25

    This study was undertaken to evaluate the possibility that an analytical ion-pair HPLC procedure used to determine phytic acid (IP6) and its degradation products (IP3-IP5) can be transformed to a preparative purification method. A commercial phytic acid (CPA) preparation was separated into its component fractions of IP3, IP4, IPS, and IP6 on two C18 columns (1.8 and 4.2 ml) using 51% methanol containing 0.6-1% tetrabutylammonium hydroxide as ion-pair reagent and 0-0.025 M formic acid (pH 4.3) as eluent at 1.7 and 3.0 cm/min linear velocity, respectively, and 40 degrees C. Elution was monitored at 40 degrees C by a refractive index detector. Reproducible separation of CPA into four well-resolved peaks on these columns was possible after optimizing method variables, particularly the concentration of ion-pair reagent in the injected sample (>1.5%). The same separations were obtained after CPA loads were scaled up 25 times on a steel column (15 cm x 19 mm I.D.), packed with Ethyl C2 sorbent (10 microm) and on a 25 cm x 21.2 mm I.D. C18 column, but at a reduced linear velocity to increase the resolution. Therefore, this optimization of separation not only is useful for analysis of phytic acid and its degradation products but also it provides key parameters for scale up for further fractionation and characterization.

  1. Inositol kinase and its product accelerate wound healing by modulating calcium levels, Rho GTPases, and F-actin assembly

    PubMed Central

    Soto, Ximena; Li, Jingjing; Lea, Robert; Dubaissi, Eamon; Papalopulu, Nancy; Amaya, Enrique

    2013-01-01

    Wound healing is essential for survival. We took advantage of the Xenopus embryo, which exhibits remarkable capacities to repair wounds quickly and efficiently, to investigate the mechanisms responsible for wound healing. Previous work has shown that injury triggers a rapid calcium response, followed by the activation of Ras homolog (Rho) family guanosine triphosphatases (GTPases), which regulate the formation and contraction of an F-actin purse string around the wound margin. How these processes are coordinated following wounding remained unclear. Here we show that inositol-trisphosphate 3-kinase B (Itpkb) via its enzymatic product inositol 1,3,4,5-tetrakisphosphate (InsP4) plays an essential role during wound healing by modulating the activity of Rho family GTPases and F-actin ring assembly. Furthermore, we show that Itpkb and InsP4 modulate the speed of the calcium wave, which propagates from the site of injury into neighboring uninjured cells. Strikingly, both overexpression of itpkb and exogenous application of InsP4 accelerate the speed of wound closure, a finding that has potential implications in our quest to find treatments that improve wound healing in patients with acute or chronic wounds. PMID:23776233

  2. Phosphate removal using sludge from fuller's earth production.

    PubMed

    Moon, Yong Hee; Kim, Jae Gon; Ahn, Joo Sung; Lee, Gyoo Ho; Moon, Hi-Soo

    2007-05-08

    This study assesses the phosphate removal capacity and mechanism of precipitation or adsorption from aqueous solutions in batch experiments by an industrial sludge containing gypsum (CaSO(4).2H(2)O) obtained as a by-product from a fuller's earth process. The potential capacity for phosphate removal was tested using various solution concentrations, pH values, reaction times, and amount of sludge. The maximum phosphate adsorption capacity calculated using the Langmuir equation was 2.0 g kg(-1). The pH for the maximum adsorption by the sludge was neutral to alkaline (pH 7-12). Over 99% of phosphate was removed from a phosphate solution of 30 mg L(-1) using 0.15 g of sludge in a 9-h reaction. Sulfate (SO(4)(2-)) concentration increased with increasing initial phosphate concentration, possibly because of dissolution of gypsum and adsorption of both sulfate and phosphate. At high phosphate concentration (>1000 mg L(-1)), relative constant concentration of Ca(2+) was not consistent with adsorption of the most important phosphate removal mechanism. Results suggest that precipitation of calcium phosphate is principally responsible for phosphate removal under its high concentration. Agglomerated precipitate in the reaction sludge was observed by SEM and identified as brushite (CaHPO(4).2H(2)O) by XRD, FT-IR, and DTA. Based on thermodynamic considerations, it is suggested that the brushite will readily transform to more stable phases, such as hydroxyapatite (Ca(5)(PO(4))(3).OH).

  3. The RpiR-like repressor IolR regulates inositol catabolism in Sinorhizobium meliloti.

    PubMed

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

    2011-10-01

    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.

  4. Leukotriene B/sub 4/ (LTB/sub 4/) induces formation of inositol-phosphates (IP's) in rat peritoneal polymorphonuclear leukocytes (PMN's)

    SciTech Connect

    Chi-Rosso, G.; Crooke, S.T.; Mong, S.

    1986-05-01

    LTB/sub 4/ induced rapid breakdown of prelabeled inositol-phospholipids (PI) in rat PMN. Formation of (/sup 3/H)-inositol-trisphosphate ((/sup 3/H)-IP/sub 3/) was rapid, with a peak of 250-300% of the control level, after 5-15 sec of stimulation with LTB/sub 4/. Accumulation of (/sup 3/H)-inositol-bisphosphate ((/sup 3/H)-IP/sub 2/) was rapid, peaking after 30 sec of stimulation. (/sup 3/H)-inositol-monophosphate ((/sup 3/H)-IP/sub 1/) accumulated gradually in the presence of LiCl. The kinetics of (/sup 3/H)-IP/sub 3/, (/sup 3/H)-IP/sub 2/ and (/sup 3/H)-IP/sub 1/ accumulation suggested that LTB/sub 4/ may interact with receptors in PMNs, activate phospholipase C which, in turn, induces hydrolysis of PI. The agonist activities of several LTB/sub 4/ analogs were employed to investigate the structure activity relationship of LTB/sub 4/ receptor mediated activation of PI hydrolysis. Increases in (/sup 3/H)-IP/sub 3/ formation were dependent upon the concentration of LTB/sub 4/ and the agonist analogs. The rank order potency of these analogs were equivalent to that of the pharmacological activity of LTB/sub 4/ agonists in the chemotaxis assay. Furthermore, the Islet activation protein (IAP) inhibited LTB/sub 4/ induced (/sup 3/H)-IP/sub 3/ formation. The tumor promoting phorbomyristate ester also inhibited LTB/sub 4/ induced (/sup 3/H)-IP/sub 3/ formation. These results suggest LTB/sub 4/ may interact with receptors in rat PMNs, activate G/sub i/ protein regulated phospholipase C and induce (/sup 3/H)-IP/sub 3/ formation.

  5. Immobilization of fission products in phosphate ceramic waste forms

    SciTech Connect

    Singh, D.

    1996-10-01

    The goal of this project is to develop and demonstrate the feasibility of a novel low-temperature solidification/stabilization (S/S) technology for immobilizing waste streams containing fission products such as cesium, strontium, and technetium in a chemically bonded phosphate ceramic. This technology can immobilize partitioned tank wastes and decontaminate waste streams containing volatile fission products.

  6. Immobilization of fission products in phosphate ceramic waste forms

    SciTech Connect

    Singh, D.; Wagh, A.

    1997-10-01

    Chemically bonded phosphate ceramics (CBPCs) have several advantages that make them ideal candidates for containing radioactive and hazardous wastes. In general, phosphates have high solid-solution capacities for incorporating radionuclides, as evidenced by several phosphates (e.g., monazites and apatites) that are natural analogs of radioactive and rare-earth elements. The phosphates have high radiation stability, are refractory, and will not degrade in the presence of internal heating by fission products. Dense and hard CBPCs can be fabricated inexpensively and at low temperature by acid-base reactions between an inorganic oxide/hydroxide powder and either phosphoric acid or an acid-phosphate solution. The resulting phosphates are extremely insoluble in aqueous media and have excellent long-term durability. CBPCs offer the dual stabilization mechanisms of chemical fixation and physical encapsulation, resulting in superior waste forms. The goal of this task is develop and demonstrate the feasibility of CBPCs for S/S of wastes containing fission products. The focus of this work is to develop a low-temperature CBPC immobilization system for eluted {sup 99}Tc wastes from sorption processes.

  7. 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.

  8. Effect of cellular inositol content on ethanol tolerance of Saccharomyces cerevisiae in sake brewing.

    PubMed

    Furukawa, Keiji; Kitano, Hideyuki; Mizoguchi, Haruhiko; Hara, Shodo

    2004-01-01

    The effect of cellular inositol content on the ethanol tolerance of sake yeast was investigated. In a static culture of strain K901 in a synthetic medium, when cells were grown in the presence of inositol in limited amount (L-cells), the inositol content of cells decreased by one-third that of cells grown in the presence of inositol in sufficient amount (H-cells). L-cells exhibited a higher death rate constant than H-cells in the presence of 12-20% ethanol, while no difference in specific ethanol production rate in the presence of 0-18% ethanol between the two cell types was observed. L-cells leaked more intracellular components, such as nucleotides, phosphate and potassium, in the presence of ethanol than H-cells. L-cells exhibited a lower intracellular pH value than H-cells, which represented the lowering of cell vitality by the decrease in H(+) extrusion activity. Furthermore, the plasma membrane H(+)-ATPase activity of L-cells was approximately one-half of that of H-cells. Therefore, it was considered that the decrease in viability in the presence of ethanol due to inositol limitation results from the lowering of H(+)-ATPase activity, which maintains the permeability barrier of the yeast membrane, ensuring the homeostasis of ions in the cytoplasm of yeast cells. It is assumed that the lowering of H(+)-ATPase activity due to inositol limitation is caused by the change in lipid environment of the enzyme, which is affected by inositol-containing glycerophospholipids such as phosphatidylinositol (PI), because in the PI-saturated mixed micellar assay system, the difference in H(+)-ATPase activity between L- and H-cells disappeared. In the early stage of sake mash, inositol limitation lowers the ethanol tolerance due to the decrease in H(+)-ATPase activity as in static culture. In the final stage of sake mash, the disruption of the ino1 gene responsible for inositol synthesis, resulted in a decrease in cell density. Furthermore, the ino1 disruptant, which was not

  9. Cancer inhibition by inositol hexaphosphate (IP6) and inositol: from laboratory to clinic.

    PubMed

    Vucenik, Ivana; Shamsuddin, AbulKalam M

    2003-11-01

    Inositol hexaphosphate (IP6) is a naturally occurring polyphosphorylated carbohydrate that is present in substantial amounts in almost all plant and mammalian cells. It was recently recognized to possess multiple biological functions. A striking anticancer effect of IP6 was demonstrated in different experimental models. Inositol is also a natural constituent possessing moderate anticancer activity. The most consistent and best anticancer results were obtained from the combination of IP6 plus inositol. In addition to reducing cell proliferation, IP6 increases differentiation of malignant cells, often resulting in a reversion to normal phenotype. Exogenously administered IP6 is rapidly taken into the cells and dephosphorylated to lower-phosphate inositol phosphates, which further interfere with signal transduction pathways and cell cycle arrest. Enhanced immunity and antioxidant properties can also contribute to tumor cell destruction. However, the molecular mechanisms underlying this anticancer action are not fully understood. Because it is abundantly present in regular diet, efficiently absorbed from the gastrointestinal tract, and safe, IP6 holds great promise in our strategies for the prevention and treatment of cancer. IP6 plus inositol enhances the anticancer effect of conventional chemotherapy, controls cancer metastases, and improves the quality of life, as shown in a pilot clinical trial. The data strongly argue for the use of IP6 plus inositol in our strategies for cancer prevention and treatment. However, the effectiveness and safety of IP6 plus inositol at therapeutic doses needs to be determined in phase I and phase II clinical trials in humans.

  10. Soluble phosphate fertilizer production using acid effluent from metallurgical industry.

    PubMed

    Mattiello, Edson M; Resende Filho, Itamar D P; Barreto, Matheus S; Soares, Aline R; Silva, Ivo R da; Vergütz, Leonardus; Melo, Leônidas C A; Soares, Emanuelle M B

    2016-01-15

    Preventive and effective waste management requires cleaner production strategies and technologies for recycling and reuse. Metallurgical industries produce a great amount of acid effluent that must be discarded in a responsible manner, protecting the environment. The focus of this study was to examine the use of this effluent to increase reactivity of some phosphate rocks, thus enabling soluble phosphate fertilizer production. The effluent was diluted in deionized water with the following concentrations 0; 12.5; 25; 50; 75% (v v(-1)), which were added to four natural phosphate rocks: Araxá, Patos, Bayovar and Catalão and then left to react for 1 h and 24 h. There was an increase in water (PW), neutral ammonium citrate (PNAC) and citric acid (PCA) soluble phosphorus fractions. Such increases were dependent of rock type while the reaction time had no significant effect (p < 0.05) on the chemical and mineralogical phosphate characteristics. Phosphate fertilizers with low toxic metal concentrations and a high level of micronutrients were produced compared to the original natural rocks. The minimum amount of total P2O5, PNAC and PW, required for national legislation for phosphate partially acidulated fertilizer, were met when using Catalão and the effluent at the concentration of 55% (v v(-1)). Fertilizer similar to partially acidulated phosphate was obtained when Bayovar with effluent at 37.5% (v v(-1)) was used. Even though fertilizers obtained from Araxá and Patos did not contain the minimum levels of total P2O5 required by legislation, they can be used as a nutrient source and for acid effluent recycling and reuse.

  11. Effect of Phosphate Concentration on Production of Tetrodotoxin by Alteromonas tetraodonis

    PubMed Central

    Gallacher, Susan; Birkbeck, T. H.

    1993-01-01

    Tetrodotoxin production by Alteromonas tetraodonis occurred during the stationary phase of growth and was regulated by phosphate concentration; toxin production was repressed if phosphate was added at the onset of stationary phase and was over 100-fold greater in phosphate-limited cultures than in cultures in which phosphate was not limiting. PMID:8285702

  12. Biochemical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): catalytic properties.

    PubMed

    Wyss, M; Brugger, R; Kronenberger, A; Rémy, R; Fimbel, R; Oesterhelt, G; Lehmann, M; van Loon, A P

    1999-02-01

    Supplementation with phytase is an effective way to increase the availability of phosphorus in seed-based animal feed. The biochemical characteristics of an ideal phytase for this application are still largely unknown. To extend the biochemical characterization of wild-type phytases, the catalytic properties of a series of fungal phytases, as well as Escherichia coli phytase, were determined. The specific activities of the fungal phytases at 37 degreesC ranged from 23 to 196 U. (mg of protein)-1, and the pH optima ranged from 2.5 to 7.0. When excess phytase was used, all of the phytases were able to release five phosphate groups of phytic acid (myo-inositol hexakisphosphate), which left myo-inositol 2-monophosphate as the end product. A combination consisting of a phytase and Aspergillus niger pH 2.5 acid phosphatase was able to liberate all six phosphate groups. When substrate specificity was examined, the A. niger, Aspergillus terreus, and E. coli phytases were rather specific for phytic acid. On the other hand, the Aspergillus fumigatus, Emericella nidulans, and Myceliophthora thermophila phytases exhibited considerable activity with a broad range of phosphate compounds, including phenyl phosphate, p-nitrophenyl phosphate, sugar phosphates, alpha- and beta-glycerophosphates, phosphoenolpyruvate, 3-phosphoglycerate, ADP, and ATP. Both phosphate liberation kinetics and a time course experiment in which high-performance liquid chromatography separation of the degradation intermediates was used showed that all of the myo-inositol phosphates from the hexakisphosphate to the bisphosphate were efficiently cleaved by A. fumigatus phytase. In contrast, phosphate liberation by A. niger or A. terreus phytase decreased with incubation time, and the myo-inositol tris- and bisphosphates accumulated, suggesting that these compounds are worse substrates than phytic acid is. To test whether broad substrate specificity may be advantageous for feed application, phosphate

  13. Synthetic inositol phosphate analogs reveal that PPIP5K2 has a surface-mounted substrate capture site that is a target for drug discovery.

    PubMed

    Wang, Huanchen; Godage, Himali Y; Riley, Andrew M; Weaver, Jeremy D; Shears, Stephen B; Potter, Barry V L

    2014-05-22

    Diphosphoinositol pentakisphosphate kinase 2 (PPIP5K2) is one of the mammalian PPIP5K isoforms responsible for synthesis of diphosphoinositol polyphosphates (inositol pyrophosphates; PP-InsPs), regulatory molecules that function at the interface of cell signaling and organismic homeostasis. The development of drugs that inhibit PPIP5K2 could have both experimental and therapeutic applications. Here, we describe a synthetic strategy for producing naturally occurring 5-PP-InsP4, as well as several inositol polyphosphate analogs, and we study their interactions with PPIP5K2 using biochemical and structural approaches. These experiments uncover an additional ligand-binding site on the surface of PPIP5K2, adjacent to the catalytic pocket. This site facilitates substrate capture from the bulk phase, prior to transfer into the catalytic pocket. In addition to demonstrating a "catch-and-pass" reaction mechanism in a small molecule kinase, we demonstrate that binding of our analogs to the substrate capture site inhibits PPIP5K2. This work suggests that the substrate-binding site offers new opportunities for targeted drug design.

  14. 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

  15. Analysis of Dictyostelium discoideum Inositol Pyrophosphate Metabolism by Gel Electrophoresis

    PubMed Central

    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 revealed

  16. A femtomole-sensitivity mass assay for inositol hexakisphosphate.

    PubMed

    Letcher, Andrew J; Schell, Michael J; Irvine, Robin F

    2010-01-01

    Inositol hexakisphosphate (InsP(6)) is an important component of cells, and its mass levels are usually assayed by either (a) equilibrium labelling of cell cultures with radiolabelled inositol or (b) by a variety of mass assays of differing sensitivities and ambiguities. Here, we describe a mass assay for InsP(6) that is based on phosphorylating InsP(6) with [(32)P]-ATP to 5-(PP)InsP(5) using a recombinant Giardia InsP(6) kinase and quantification of the radiolabelled 5-[(32)P](PP)InsP(5) product by anion exchange HPLC with an internal [(3)H]-(PP)InsP(5) standard. Interference with the enzyme reaction by other factors in the tissue extract is corrected for by assay of identical aliquots of tissue spiked with known amounts of InsP(6). This assay only measures InsP(6) (and not other inositol phosphates), and although it is simple in principle and requires no dedicated or specialised equipment, it is quite time-consuming. But the assay is unambiguous and is capable of quantifying accurately as little as 10 fmol of InsP(6) in a cell extract.

  17. Enzymes of myo-inositol and inositol lipid metabolism in rats with streptozotocin-induced diabetes.

    PubMed Central

    Whiting, P H; Palmano, K P; Hawthorne, J N

    1979-01-01

    Diabetes, with only mild ketosis, was induced in male rats by a single injection of streptozotocin. After 12 weeks the specific activities of enzymes concerned with the metabolism of inositol and of inositol lipids were measured in various tissues. Inositol 1-phosphate synthase (EC 5.5.1.4) was most active in testis and the activity was significantly less in diabetic rats than in controls on a similar diet. Inositol oxygenase (EC 1.13.99.1), which converts myo-inositol into glucuronic acid, was also less active in kidney from diabetic animals. CDP-diacylglycerol-inositol phosphatidyltransferase (EC 2.7.8.11) and phosphatidylinositol 4-phosphate kinase (EC 2.7.1.68) showed decreased specific activities in brain and sciatic nerve of diabetic rats. By contrast the diabetic state did not affect the specific activities of phosphatidylinositol kinase (EC 2.7.1.67) or phosphatidylinositol 4,5-bisphosphate phosphatase (EC 3.1.3.36) in these tissues. The results are discussed in relation to diabetic neuropathy. PMID:224862

  18. Chronic treatment with lithium and pretreatment with excess inositol reduce inositol pool size in astrocytes by different mechanisms.

    PubMed

    Wolfson, M; Hertz, E; Belmaker, R H; Hertz, L

    1998-03-16

    Chronic treatment with a lithium salt is the classical treatment for manic-depressive disorder. It is hypothesized that the therapeutic action of lithium is caused by its inhibition of inositol phosphatases which leads to a relative deficiency of inositol and, therefore, an impairment of inositol recycling and production of precursor for the second messengers inositol triphosphate (IP3) and diacylglycerol (DAG). However, peculiarly enough, treatment with high doses of inositol also has an antidepressant effect. In the present work, we have studied the acute and chronic effects of lithium and of excess inositol, in separation or together, on accumulation of 50 microM [3H]inositol (a physiologically relevant concentration) into primary cultures of mouse astrocytes. Two parameters were investigated: (1) rate of unidirectional uptake across the cell membrane (measured during short-term exposure to the radioisotope), and (2) magnitude of the intracellular pool of inositol, equilibrating with extracellular inositol (measured during long-term exposure to the radioisotope). Inositol uptake was highly concentrative and occurred with a Km of approximately 500 microM and a Vmax of 1.5 nmol/min/mg protein. The uptake rate was not affected by either acute or chronic treatment with LiCl (or both), but it was substantially reduced ('down-regulated') after pretreatment with a high concentration of inositol. The inositol pool size was decreased to a similar extent as the uptake rate by previous exposure to excess inositol. In spite of the fact that inositol uptake rate was unaffected by lithium, the magnitude of the inositol pool was significantly decreased by chronic treatment with a pharmacologically relevant concentration of LiCl (1 mM), but not by treatment with lower concentrations. This decrease is likely to reflect a reduction in either inositol synthesis or replenishment of inositol from IP3, due to the inhibition of inositol phosphatases by the lithium ion. In agreement

  19. The Endoplasmic Reticulum Stress Sensor Inositol-Requiring Enzyme 1α Augments Bacterial Killing through Sustained Oxidant Production.

    PubMed

    Abuaita, Basel H; Burkholder, Kristin M; Boles, Blaise R; O'Riordan, Mary X

    2015-07-14

    Bacterial infection can trigger cellular stress programs, such as the unfolded protein response (UPR), which occurs when misfolded proteins accumulate within the endoplasmic reticulum (ER). Here, we used the human pathogen methicillin-resistant Staphylococcus aureus (MRSA) as an infection model to probe how ER stress promotes antimicrobial function. MRSA infection activated the most highly conserved unfolded protein response sensor, inositol-requiring enzyme 1α (IRE1α), which was necessary for robust bacterial killing in vitro and in vivo. The macrophage IRE1-dependent bactericidal activity required reactive oxygen species (ROS). Viable MRSA cells excluded ROS from the nascent phagosome and strongly triggered IRE1 activation, leading to sustained generation of ROS that were largely Nox2 independent. In contrast, dead MRSA showed early colocalization with ROS but was a poor activator of IRE1 and did not trigger sustained ROS generation. The global ROS stimulated by IRE1 signaling was necessary, but not sufficient, for MRSA killing, which also required the ER resident SNARE Sec22B for accumulation of ROS in the phagosomal compartment. Taken together, these results suggest that IRE1-mediated persistent ROS generation might act as a fail-safe mechanism to kill bacterial pathogens that evade the initial macrophage oxidative burst. Cellular stress programs have been implicated as important components of the innate immune response to infection. The role of the IRE1 pathway of the ER stress response in immune secretory functions, such as antibody production, is well established, but its contribution to innate immunity is less well defined. Here, we show that infection of macrophages with viable MRSA induces IRE1 activation, leading to bacterial killing. IRE1-dependent bactericidal activity required generation of reactive oxygen species in a sustained manner over hours of infection. The SNARE protein Sec22B, which was previously demonstrated to control ER

  20. Calcium, phosphate and calcium phosphate product are markers of outcome in patients with chronic heart failure.

    PubMed

    Cubbon, Richard M; Thomas, Ceri Haf; Drozd, Michael; Gierula, John; Jamil, Haqeel A; Byrom, Rowenna; Barth, Julian H; Kearney, Mark T; Witte, Klaus K A

    2015-04-01

    Serum calcium (Ca) and inorganic phosphate (Pi) concentrations and calcium-phosphate product (CPP) levels are positively associated with worse outcomes in patients with chronic kidney disease, but there are few data for Pi or Ca and none for CPP in patients with chronic heart failure (CHF). Unselected, consecutive patients with CHF (left ventricular ejection fraction, LVEF ≤45%) were enrolled in a prospective observational study for the occurrence of hospitalisation and mortality. Blood samples were collected at the time of recruitment and analysed immediately. Patients (n = 713) were on contemporary optimal treatment and mean (standard error, SE) follow-up was 765 (18.9) days. Mean (SE) Ca was 2.29 (0.004) mmol/l. Median (interquartile range, IQR) Pi was 1.11 (0.98-1.23) mmol/l and median CPP 2.53 (2.21-2.88) mmol(2)/l(2). LVEF correlated inversely with Ca, natural log-transformed (Ln)Pi, and LnCPP. There was no difference in CPP between classes of symptom severity or diabetes status. Ca and LnCPP (but not LnPi) were associated with total mortality. Ca was significantly associated with progressive HF and non-cardiovascular death but not with sudden death. Binary logistic regression analyses showed that LnPi and LnCPP were associated with risk of hospitalisation. Ca, Pi and CPP could be useful additional variables in determining risk in CHF patients. Further work is required to elucidate the mechanisms underlying the adverse influence and determine whether lowering phosphate levels per se in CHF patients is of benefit.

  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. 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.

  3. Effects of prostaglandin F2 alpha and a gonadotropin-releasing hormone agonist on inositol phospholipid metabolism in isolated rat corpora lutea of various ages

    SciTech Connect

    Lahav, M.; West, L.A.; Davis, J.S.

    1988-08-01

    The sensitivity of rat corpora lutea to luteolytic agents increases with luteal age. We examined the effect of prostaglandin F2 alpha (PGF2 alpha) and (D-Ala6,Des-Gly10)GnRH ethylamide (GnRHa) on inositol phospholipid metabolism in day 2 and day 7 corpora lutea from PMSG-treated rats. Isolated corpora lutea were incubated with 32PO4 or (3H)inositol and were treated with LH, PGF2 alpha, or GnRHa. Phospholipids were purified by TLC, and the water-soluble products of phospholipase-C activity (inositol phosphates) were isolated by ion exchange chromatography. In day 2 corpora lutea, PGF2 alpha, (10 microM) and GnRHa (100 ng/ml) significantly increased 32PO4 incorporation into phosphatidic acid (PA) and phosphatidylinositol (PI), but not into other fractions. LH provoked slight increases in PA. Results were similar with 30 min of prelabeling or simultaneous addition of 32PO4 and stimulants. In other experiments, PGF2 alpha and GnRHa provoked rapid increases (1-5 min) in the accumulation of inositol mono-, bis-, and trisphosphates. LH did not significantly increase inositol phosphate accumulation, but stimulated cAMP accumulation in 2-day-old corpora lutea. Inositol phospholipid metabolism was increased in day 7 corpora lutea compared to that in day 2 corpora lutea. This increase was associated with increased incorporation of 32PO4 into PA and PI and increased accumulation of (3H)inositol phosphates. In day 7 corpora lutea, which are very sensitive to the luteolytic effect of PGF2 alpha, the PG-induced increase in PA labeling was small and inconsistent, whereas PI labeling was unaffected in 30-min incubations. GnRHa was without effect in such corpora lutea. LH, PGF2 alpha, or GnRHa did not increase inositol phosphate accumulation in 7-day-old corpora lutea. These studies demonstrate that the transformation of young (day 2) to mature (day 7) corpora lutea is associated with an increase in luteal inositol phospholipid metabolism.

  4. Modulation of basal nitric oxide-dependent cyclic-GMP production by ambient glucose, myo-inositol, and protein kinase C in SH-SY5Y human neuroblastoma cells.

    PubMed Central

    Shindo, H; Thomas, T P; Larkin, D D; Karihaloo, A K; Inada, H; Onaya, T; Stevens, M J; Greene, D A

    1996-01-01

    Defective tissue perfusion and nitric oxide production and altered myo-inositol metabolism and protein kinase C activation have been invoked in the pathogenesis of diabetic complications including neuropathy. The precise cellular compartmentalization and mechanistic interrelationships of these abnormalities remain obscure, and nitric oxide possesses both neurotransmitter and vasodilator activity. Therefore the effects of ambient glucose and myo-inositol on nitric oxide-dependent cGMP production and protein kinase C activity were studied in SH-SY5Y human neuroblastoma cells, a cell culture model for peripheral cholinergic neurons. D-Glucose lowered cellular myo-inositol content, phosphatidylinositol synthesis, and phosphorylation of an endogenous protein kinase C substrate, and specifically reduced nitric oxide-dependent cGMP production a time- and dose-dependent manner with an apparent IC50 of approximately 30 mM. The near maximal decrease in cGMP induced by 50 mM D-glucose was corrected by the addition of protein kinase C agonists or 500 microM myo-inositol to the culture medium, and was reproduced by protein kinase C inhibition or downregulation, or by myo-inositol deficient medium. Sodium nitroprusside increased cGMP in a dose-dependent fashion, with low concentrations (1 microM) counteracting the effects of 50 mM D-glucose or protein kinase C inhibition. The demonstration that elevated D-glucose diminishes basal nitric oxide-dependent cGMP production by myo-inositol depletion and protein kinase C inhibition in peripheral cholinergic neurons provides a potential metabolic basis for impaired nitric oxide production, nerve blood flow, and nerve impulse conduction in diabetes. PMID:8609230

  5. A Novel Inositol Pyrophosphate Phosphatase in Saccharomyces cerevisiae

    PubMed Central

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

    2016-01-01

    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, the Saccharomyces cerevisiae homolog 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-IP5 or IP7) in vitro. In vivo, siw14Δ yeast mutants possess increased IP7 levels, whereas heterologous SIW14 overexpression eliminates IP7 from cells. IP7 levels increased proportionately when siw14Δ was combined with ddp1Δ or vip1Δ, 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 IP7 isoform 5PP-IP5 to IP6. PMID:26828065

  6. 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

  7. Dose assessment to workers in a dicalcium phosphate production plant.

    PubMed

    Mulas, D; Garcia-Orellana, J; Casacuberta, N; Hierro, A; Moreno, V; Masqué, P

    2016-12-01

    The production of dicalcium phosphate (DCP) uses phosphate rock (PR) as a raw material. Sedimentary phosphate rocks are enriched with relevant concentrations of natural radionuclides from the (238)U decay chain (around 10(3) Bq·kg(-1)), leading to the need of controlling potential exposures to radiation of workers and members of the public in accordance with IAEA safety standards. Indeed, phosphate industries are classified as Naturally Occurring Radioactive Material (NORM) industries. Thus, the aim of this work is to assess the radiological risk of the workers in a DCP production plant located in the Iberian Peninsula (South-West Europe), which digests PR with hydrochloric acid. In the present study (238)U, (230)Th, (222)Rn, (210)Pb and (210)Po concentrations in aerosols (indoor and outdoor areas) are reported. Aerosols showed concentrations between 0.42-92 mBq·m(-3) for (238)U, 0.24-33 mBq·m(-3) for (230)Th, 0.67-147 mBq·m(-3) for (210)Pb and 0.09-34 mBq·m(-3) for (210)Po. Long-term exposure (four months) of passive (222)Rn detectors provided concentrations that ranged from detection limit (< DL) to 121 Bq·m(-3) in outdoor areas and from < DL to 211 Bq·m(-3) in indoor areas, similar to concentrations obtained from short-term measurements with active detectors from < DL to 117 Bq·m(-3) in outdoor areas and from < DL to 318 Bq·m(-3) in indoor places. (226)Ra accumulation in ebonite and pipe scales were the most important contributions to the ambient dose equivalent H*(10), resulting in 0.07 (background)-27 μSv·h(-1) with a median value of 1.1 μSv·h(-1). Average (222)Rn air concentrations were lower than the 300 Bq·m(-3) limit and therefore, according to European Directive 2013/59/EURATOM, (222)Rn concentration is excluded from the worker operational annual effective dose. Thus, considering the inhalation of aerosols and the external dose sources, the total effective dose determined for plant operators was 0.37 mSv·y(-1). Copyright © 2016 Elsevier

  8. Salt-induced abnormalities on root tip mitotic cells of Allium cepa: prevention by inositol pretreatment.

    PubMed

    Chatterjee, Jolly; Majumder, Arun Lahiri

    2010-09-01

    Salt-induced growth reduction of plants is a well-known phenomenon which poses major problem in crop productivity in places where vast majority of land plants are affected by salt. In this report, studies were carried out to reveal the effect of salt injury on the cell division pattern in roots and the role of myo-inositol in preventing the salt-induced ion disequilibrium on the chromosome and DNA degradation in roots. Present study revealed induction of various chromosomal abnormalities on the root tip mitotic cells of Allium cepa by treatment with different concentrations of NaCl (0-500 mM) for 24 h as also the amelioration of such effect by prior treatment of the roots with different concentration of myo-inositol (0-300 mM). Results showed that a narrow albeit definite range of extracellular myo-inositol (100-150 mM) is effective in preventing internucleosomal fragmentation which is the early response in roots under salt stress. Transgenic tobacco plants overexpressing Oryza (OsINO1) as well as Porteresia (PcINO1) cytosolic L: -myo-inositol-1-phosphate synthase coding genes can withstand and retain their chromosomal and DNA integrity in 100 mM NaCl solution and can subsequently prevent DNA fragmentation, caused by intracellular endonuclease activity at this salt concentration.

  9. Resveratrol Stimulates Sphingosine-1-Phosphate Signaling of Cathelicidin Production

    PubMed Central

    Park, Kyungho; Elias, Peter M.; Hupe, Melanie; Borkowski, Andrew W.; Gallo, Richard L.; Shin, Kyong-Oh; Lee, Yong-Moon; Holleran, Walter M.; Uchida, Yoshikazu

    2013-01-01

    We recently discovered a regulatory mechanism that stimulates production of the multifunctional antimicrobial peptide, cathelicidin antimicrobial peptide (CAMP). In response to subtoxic levels of ER stress, increased sphingosine-1-phosphate (S1P) production activates an NFκB→C/EBPα dependent pathway that enhances CAMP production in cultured human keratinocytes. Since the multifunctional stilbenoid compound, resveratrol (RESV), increases ceramide (Cer) levels, a precursor of S1P, we hypothesized and assessed whether RESV could exploit the same pathway to regulate CAMP production. Accordingly, RESV significantly increased Cer and S1P levels in cultured keratinocytes, paralleled by increased CAMP mRNA/protein expression. Furthermore, topical RESV also increased murine CAMP mRNA/protein expression in mouse skin. Conversely, blockade of Cer→sphingosine→S1P metabolic conversion, with specific inhibitors of ceramidase or sphingosine kinase, attenuated the expected RESV-mediated increase in CAMP expression. The RESV-induced increase in CAMP expression required both NF-κB and C/EBPα transactivation. Moreover, conditioned media from keratinocyte treated with RESV significantly suppressed Staphylococcus aureus growth. Finally, topical RESV, if not coapplied with a specific inhibitor of sphingosine kinase, blocked Staphylococcus aureus invasion into murine skin. These results demonstrate that the dietary stilbenoid, RESV, stimulates S1P signaling of CAMP production through an NF-κB→C/EBPα-dependent mechanism, leading to enhanced antimicrobial defense against exogenous microbial pathogens. PMID:23856934

  10. Inositol phosphatase activity of the Escherichia coli agp-encoded acid glucose-1-phosphatase.

    PubMed

    Cottrill, Michael A; Golovan, Serguei P; Phillips, John P; Forsberg, Cecil W

    2002-09-01

    When screening an Escherichia coli gene library for myo-inositol hexakisphosphate (InsP6) phosphatases (phytases), we discovered that the agp-encoded acid glucose-1-phosphatase also possesses this activity. Purified Agp hydrolyzes glucose-1-phosphate, p-nitrophenyl phosphate, and InsP6 with pH optima, 6.5, 3.5, and 4.5, respectively, and was stable when incubated at pH values ranging from 3 to 10. Glucose-1-phosphate was hydrolyzed most efficiently at 55 degrees C. while InsP6 and p-nitrophenyl phosphate were hydrolyzed maximally at 60 degrees C. The Agp exhibited Km values of (0.39 mM, 13 mM, and 0.54 mM for the hydrolysis of glucose-1-phosphate, p-nitrophenyl phosphate, and InsP6, respectively. High-pressure liquid chromatography (HPLC) analysis of inositol phosphate hydrolysis products of Agp demonstrated that the enzyme catalyzes the hydrolysis of phosphate from each of InsP6, D-Ins(1,2,3,4,5)P5, Ins(1,3,4,5,6)P5, and Ins(1,2,3,4,6)P5, producing D/L-Ins(1,2,4,5,6)P5. D-Ins(1,2,4,5)P4, D/L-Ins(1,4,5,6)P4 and D/L-Ins(1,2,4,6)P4, respectively. These data support the contention that Agp is a 3-phosphatase.

  11. Fact Sheet - Phosphate Fertilizer Production Plants and Phosphoric Acid Manufacturing Plants NESHAP

    EPA Pesticide Factsheets

    Fact sheet summarizing National Emission Standards for Hazardous Air Pollutants (NESHAP) for Phosphate Fertilizer Production Plants and Phosphoric Acid Manufacturing Plants (40 CFR 63 Subparts AA and BB).

  12. Role of an Expanded Inositol Transporter Repertoire in Cryptococcus neoformans Sexual Reproduction and Virulence

    PubMed Central

    Xue, Chaoyang; Liu, Tongbao; Chen, Lydia; Li, Wenjun; Liu, Iris; Kronstad, James W.; Seyfang, Andreas; Heitman, Joseph

    2010-01-01

    ABSTRACT Cryptococcus neoformans and Cryptococcus gattii are globally distributed human fungal pathogens and the leading causes of fungal meningitis. Recent studies reveal that myo-inositol is an important factor for fungal sexual reproduction. That C. neoformans can utilize myo-inositol as a sole carbon source and the existence of abundant inositol in the human central nervous system suggest that inositol is important for Cryptococcus development and virulence. In accord with this central importance of inositol, an expanded myo-inositol transporter (ITR) gene family has been identified in Cryptococcus. This gene family contains two phylogenetically distinct groups, with a total of 10 or more members in C. neoformans and at least six members in the sibling species C. gattii. These inositol transporter genes are differentially expressed under inositol-inducing conditions based on quantitative real-time PCR analyses. Expression of ITR genes in a Saccharomyces cerevisiae itr1 itr2 mutant lacking inositol transport can complement the slow-growth phenotype of this strain, confirming that ITR genes are bona fide inositol transporters. Gene mutagenesis studies reveal that the Itr1 and Itr1A transporters are important for myo-inositol stimulation of mating and that functional redundancies among the myo-inositol transporters likely exist. Deletion of the inositol 1-phosphate synthase gene INO1 in an itr1 or itr1a mutant background compromised virulence in a murine inhalation model, indicating the importance of inositol sensing and acquisition for fungal infectivity. Our study provides a platform for further understanding the roles of inositol in fungal physiology and virulence. PMID:20689743

  13. 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.

  14. Structural analysis of a multifunctional, tandemly repeated inositol polyphosphatase.

    PubMed

    Gruninger, Robert J; Selinger, L Brent; Mosimann, Steven C

    2009-09-11

    Mitsuokella multacida expresses a unique inositol polyphosphatase (PhyAmm) that is composed of tandem repeats (TRs). Each repeat possesses a protein tyrosine phosphatase (PTP) active-site signature sequence and fold. Using a combination of structural, mutational, and kinetic studies, we show that the N-terminal (D1) and C-terminal (D2) active sites of the TR have diverged and possess significantly different specificities for inositol polyphosphate. Structural analysis and molecular docking calculations identify steric and electrostatic differences within the substrate binding pocket of each TR that may be involved in the altered substrate specificity. The implications of our results for the biological function of related PTP-like phytases are discussed. Finally, the structures and activities of PhyAmm and tandemly repeated receptor PTPs are compared and discussed. To our knowledge, this is the first example of an inositol phosphatase with tandem PTP domains possessing substrate specificity for different inositol phosphates.

  15. Radioactive contamination in Croatia by phosphate fertilizer production.

    PubMed

    Bituh, Tomislav; Marovic, Gordana; Franic, Zdenko; Sencar, Jasminka; Bronzovic, Maja

    2009-03-15

    The contents of natural radionuclides (radium, uranium and potassium) were measured in the area of a phosphate fertilizer factory in central Croatia, as a part of extended and still ongoing monitoring program of radioactive contamination of human environment in Croatia that is performed by the Radiation Protection Unit of the Institute for Medical Research and Occupational Health in Zagreb. Activity concentrations in all analysed media (waste water, trickling water from piezometers, phosphogypsum deposit and final products) considerably fluctuated, especially in phosphogypsum and waste water. Mean (226)Ra activity concentration in waste phosphogypsum was measured to be 483+/-190 Bqkg(-1). Based on that value, it was estimated that 4 million m(3) of phosphogypsum that have been deposited up to now contain about 4.3 x 10(12) Bq, i.e. about 200 g of (226)Ra. However, effective dose for an adult that would be incurred by consumption of water from nearby wells was estimated to be 5.3+/-1.3 microSv. The results show that (226)Ra activities cause effective doses, which are below the recommended maximum as the estimated annual (226)Ra effective dose does not exceed 0.1 mSv as recommended by the World Health Organization.

  16. Production of Calcium Phosphate Nanoparticles by Laser Ablation in Liquid

    NASA Astrophysics Data System (ADS)

    Boutinguiza, M.; Pou, J.; Lusquiños, F.; Comesaña, R.; Riveiro, A.

    Calcium phosphate nanoparticles represent an important object of investigation in the field of biomaterials due to the new properties obtainable at nanoscale. In this work calcium phosphate nanoparticles are obtained by laser ablation of hydroxyapatite (HA) targets in water and in ambient conditions; on the other hand particles of HA are reduced to nanoscale by laser-induced fracture. The results show that nanometric particles of calcium phosphate can be obtained. The morphology and the composition of the obtained particles were characterized by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX) and conventional and high resolution transmission electron microscopy (TEM, HRTEM).

  17. Insulin stimulates the biosynthesis of chiro-inositol-containing phospholipids in a rat fibroblast line expressing the human insulin receptor.

    PubMed Central

    Pak, Y; Paule, C R; Bao, Y D; Huang, L C; Larner, J

    1993-01-01

    HIRc-B cells (rat fibroblasts expressing the human insulin receptor) were incubated with myo-[3H]inositol for 48 hr, and the biosynthesis of chiro-[3H]inositol was investigated in the absence and presence of insulin following a time course up to 60 min. After phase separation, treatment with insulin for 15 min caused a 2.2-fold increase in the specific radioactivity of chiro-[3H]inositol-containing phospholipids in contrast to a 1.2-fold increase in the specific radioactivity of myo-[3H]inositol-containing phospholipids. No insulin-mediated change in the specific radioactivity was observed in the inositol phosphates or free inositols. Further detailed analysis of individual [3H]inositol-containing phospholipids demonstrated marked increases in specific activity of the chiro-[3H]inositol phospholipids after 15 min of incubation with insulin: phosphatidylinositol 4-phosphate and 4,5-bisphosphate, 4.2-fold; lysophosphatidylinositol, 1.5-fold; phosphatidylinositol, 3.2-fold. In contrast, myo-[3H]inositol-containing phospholipids demonstrated relatively small increases (1.1- to 1.4-fold) after 5 min of incubation with insulin. These findings indicate that insulin stimulates de novo synthesis of chiro-inositol-containing phospholipids at the inositol phospholipid level. Images Fig. 2 Fig. 3 PMID:8356081

  18. myo-Inositol homeostasis in foetal rabbit lung

    PubMed Central

    Bleasdale, John E.; Maberry, Mark C.; Quirk, J. Gerald

    1982-01-01

    In several species, lung maturation is accompanied by a decline in the phosphatidylinositol content of lung surfactant and a concomitant increase in its phosphatidylglycerol content. To examine the possibility that this developmental change is influenced by the availability of myo-inositol, potential sources of myo-inositol for the developing rabbit lung were investigated. On day 28 of gestation the myo-inositol content of foetal rabbit lung tissue (2.3±0.5μmol/g of tissue) was not significantly different from that of adult lung tissue but the activity of d-glucose 6-phosphate:1l-myo-inositol 1-phosphate cyclase (cyclase) in foetal lung tissue (81.0±9.0nmol·h−1·g of tissue−1) was higher than that found in adult lung tissue (23.2±1.0nmol·h−1·g of tissue−1). Day 28 foetal rabbit lung tissue was found also to take up myo-inositol by a specific, energy-dependent, Na+-requiring mechanism. Half-maximal uptake of myo-inositol by foetal rabbit lung slices was observed when the concentration of myo-inositol in the incubation medium was 85μm. When the myo-inositol concentration was 1mm (but not 100μm) the addition of glucose (5.5mm) stimulated myo-inositol uptake. myo-Inositol uptake was observed also in adult rabbit lung and was found to be sub-maximal at the concentration of myo-inositol found in adult rabbit serum. The concentration of myo-inositol in the serum of pregnant adult rabbits (47.5±5.5μm) was significantly lower than that of non-pregnant adult female rabbits (77.9±9.2μm). On day 28 of gestation the concentration of myo-inositol in foetal serum (175.1±12.0μm) was much less than on day 25, but more than that found on day 30. A transient post-partum increase in the concentration of myo-inositol in serum was followed by a rapid decline. Much of the myo-inositol in foetal rabbit serum probably originates from the placenta, where on day 28 of gestation a high cyclase activity (527±64nmol·h−1·g of tissue−1) was measured. The gestational

  19. 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...

  20. 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 1...

  1. 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...

  2. 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 1...

  3. 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 1...

  4. Polymeric calcium phosphate cements: analysis of reaction products and properties.

    PubMed

    Miyazaki, K; Horibe, T; Antonucci, J M; Takagi, S; Chow, L C

    1993-01-01

    Chemical and mechanical properties of water-based polymeric calcium phosphate cements (PCPC) were investigated. These cements were derived from mixing several types of water-soluble polymers, e.g., gelatin, poly(vinyl alcohol) (PVA), and poly(alkenoic acids) such as poly(acrylic acid), with a calcium phosphate cement (CPC) mixture consisting of equimolar amounts of tetracalcium phosphate (TTCP) and anhydrous dicalcium phosphate (DCPA) as well as several other TTCP-containing mixtures. Cement formation was observed with all of the PCPCs. With the gelatin and PVA cements, significant amounts of hydroxyapatite (HA) formation were observed within 24 h. Their setting times and mechanical properties were similar to those of the purely inorganic CPC that is derived from the reaction of TTCP and DCPA in water. Although the mechanical properties of a gelatin-CPC cement were only slightly improved, its handling characteristics were superior to that of CPC. Significantly faster setting and stronger cements were obtained using polycarboxylic acid polymers with CPC. However, only small amounts of HA were observed in these types of polymeric cements even after 1 mon storage in distilled water at 37 degrees C. This research demonstrates the feasibility of preparing several new types of dental cements based on the interaction of water-soluble polymers with a self-setting calcium phosphate powder mixture.

  5. Brain inositol is a novel stimulator for promoting Cryptococcus penetration of the blood-brain barrier.

    PubMed

    Liu, Tong-Bao; Kim, Jong-Chul; Wang, Yina; Toffaletti, Dena L; Eugenin, Eliseo; Perfect, John R; Kim, Kee Jun; Xue, Chaoyang

    2013-01-01

    Cryptococcus neoformans is the most common cause of fungal meningitis, with high mortality and morbidity. The reason for the frequent occurrence of Cryptococcus infection in the central nervous system (CNS) is poorly understood. The facts that human and animal brains contain abundant inositol and that Cryptococcus has a sophisticated system for the acquisition of inositol from the environment suggests that host inositol utilization may contribute to the development of cryptococcal meningitis. In this study, we found that inositol plays an important role in Cryptococcus traversal across the blood-brain barrier (BBB) both in an in vitro human BBB model and in in vivo animal models. The capacity of inositol to stimulate BBB crossing was dependent upon fungal inositol transporters, indicated by a 70% reduction in transmigration efficiency in mutant strains lacking two major inositol transporters, Itr1a and Itr3c. Upregulation of genes involved in the inositol catabolic pathway was evident in a microarray analysis following inositol treatment. In addition, inositol increased the production of hyaluronic acid in Cryptococcus cells, which is a ligand known to binding host CD44 receptor for their invasion. These studies suggest an inositol-dependent Cryptococcus traversal of the BBB, and support our hypothesis that utilization of host-derived inositol by Cryptococcus contributes to CNS infection.

  6. 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.

  7. The absence of expression of the three isoenzymes of the inositol 1,4,5-trisphosphate 3-kinase does not prevent the formation of inositol pentakisphosphate and hexakisphosphate in mouse embryonic fibroblasts.

    PubMed

    Leyman, Alexandre; Pouillon, Valérie; Bostan, Alionka; Schurmans, Stéphane; Erneux, Christophe; Pesesse, Xavier

    2007-07-01

    The activation of phospholipase C leads to the formation of both I(1,4,5)P(3) and diacylglycerol (DAG). I(1,4,5)P(3) can be metabolized by dephosphorylation catalyzed by Type I I(1,4,5)P(3) 5-phosphatase and by enzymatic phosphorylation to various inositol phosphates. This last step is catalyzed by three mammalian isoenzymes that specifically phosphorylate the 3-phosphate position of the inositol ring Itpka, Itpkb and Itpkc and a less specific enzyme Ipmk (or inositol multikinase) that phosphorylates I(1,4,5)P(3) at the D-3 and D-6 positions. This study was performed in mice cells in order to understand the synthetic pathway of IP5 and IP6 following PLC stimulation and possible link with Itpk activity. Mouse embryonic fibroblasts (MEF) were prepared from Itpkb(-/-) Itpkc(-/-) mice. Western blot and RT-PCR analysis show that the cells do not express Itpka. In contrast, they do express Ipmk. The cells still produce IP5 and IP6. Our data show that the absence of expression of the three isoenzymes of Itpk does not prevent the formation of IP5 and IP6, at least in mouse embryonic fibroblasts. The nuclear Ipmk plays therefore a critical role in the metabolism of I(1,4,5)P(3) and production of highly phosphorylated IP5 and IP6.

  8. Methane production by phosphate-starved SAR11 chemoheterotrophic marine bacteria.

    PubMed

    Carini, Paul; White, Angelicque E; Campbell, Emily O; Giovannoni, Stephen J

    2014-07-07

    The oxygenated surface waters of the world's oceans are supersaturated with methane relative to the atmosphere, a phenomenon termed the 'marine methane paradox'. The production of methylphosphonic acid (MPn) by marine archaea related to Nitrosopumilus maritimus and subsequent decomposition of MPn by phosphate-starved bacterioplankton may partially explain the excess methane in surface waters. Here we show that Pelagibacterales sp. strain HTCC7211, an isolate of the SAR11 clade of marine α-proteobacteria, produces methane from MPn, stoichiometric to phosphorus consumption, when starved for phosphate. Gene transcripts encoding phosphonate transport and hydrolysis proteins are upregulated under phosphate limitation, suggesting a genetic basis for the methanogenic phenotype. Strain HTCC7211 can also use 2-aminoethylphosphonate and assorted phosphate esters for phosphorus nutrition. Despite strain-specific differences in phosphorus utilization, these findings identify Pelagibacterales bacteria as a source of biogenic methane and further implicate phosphate starvation of chemoheterotrophic bacteria in the long-observed methane supersaturation in oxygenated waters.

  9. Biphasic products of dicalcium phosphate-rich cement with injectability and nondispersibility.

    PubMed

    Ko, Chia-Ling; Chen, Jian-Chih; Hung, Chun-Cheng; Wang, Jen-Chyan; Tien, Yin-Chun; Chen, Wen-Cheng

    2014-06-01

    In this study, a calcium phosphate cement was developed using tetracalcium phosphate and surface-modified dicalcium phosphate anhydrous (DCPA). This developed injectable bone graft substitute can be molded to the shape of the bone cavity and set in situ through the piping system that has an adequate mechanical strength, non-dispersibility, and biocompatibility. The materials were based on the modified DCPA compositions of calcium phosphate cement (CPC), where the phase ratio of the surface-modified DCPA is higher than that of the conventional CPC for forming dicalcium phosphate (DCP)-rich cement. The composition and morphology of several calcium phosphate cement specimens during setting were analyzed via X-ray diffractometry and transmission electron microscopy coupled with an energy dispersive spectroscopy system. The compressive strength of DCP-rich CPCs was greater than 30MPa after 24h of immersion in vitro. The reaction of the CPCs produced steady final biphasic products of DCPs with apatite. The composites of calcium phosphate cements derived from tetracalcium phosphate mixed with surface-modified DCPA exhibited excellent mechanical properties, injectability, and interlocking forces between particles, and they also featured nondispersive behavior when immersed in a physiological solution.

  10. Distribution of natural radionuclides in the production and use of phosphate fertilizers in Brazil.

    PubMed

    Saueia, C H R; Mazzilli, B P

    2006-01-01

    The Brazilian phosphate fertilizer is obtained by wet reaction of igneous phosphate rock with concentrated sulphuric acid, giving as final product, phosphoric acid and dehydrated calcium sulphate (phosphogypsum) as by-products. Phosphoric acid is the starting material for triple superphosphate (TSP), single superphosphate (SSP), monoammonium phosphate (MAP) and diammonium phosphate (DAP). The phosphate rock used as raw material presents in its composition radionuclides of the U and Th natural series. Taking this into account, the main aim of this paper is to evaluate the fluxes of natural radionuclides and radioactive disequilibria involved in the Brazilian industrial process of phosphoric acid production; to determine the content of radioactivity in several commercial fertilizers produced by this industry; to estimate their radiological impact in crop soils and the long term exposure due to their application. Radiological characterization of phosphate rock, phosphogypsum and phosphate fertilizers was performed by alpha and gamma spectrometry. The fertilizer samples, which are derived directly from phosphoric acid, MAP and DAP, presented in their composition low activity concentrations for 226Ra, 228Ra and 210Pb. As for U and Th, the concentrations found in MAP and DAP are more significant, up to 822 and 850Bqkg(-1), respectively. SSP and TSP, which are obtained by mixing phosphoric acid with different amounts of phosphate rock, presented higher concentrations of radionuclides, up to 1158Bqkg(-1) for (238)U, 1167Bqkg(-1) for (234)U, 1169Bqkg(-1) for 230Th, 879Bqkg(-1) for 226Ra, 1255Bqkg(-1) for 210Pb, 521Bqkg(-1) for 232Th, 246Bqkg(-1) for 228Ra and 302Bqkg(-1) for 228Th. Long term exposure due to successive fertilizer applications was evaluated. Internal doses due to the application of phosphate fertilizer for 10, 50 and 100 years were below 1mSvy(-1), showing that the radiological impact of such practice is negligible.

  11. 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

  12. Protein pyrophosphorylation by inositol pyrophosphates is a posttranslational event

    PubMed Central

    Bhandari, Rashna; Saiardi, Adolfo; Ahmadibeni, Yousef; Snowman, Adele M.; Resnick, Adam C.; Kristiansen, Troels Z.; Molina, Henrik; Pandey, Akhilesh; Werner, J. Kent; Juluri, Krishna R.; Xu, Yong; Prestwich, Glenn D.; Parang, Keykavous; Snyder, Solomon H.

    2007-01-01

    In a previous study, we showed that the inositol pyrophosphate diphosphoinositol pentakisphosphate (IP7) physiologically phosphorylates mammalian and yeast proteins. We now report that this phosphate transfer reflects pyrophosphorylation. Thus, proteins must be prephosphorylated by ATP to prime them for IP7 phosphorylation. IP7 phosphorylates synthetic phosphopeptides but not if their phosphates have been masked by methylation or pyrophosphorylation. Moreover, IP7 phosphorylated peptides are more acid-labile and more resistant to phosphatases than ATP phosphorylated peptides, indicating a different type of phosphate bond. Pyrophosphorylation may represent a novel mode of signaling to proteins. PMID:17873058

  13. Inositol polyphosphate phosphatases in human disease.

    PubMed

    Hakim, Sandra; Bertucci, Micka C; Conduit, Sarah E; Vuong, David L; Mitchell, Christina A

    2012-01-01

    Phosphoinositide signalling molecules interact with a plethora of effector proteins to regulate cell proliferation and survival, vesicular trafficking, metabolism, actin dynamics and many other cellular functions. The generation of specific phosphoinositide species is achieved by the activity of phosphoinositide kinases and phosphatases, which phosphorylate and dephosphorylate, respectively, the inositol headgroup of phosphoinositide molecules. The phosphoinositide phosphatases can be classified as 3-, 4- and 5-phosphatases based on their specificity for dephosphorylating phosphates from specific positions on the inositol head group. The SAC phosphatases show less specificity for the position of the phosphate on the inositol ring. The phosphoinositide phosphatases regulate PI3K/Akt signalling, insulin signalling, endocytosis, vesicle trafficking, cell migration, proliferation and apoptosis. Mouse knockout models of several of the phosphoinositide phosphatases have revealed significant physiological roles for these enzymes, including the regulation of embryonic development, fertility, neurological function, the immune system and insulin sensitivity. Importantly, several phosphoinositide phosphatases have been directly associated with a range of human diseases. Genetic mutations in the 5-phosphatase INPP5E are causative of the ciliopathy syndromes Joubert and MORM, and mutations in the 5-phosphatase OCRL result in Lowe's syndrome and Dent 2 disease. Additionally, polymorphisms in the 5-phosphatase SHIP2 confer diabetes susceptibility in specific populations, whereas reduced protein expression of SHIP1 is reported in several human leukaemias. The 4-phosphatase, INPP4B, has recently been identified as a tumour suppressor in human breast and prostate cancer. Mutations in one SAC phosphatase, SAC3/FIG4, results in the degenerative neuropathy, Charcot-Marie-Tooth disease. Indeed, an understanding of the precise functions of phosphoinositide phosphatases is not only

  14. Stereo- and regiospecificity of yeast phytases-chemical synthesis and enzymatic conversion of the substrate analogues neo- and L-chiro-inositol hexakisphosphate.

    PubMed

    Adelt, Stephan; Podeschwa, Michael; Dallmann, Guido; Altenbach, Hans-Josef; Vogel, Günter

    2003-02-01

    Phytases are enzymes that catalyze the hydrolysis of phosphate esters in myo-inositol hexakisphosphate (phytic acid). The precise routes of enzymatic dephosphorylation by phytases of the yeast strains Saccharomyces cerevisiae and Pichia rhodanensis have been investigated up to the myo-inositol trisphosphate level, including the absolute configuration of the intermediates. Stereoselective assignment of the myo-inositol pentakisphosphates (D-myo-inositol 1,2,4,5,6-pentakisphosphate and D-myo-inositol 1,2,3,4,5-pentakisphosphate) generated was accomplished by a new method based on enantiospecific enzymatic conversion and HPLC analysis. Via conduritol B or E derivatives the total syntheses of two epimers of myo-inositol hexakisphosphate, neo-inositol hexakisphosphate and L-chiro-inositol hexakisphosphate were performed to examine the specificity of the yeast phytases with these substrate analogues. A comparison of kinetic data and the degradation pathways determined gave the first hints about the molecular recognition of inositol hexakisphosphates by the enzymes. Exploitation of the high stereo- and regiospecificity observed in the dephosphorylation of neo- and L-chiro-inositol hexakisphosphate made it possible to establish enzyme-assisted steps for the synthesis of D-neo-inositol 1,2,5,6-tetrakisphosphate, L-chiro-inositol 1,2,3,5,6-pentakisphosphate and L-chiro-inositol 1,2,3,6-tetrakisphosphate.

  15. Organic acid production in vitro and plant growth promotion in maize under controlled environment by phosphate-solubilizing fluorescent Pseudomonas

    PubMed Central

    2009-01-01

    Background Phosphorus deficiency is a major constraint to crop production due to rapid binding of the applied phosphorus into fixed forms not available to the plants. Microbial solubilization of inorganic phosphates has been attributed mainly to the production of organic acids. Phosphate-solubilizing microorganisms enhance plant growth under conditions of poor phosphorus availability by solubilizing insoluble phosphates in the soil. This paper describes the production of organic acids during inorganic phosphate solubilization and influence on plant growth as a function of phosphate solubilization by fluorescent Pseudomonas. Results Nineteen phosphate-solubilizing fluorescent Pseudomonas strains of P. fluorescens, P. poae, P. trivialis, and Pseudomonas spp. produced gluconic acid, oxalic acid, 2-ketogluconic acid, lactic acid, succinic acid, formic acid, citric acid and malic acid in the culture filtrates during the solubilization of tricalcium phosphate, Mussoorie rock phosphate, Udaipur rock phosphate and North Carolina rock phosphate. The strains differed quantitatively and qualitatively in the production of organic acids during solubilization of phosphate substrates. Cluster analysis based on organic acid profiling revealed inter-species and intra-species variation in organic acids produced by Pseudomonas strains. The phosphate-solubilizing bacterial treatments P. trivialis BIHB 745, P. trivialis BIHB 747, Pseudomonas sp. BIHB 756 and P. poae BIHB 808 resulted in significantly higher or statistically at par growth and total N, P and K content over single super phosphate treatment in maize. These treatments also significantly affected pH, organic matter, and N, P, and K content of the soil. Conclusion The results implied that organic acid production by Pseudomonas strains is independent of their genetic relatedness and each strain has its own ability of producing organic acids during the solubilization of inorganic phosphates. Significant difference in plant

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

    USDA-ARS?s Scientific Manuscript database

    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...

  17. Analysis of myo-inositol hexakisphosphate hydrolysis by Bacillus phytase: indication of a novel reaction mechanism.

    PubMed

    Kerovuo, J; Rouvinen, J; Hatzack, F

    2000-12-15

    Phytic acid (myo-inositol hexakisphosphate, InsP(6)) hydrolysis by Bacillus phytase (PhyC) was studied. The enzyme hydrolyses only three phosphates from phytic acid. Moreover, the enzyme seems to prefer the hydrolysis of every second phosphate over that of adjacent ones. Furthermore, it is very likely that the enzyme has two alternative pathways for the hydrolysis of phytic acid, resulting in two different myo-inositol trisphosphate end products: Ins(2,4,6)P(3) and Ins(1,3,5)P(3). These results, together with inhibition studies with fluoride, vanadate, substrate and a substrate analogue, indicate a reaction mechanism different from that of other phytases. By combining the data presented in this study with (1) structural information obtained from the crystal structure of Bacillus amyloliquefaciens phytase [Ha, Oh, Shin, Kim, Oh, Kim, Choi and Oh (2000) Nat. Struct. Biol. 7, 147-153], and (2) computer-modelling analyses of enzyme-substrate complexes, a novel mode of phytic acid hydrolysis is proposed.

  18. Inhibition of the formation of oral calcium phosphate precipitates: the possible effects of certain honeybee products.

    PubMed

    Hidaka, S; Okamoto, Y; Ishiyama, K; Hashimoto, K

    2008-08-01

    We studied the effects of honeybee products on the in vitro formation of calcium phosphate precipitates. Screening tests of the in vitro formation of calcium phosphate precipitates using 20 types of honey and four types of propolis were carried out using the pH drop method. The inhibitory effect on the rate of amorphous calcium phosphate transformation to hydroxyapatite and on the induction time varied greatly among the 20 types of honey and four types of propolis. We classified them according to their effects on decreasing the rate of amorphous calcium phosphate transformation to hydroxyapatite and/or increasing the induction time. Two of the 20 honeys showed little or no inhibition, either on the rate of amorphous calcium phosphate transformation to hydroxyapatite or on the induction time. Six of the honeys reduced the rate of amorphous calcium phosphate transformation to hydroxyapatite by 12-35% and with a 2.5- to 4.4-fold increase in the induction time. The remaining 12 honeys showed even greater activity. Because four of these 12 honeys had an inhibitory effect on the rate of amorphous calcium phosphate formation, they were excluded as candidates for anticalculus agents. Furthermore, three of the four types of propolis showed an inhibitory effect that was the same as or greater than 1-hydroxyethylidene- 1,1-bisphosphonate. These results suggest that eight honeys and three types of propolis may have potential as anticalculus agents in toothpastes and mouthwashes.

  19. Specificity determinants in phosphoinositide dephosphorylation: crystal structure of an archetypal inositol polyphosphate 5-phosphatase.

    PubMed

    Tsujishita, Y; Guo, S; Stolz, L E; York, J D; Hurley, J H

    2001-05-04

    Inositol polyphosphate 5-phosphatases are central to intracellular processes ranging from membrane trafficking to Ca(2+) signaling, and defects in this activity result in the human disease Lowe syndrome. The 1.8 resolution structure of the inositol polyphosphate 5-phosphatase domain of SPsynaptojanin bound to Ca(2+) and inositol (1,4)-bisphosphate reveals a fold and an active site His and Asp pair resembling those of several Mg(2+)-dependent nucleases. Additional loops mediate specific inositol polyphosphate contacts. The 4-phosphate of inositol (1,4)-bisphosphate is misoriented by 4.6 compared to the reactive geometry observed in the apurinic/apyrimidinic endonuclease 1, explaining the dephosphorylation site selectivity of the 5-phosphatases. Based on the structure, a series of mutants are described that exhibit altered substrate specificity providing general determinants for substrate recognition.

  20. Phosphate enhances levan production in the endophytic bacterium Gluconacetobacter diazotrophicus Pal5

    PubMed Central

    Idogawa, Nao; Amamoto, Ryuta; Murata, Kousaku; Kawai, Shigeyuki

    2014-01-01

    Gluconacetobacter diazotrophicus is a gram-negative and endophytic nitrogen-fixing bacterium that has several beneficial effects in host plants; thus, utilization of this bacterium as a biofertilizer in agriculture may be possible. G. diazotrophicus synthesizes levan, a D-fructofuranosyl polymer with β-(2→6) linkages, as an exopolysaccharide and the synthesized levan improves the stress tolerance of the bacterium. In this study, we found that phosphate enhances levan production by G. diazotrophicus Pal5, a wild type strain that showed a stronger mucous phenotype on solid medium containing 28 mM phosphate than on solid medium containing 7 mM phosphate. A G. diazotrophicus Pal5 levansucrase disruptant showed only a weak mucous phenotype regardless of the phosphate concentration, indicating that the mucous phenotype observed on 28 mM phosphate medium was caused by levan. To our knowledge, this is the first report of the effect of a high concentration of phosphate on exopolysaccharide production. PMID:24717418

  1. Do mammals make all their own inositol hexakisphosphate?

    PubMed

    Letcher, Andrew J; Schell, Michael J; Irvine, Robin F

    2008-12-01

    A highly specific and sensitive mass assay for inositol hexakisphosphate (InsP6) was characterized. This centres around phosphorylating InsP6 with [32P]ATP using a recombinant InsP6 kinase from Giardia lambia, followed by HPLC of the 32P-labelled products with an internal [3H]InsP7 standard. This assay was used to quantify InsP6 levels in a variety of biological samples.Concentrations of InsP6 in rat tissues varied from 10-20 microM (assuming 64% of wet weight of tissue is cytosol water), whereas using the same assumption axenic Dictyostelium discoideum cells contained 352 +/- 11 microM InsP6. HeLa cells were seeded at low density and grown to confluence, at which point they contained InsP6 levels per mg of protein similar to rat tissues. This amounted to 1.952 +/- 0.117 nmol InsP6 per culture dish, despite the cells being grown in serum shown to contain no detectable(less than 20 pmol per dish) InsP6. These results demonstrate that mammalian cells synthesize all their own InsP6. Human blood was analysed, and although the white cell fraction contained InsP6 at a concentration comparable with other tissues, in serum and platelet-free plasma no InsP6 was detected (<1 nM InsP6). Human urine was also examined, and also contained no detectable (<5 nM) InsP6. These results suggest that dietary studies purporting to measure InsP6 at micromolar concentrations in human plasma or urine may not have been quantifying this inositol phosphate. Therefore claims that administrating InsP6 in the diet or applying it topically can produce health benefits by increasing extracellular InsP6 levels may require reassessment.

  2. Production of high-quality liquid fertilizers from wet-process acid via urea phosphate

    SciTech Connect

    Hodge, C.A.; Motes, T.W.

    1991-07-01

    A pilot-plant process is described that purifies wet-process phosphoric acid for the production of a high-quality urea-ammonium polyphosphate base solution. An intermediate product, crystalline urea phosphate, is produced from urea and merchant-grade (54% P{sub 2}O{sub 5}) wet-process phosphoric acid. The urea phosphate crystals contain only about 15 to 20% of the objectional impurities (iron, aluminum, and magnesium) originally contained in the feed wet-process acid. The urea phosphate crystals are pyrolyzed, converting orthophosphate to polyphosphate with very little energy consumption. The resulting melt is dissolved and neutralized with ammonia to produce 14-29-0 liquid product of high polyphosphate content.

  3. Production of high-quality liquid fertilizers from wet-process acid via urea phosphate

    SciTech Connect

    Hodge, C.A.; Motes, T.W.

    1991-07-01

    A pilot-plant process is described that purifies wet-process phosphoric acid for the production of a high-quality urea-ammonium polyphosphate base solution. An intermediate product, crystalline urea phosphate, is produced from urea and merchant-grade (54% P[sub 2]O[sub 5]) wet-process phosphoric acid. The urea phosphate crystals contain only about 15 to 20% of the objectional impurities (iron, aluminum, and magnesium) originally contained in the feed wet-process acid. The urea phosphate crystals are pyrolyzed, converting orthophosphate to polyphosphate with very little energy consumption. The resulting melt is dissolved and neutralized with ammonia to produce 14-29-0 liquid product of high polyphosphate content.

  4. PROCESS USING BISMUTH PHOSPHATE AS A CARRIER PRECIPITATE FOR FISSION PRODUCTS AND PLUTONIUM VALUES

    DOEpatents

    Finzel, T.G.

    1959-03-10

    A process is described for separating plutonium from fission products carried therewith when plutonium in the reduced oxidation state is removed from a nitric acid solution of irradiated uranium by means of bismuth phosphate as a carrier precipitate. The bismuth phosphate carrier precipitate is dissolved by treatment with nitric acid and the plutonium therein is oxidized to the hexavalent oxidation state by means of potassium dichromate. Separation of the plutonium from the fission products is accomplished by again precipitating bismuth phosphate and removing the precipitate which now carries the fission products and a small percentage of the plutonium present. The amount of plutonium carried in this last step may be minimized by addition of sodium fluoride, so as to make the solution 0.03N in NaF, prior to the oxidation and prccipitation step.

  5. Participation of phosphatidyl inositol 3-kinase/protein kinase B and ERK1/2 pathways in interleukin-1beta stimulation of lactate production in Sertoli cells.

    PubMed

    Riera, María Fernanda; Galardo, María Noel; Pellizzari, Eliana Herminia; Meroni, Silvina Beatriz; Cigorraga, Selva Beatriz

    2007-04-01

    Interleukin-1beta (IL1beta ) belongs to a set of intratesticular regulators that provide the fine-tuning of cellular processes implicated in the maintenance of spermatogenesis. The aim of the present study was to analyze the signaling pathways that may participate in IL1beta regulation of Sertoli cell function. Sertoli cell cultures from 20-day-old rat were used. Stimulation of the cultures with IL1beta showed increments in phosphorylated protein kinase B (PKB), P70S6K, and ERK1/2 levels. A phosphatidyl inositol 3-kinase (PI3K) inhibitor (wortmannin (W)), a mammalian target of rapamycin inhibitor (rapamycin (R)), and a MEK inhibitor (PD98059 (PD)) were utilized to evaluate the participation of PI3K/PKB, P70S6K, and ERK1/2 pathways in the regulation of lactate production by IL1beta . PD and W, but not R, decreased IL1beta-stimulated lactate production. The participation of these pathways in the regulation of glucose uptake and lactate dehydrogenase (LDH) A mRNA levels by IL1beta was also analyzed. It was observed that W decreased IL1beta-stimulated glucose uptake, whereas PD and R did not modify it. On the other hand, PD decreased the stimulation of LDH A mRNA levels by IL1beta , whereas W and R did not modify it. In summary, results presented herein demonstrate that IL1beta stimulates PI3K/PKB-, P70S6K-, and ERK1/2-dependent pathways in rat Sertoli cells. Moreover, these results show that while IL1beta utilizes the PI3K/PKB pathway to regulate glucose transport, it utilizes the ERK1/2 pathway to regulate LDH A mRNA levels. This study reveals that IL1beta utilizes different signal transduction pathways to modify the biochemical steps that are important to regulate lactate production in rat Sertoli cells.

  6. 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. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

  7. Plant inositol monophosphatase is a lithium-sensitive enzyme encoded by a multigene family.

    PubMed Central

    Gillaspy, G E; Keddie, J S; Oda, K; Gruissem, W

    1995-01-01

    myo-Inositol monophosphatase (IMP) is a soluble, Li(+)-sensitive protein that catalyzes the removal of a phosphate from myo-inositol phosphate substrates. IMP is required for de novo inositol synthesis from glucose 6-phosphate and for breakdown of inositol trisphosphate, a second messenger generated by the phosphatidylinositol signaling pathway. We cloned the IMP gene from tomato (LeIMP) and show that the plant enzyme is encoded by a small gene family. Three different LeIMP cDNAs encode distinct but highly conserved IMP enzymes that are catalytically active in vitro. Similar to the single IMP from animals, the activities of all three LeIMPs are inhibited by low concentrations of LiCl. LeIMP mRNA levels are developmentally regulated in seedlings and fruit and in response to light. Immunoblot analysis detected three proteins of distinct molecular masses (30, 29, and 28 kD) in tomato; these correspond to the predicted molecular masses of the LeIMPs encoded by the genes. Immunoreactive proteins in the same size range are also present in several other plants. Immunolocalization studies indicated that many cell types within seedlings accumulate LeIMP proteins. In particular, cells associated with the vasculature express high levels of LeIMP protein; this may indicate a coordinate regulation between phloem transport and synthesis of inositol. The presence of three distinct enzymes in tomato most likely reflects the complexity of inositol utilization in higher plants. PMID:8718627

  8. Low temperature method for the production of calcium phosphate fillers

    PubMed Central

    Calafiori, Anna Rita; Marotta, Marcello; Nastro, Alfonso; Martino, Guglielmo

    2004-01-01

    Background Calcium phosphate manufactured samples, prepared with hydroxyapatite, are used as either spacers or fillers in orthopedic surgery, but these implants have never been used under conditions of mechanical stress. Similar conditions also apply with cements. Many authors have postulated that cements are a useful substitute material when implanted in vivo. The aim of this research is to develop a low cristalline material similar to bone in porosity and cristallinity. Methods Commercial hydroxyapatite (HAp) and monetite (M) powders are mixed with water and compacted to produce cylindrical samples. The material is processed at a temperature of 37–120 degrees C in saturated steam to obtain samples that are osteoconductive. The samples are studied by X-ray powder diffraction (XRD), Vickers hardness test (HV), scanning electron microscopy (SEM), and porosity evaluation. Results The X-ray diffractions of powders from the samples show patterns typical of HAp and M powders. After thermal treatment, no new crystal phase is formed and no increase of the relative intensity of the peaks is obtained. Vicker hardness data do not show any relationship with treatment temperature. The total porosity decreases by 50–60% according to the specific thermal treatment. Scanning electron microscopy of the surfaces of the samples with either HAp 80%-M 20% (c) or Hap 50%-M 50% (f), show cohesion of the powder grains. Conclusions The dissolution-reprecipitation process is more intesive in manufactured samples (c) and (f), according to Vickers hardness data. The process occurs in a steam saturated environment between 37 degrees and 120 degrees C. (c) (f) manufactured samples show pore dimension distributions useful to cellular repopulation in living tissues. PMID:15035671

  9. Gluconic acid production and phosphate solubilization by the plant growth-promoting bacterium Azospirillum spp.

    NASA Astrophysics Data System (ADS)

    Rodriguez, Hilda; Gonzalez, Tania; Goire, Isabel; Bashan, Yoav

    2004-11-01

    In vitro gluconic acid formation and phosphate solubilization from sparingly soluble phosphorus sources by two strains of the plant growth-promoting bacteria A. brasilense (Cd and 8-I) and one strain of A. lipoferum JA4 were studied. Strains of A. brasilense were capable of producing gluconic acid when grown in sparingly soluble calcium phosphate medium when their usual fructose carbon source is amended with glucose. At the same time, there is a reduction in pH of the medium and release of soluble phosphate. To a greater extent, gluconic acid production and pH reduction were observed for A. lipoferum JA4. For the three strains, clearing halos were detected on solid medium plates with calcium phosphate. This is the first report of in vitro gluconic acid production and direct phosphate solubilization by A. brasilense and the first report of P solubilization by A. lipoferum. This adds to the very broad spectrum of plant growth-promoting abilities of this genus.

  10. 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

  11. 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

    Mills, Stephen J; Silvander, Camilla; Cozier, Gyles; Trésaugues, Lionel; Nordlund, Pär; Potter, Barry V L

    2016-03-08

    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.

  12. Enzymic synthesis of indol-3-ylacetyl-myo-inositol galactoside.

    PubMed

    Corcuera, L J; Michalczuk, L; Bandurski, R S

    1982-11-01

    Extracts of immature kernels of Zea mays catalysed the synthesis of indol-3-ylacetyl-myo-inositol galactoside from indol-3-ylacetyl-myo-inositol and UDP-galactose. Addition of 2-mercaptoethanol was required for stability of the catalytic activity during dialysis. The enzyme could be fractionated with (NH4)2SO4, and 55% of the activity was recovered in the 30-60%-saturation fraction. The product of the reaction contained radioactivity from UDP-[U-14C]galactose and was identified as indol-3-ylacetyl-myo-inositol galactoside by gas chromatography-mass spectrometry. Therefore a UDP-galactose:indol-3-ylacetyl-myo-inositol galactosyltransferase (indol-3-ylacetyl-myo-inositol galactoside synthase) is present in developing kernels of Zea mays. The description of this enzyme, together with the enzymes described in the accompanying paper [Michalczuk & Bandurski (1982) Biochem. J. 207, 273-281] for the synthesis of indol-3-ylacetylglucose and indol-3-ylacetyl-myo-inositol, now provides mechanisms for the biosynthesis of one-half of the low-molecular-weight esters of indol-3-ylacetic acid in Zea mays.

  13. 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.

  14. Coupling of inositol phospholipid hydrolysis to peptide hormone receptors expressed from adrenal and pituitary mRNA in Xenopus laevis oocytes

    SciTech Connect

    McIntosh, R.P.; Catt, K.J.

    1987-12-01

    The expression of several neurotransmitter and drug receptors from injected exogenous mRNA in Xenopus laevis oocytes has been demonstrated by electrophysiological measurements of ion channel activation. The expression of specific receptors for peptide hormones in such a translation system would facilitate studies on the structure and regulation of cell-surface receptors as well as their coupling to membrane transduction mechanisms. The expression of receptors for calcium-mobilizing hormones in Xenopus oocytes was sought by analysis of phospholipid turnover in hormone-stimulated oocytes. For this purpose, Xenopus oocytes were injected with mRNA extracted from bovine adrenal and pituitary glands and incubated with myo-(/sup 3/H)inositol to label plasma-membrane phosphatidylinositol phosphates. The expression of functionally active receptors for angiotensin II (AII) and thyrotropin-releasing hormone (TRH) was demonstrated by the stimulation of (/sup 3/H)inositol phosphate production by AII and TRH in the mRNA-injected, (/sup 3/H)inositol-prelabeled oocytes. The ability of AII and TRH to act by way of newly synthesized receptors from mammalian endocrine tissues to stimulate phosphatidylinositol polyphosphate hydrolysis in Xenopus oocytes suggests a generalized and conserved mechanism of receptor coupling to the transduction mechanism responsible for activation of phospholipase C in the plasma membrane.

  15. Bio-catalytic nanocompartments for in situ production of glucose-6-phosphate.

    PubMed

    Lomora, M; Gunkel-Grabole, G; Mantri, S; Palivan, C G

    2017-08-29

    Cells are sophisticated biocatalytic systems driving a complex network of biochemical reactions. A bioinspired strategy to create advanced functional systems is to design confined spaces for complex enzymatic reactions by using a combination of synthetic polymer assemblies and natural cell components. Here, we developed bio-catalytic nanocompartments that contain phosphoglucomutase protected by a biomimetic polymer membrane, which was permeabilized for reactants through insertion of an engineered α-hemolysin pore protein. These bio-catalytic nanocompartments serve for production of glucose-6-phosphate, and thus possess great potential for applications in an incomplete glycolysis, pentose phosphate pathway, or in plant biological reactions.

  16. Phosphate bonded structural products from high volume wastes

    SciTech Connect

    Singh, D.; Wagh, A.S.

    1998-12-08

    A method to produce structural products from benign waste is provided comprising mixing pretreated oxide with phosphoric acid to produce an acid solution, mixing the acid solution with waste particles to produce a slurry, and allowing the slurry to cure. The invention also provides for a structural material comprising waste particles enveloped by an inorganic binder. 1 fig.

  17. Phosphate bonded structural products from high volume wastes

    DOEpatents

    Singh, Dileep; Wagh, Arun S.

    1998-01-01

    A method to produce structural products from benign waste is provided comprising mixing pretreated oxide with phosphoric acid to produce an acid solution, mixing the acid solution with waste particles to produce a slurry, and allowing the slurry to cure. The invention also provides for a structural material comprising waste particles enveloped by an inorganic binder.

  18. Phosphate bonded structural products from high volume wastes

    DOEpatents

    Singh, D.; Wagh, A.S.

    1998-12-08

    A method to produce structural products from benign waste is provided comprising mixing pretreated oxide with phosphoric acid to produce an acid solution, mixing the acid solution with waste particles to produce a slurry, and allowing the slurry to cure. The invention also provides for a structural material comprising waste particles enveloped by an inorganic binder. 1 fig.

  19. 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

  20. PRODUCTION OF PLUTONIUM FLUORIDE FROM BISMUTH PHOSPHATE PRECIPITATE CONTAINING PLUTONIUM VALUES

    DOEpatents

    Brown, H.S.; Bohlmann, E.G.

    1961-05-01

    A process is given for separating plutonium from fission products present on a bismuth phosphate carrier. The dried carrier is first treated with hydrogen fluoride at between 500 and 600 deg C whereby some fission product fluorides volatilize away from plutonium tetrafluoride, and nonvolatile fission product fluorides are formed then with anhydrous fluorine at between 400 and 500 deg C. Bismuth and plutonium distill in the form of volatile fluorides away from the nonvolatile fission product fluorides. The bismuth and plutonium fluorides are condensed at below 290 deg C.

  1. New industrial heat pump applications to phosphate fertilizer production

    SciTech Connect

    Not Available

    1990-06-01

    In this study Process Integration techniques based on Pinch Technology have been applied to Chevron's fertilizer complex in Rock Springs, Wyoming. The objectives of the study were to: identify heat pump opportunities and to determine the cost effectiveness of heat pumping compared to other process improvements. Significance of this Work Chevron's fertilizer complex is an example of an exothermic process. The sulfuric acid plant produces more heat than is needed for the rest of the site. The complex has, therefore, no need for a heating utility. The heat created in the sulfuric acid plant is used to produce high pressure steam, which is let down through a turbo generator satisfying most of the site's electrical needs. This type of process would normally not be considered for heat pumping because there is no heating utility load to reduce. However, reducing the requirements for extraction steam will liberate more steam for power generation. Heat recovery and heat pumping, therefore, have the unusual effect of an increase in electricity production, resulting in a reduction in electricity import, rather than a reduction in fuel consumption. Heat recovery opportunities show promise at both the sulfuric acid and phosphoric acid plants. No economically attractive opportunities were found for heat pumps in the process units when they were considered individually; however, the study identified that significant energy savings can be achieved by heat integration between the sulfuric acid plant and the phosphoric acid plant. 16 figs.

  2. Sunless tanning products containing dihydroxyacetone in combination with a perfluoropolyether phosphate.

    PubMed

    Pantini, G; Ingoglia, R; Brunetta, F; Brunetta, A

    2007-06-01

    It is well known that the shelf-life of products containing dihydroxyacetone (DHA) is quite unsatisfactory, and that in this area the adjustment to an acidic pH is the way usually recommended to improve DHA stability. The adoption of this rule, however, is a challenge for the cosmetic chemist not only because of the risks of skin irritation, but also because of the strong limitations in the choice of the acidic agent. It occurs that the use of either hydroxy-acids and phosphoric acid/phosphate buffers has the opposite effect on the one intended. Recently, a perfluoropolyether phosphate (PFPE phosphate) was introduced as a new acidic agent to lower the pH without increasing the skin irritation. Starting from a rationale based on this potential, we investigated sunless tanners containing PFPE phosphate and DHA. We monitored the ageing of sunless tanners with and without PFPE phosphate, by visual observation and instrumentation (pH measures and (1)H-NMR spectroscopy). Furthermore, to evaluate whether the substances produced from the DHA degradation are potential irritants, patch tests on volunteers were used to check the safety and indirectly the stability of the compositions. In conclusion, our investigations on the guidelines to formulate DHA-containing sunless tanners: confirmed that the pH is a key parameter in formulating these compositions; proved that PFPE phosphate is a safe and compatible ingredient to lower the pH; gave some evidence of the critical importance of thickeners; suggested a strategy based on multifunctional agents to minimize the ingredients.

  3. Structural characterization of the enzyme-substrate, enzyme-intermediate, and enzyme-product complexes of thiamin phosphate synthase.

    PubMed

    Peapus, D H; Chiu, H J; Campobasso, N; Reddick, J J; Begley, T P; Ealick, S E

    2001-08-28

    Thiamin phosphate synthase catalyzes the formation of thiamin phosphate from 4-amino-5-(hydroxymethyl)-2-methylpyrimidine pyrophosphate and 5-(hydroxyethyl)-4-methylthiazole phosphate. Several lines of evidence suggest that the reaction proceeds via a dissociative mechanism. The previously determined crystal structure of thiamin phosphate synthase in complex with the reaction products, thiamin phosphate and magnesium pyrophosphate, provided a view of the active site and suggested a number of additional experiments. We report here seven new crystal structures primarily involving crystals of S130A thiamin phosphate synthase soaked in solutions containing substrates or products. We prepared S130A thiamin phosphate synthase with the intent of characterizing the enzyme-substrate complex. Surprisingly, in three thiamin phosphate synthase structures, the active site density cannot be modeled as either substrates or products. For these structures, the best fit to the electron density is provided by a model that consists of independent pyrimidine, pyrophosphate, and thiazole phosphate fragments, consistent with a carbenium ion intermediate. The resulting carbenium ion is likely to be further stabilized by proton transfer from the pyrimidine amino group to the pyrophosphate to give the pyrimidine iminemethide, which we believe is the species that is observed in the crystal structures.

  4. Overexpression of glucose-6-phosphate dehydrogenase enhances riboflavin production in Bacillus subtilis.

    PubMed

    Duan, Yun Xia; Chen, Tao; Chen, Xun; Zhao, Xue Ming

    2010-02-01

    Carbon flow in Bacillus subtilis through the pentose phosphate (PP) pathway was modulated by overexpression of glucose-6-phosphate dehydrogenase (G6PDH) under the control of the inducible Pxyl promoter in B. subtilis PY. Alteration of carbon flow into the PP pathway will affect the availability of ribulose-5-phosphate (Ru5P) and the riboflavin yield. Overexpression of G6PDH resulted in the glucose consumption rate increasing slightly, while the specific growth rate was unchanged. An improvement by 25% + or - 2 of the riboflavin production was obtained. Compared to by-products formation in flask culture, low acid production (acetate and pyruvate) and more acetoin were observed. Metabolic analysis, together with carbon flux redistribution, indicated that the PP pathway fluxes are increased in response to overexpression of G6PDH. Moreover, increased flux of the PP pathway is associated with an increased intracellular pool of Ru5P, which is a precursor for riboflavin biosynthesis. The high concentrations of Ru5P could explain the increased riboflavin production.

  5. Production of biomass/energy crops on phosphatic clay soils in central Florida

    SciTech Connect

    Stricker, J.A.; Prine, G.M.; Woodard, K.R.; Anderson, D.L.; Shibles, D.B.; Riddle, T.C.

    1993-12-31

    Phosphatic clay is a byproduct of phosphate mining. Presently more than 40,470 ha have been created, most in central Florida, and about 810 ha are being added each year. Phosphatic clays have high fertility and high water holding capacity, reducing fertilization costs and producing high yields without irrigation. Based on 10 years of research, scientists have selected tall annual-regenerating perennial C-4 grasses as having the greatest potential for biomass production in Florida. The purpose of this work was to determine the feasibility of growing these tall perennial grasses for biomass on phosphatic clay. Elephantgrass, sugarcane and energycane, and erianthus were planted in duplicate replications on phosphatic clay soil in late August, 1986. yield was measured by one harvest in December or January each year for four years. Nitrogen fertilization included 112 kg ha{sup {minus}1} the first year followed by 134 kg ha{sup {minus}1} for the next three years. Nitrogen is the only supplemental nutrient needed to grow all tall grass crops on phosphatic clay. The average annual oven dry matter yield over the 4-yr period was 36.3 Mg ha{sup {minus}1} for PI 300086 elephantgrass, 45.2 for N51 elephantgrass, 42.5 for L79-1002 energycane, 49.0 for US72-1153 energycane, 49.7 for US78-1009 sugarcane, 52.2 for US56-9 sugarcane, 56.2 for CP72-1210 sugarcane, and 48.8 for 1K-7647 erianthus. More recent work has utilized domestic sewage sludge as a nitrogen source for the tall grasses. Preliminary sugar yields of selected sugarcane accessions & sweet sorghum were 4.7 Mg ha{sup {minus}1} for CP72-1210, 12.5 for US67-2022, 3.4 for US78-1009 and 1.3 Mg ha{sup {minus}1} for sweet sorghum. The high yields of the tall grasses grown on phosphatic clay with low inputs indicate a great potential for these crops as a source of renewable energy. A sustainable cropping system may be maintained by utilizing municipal sewage sludge as a nitrogen source with tall grasses on phosphatic clay.

  6. Laboratory-Scale Bismuth Phosphate Extraction Process Simulation To Track Fate of Fission Products

    SciTech Connect

    Serne, R. JEFFREY; Lindberg, Michael J.; Jones, Thomas E.; Schaef, Herbert T.; Krupka, Kenneth M.

    2007-02-28

    Recent field investigation that collected and characterized vadose zone sediments from beneath inactive liquid disposal facilities at the Hanford 200 Areas show lower than expected concentrations of a long-term risk driver, Tc-99. Therefore laboratory studies were performed to re-create one of the three processes that were used to separate the plutonium from spent fuel and that created most of the wastes disposed or currently stored in tanks at Hanford. The laboratory simulations were used to compare with current estimates based mainly on flow sheet estimates and spotty historical data. Three simulations of the bismuth phosphate precipitation process show that less that 1% of the Tc-99, Cs-135/137, Sr-90, I-129 carry down with the Pu product and thus these isotopes should have remained within the metals waste streams that after neutralization were sent to single shell tanks. Conversely, these isotopes should not be expected to be found in the first and subsequent cycle waste streams that went to cribs. Measurable quantities (~20 to 30%) of the lanthanides, yttrium, and trivalent actinides (Am and Cm) do precipitate with the Pu product, which is higher than the 10% estimate made for current inventory projections. Surprisingly, Se (added as selenate form) also shows about 10% association with the Pu/bismuth phosphate solids. We speculate that the incorporation of some Se into the bismuth phosphate precipitate is caused by selenate substitution into crystal lattice sites for the phosphate. The bulk of the U daughter product Th-234 and Np-237 daughter product Pa-233 also associate with the solids. We suspect that the Pa daughter products of U (Pa-234 and Pa-231) would also co-precipitate with the bismuth phosphate induced solids. No more than 1 % of the Sr-90 and Sb-125 should carry down with the Pu product that ultimately was purified. Thus the current scheme used to estimate where fission products end up being disposed overestimates by one order of magnitude the

  7. Modulation of inositol polyphosphate levels regulates neuronal differentiation

    PubMed Central

    Loss, Omar; Wu, Chun Ting; Riccio, Antonella; Saiardi, Adolfo

    2013-01-01

    The binding of neurotrophins to tropomyosin receptor kinase receptors initiates several signaling pathways, including the activation of phospholipase C-γ, which promotes the release of diacylglycerol and inositol 1,4,5-trisphosphate (IP3). In addition to recycling back to inositol, IP3 serves as a precursor for the synthesis of higher phosphorylated inositols, such as inositol 1,3,4,5,6-pentakisphosphate (IP5) and inositol hexakisphosphate (IP6). Previous studies on the effect of neurotrophins on inositol signaling were limited to the analysis of IP3 and its dephosphorylation products. Here we demonstrate that nerve growth factor (NGF) regulates the levels of IP5 and IP6 during PC12 differentiation. Furthermore, both NGF and brain-derived neurotrophic factor alter IP5 and IP6 intracellular ratio in differentiated PC12 cells and primary neurons. Neurotrophins specifically regulate the expression of IP5-2 kinase (IP5-2K), which phosphorylates IP5 into IP6. IP5-2K is rapidly induced after NGF treatment, but its transcriptional levels sharply decrease in fully differentiated PC12 cells. Reduction of IP5-2K protein levels by small interfering RNA has an effect on the early stages of PC12 cell differentiation, whereas fully differentiated cells are not affected. Conversely, perturbation of IP5-2K levels by overexpression suggests that both differentiated PC12 cells and sympathetic neurons require low levels of the enzyme for survival. Therefore maintaining appropriate intracellular levels of inositol polyphosphates is necessary for neuronal survival and differentiation. PMID:23864704

  8. Impact of phosphate concentration on docosahexaenoic acid production and related enzyme activities in fermentation of Schizochytrium sp.

    PubMed

    Ren, Lu-Jing; Feng, Yun; Li, Juan; Qu, Liang; Huang, He

    2013-09-01

    Docosahexaenoic acid (DHA) is an important and widely used infant food additive. In this study, the effects of phosphate concentration on lipid and especially DHA synthesis in the oleaginous fungi Schizochytrium sp. HX-308 have been investigated in batch cultures. The maximum DHA yield (8.9 g/L) and DHA productivity (148.3 mg/L h) in 0.1 g/L KH2PO4 concentration were higher than the DHA yield (6.2 g/L) and DHA productivity (86.1 mg/L h) in 4 g/L KH2PO4 concentration. Furthermore, differences in related enzyme activities (malic enzyme, glucose-6-phosphate dehydrogenase and NAD(+)-isocitrate dehydrogenase) between phosphate-sufficient and phosphate-limitation conditions were assayed. The results showed that the phosphate-limitation condition could maintain higher activities of malic enzyme and glucose-6-phosphate dehydrogenase in addition to lower activity of NAD(+)-isocitrate dehydrogenase. In addition, glucose-6-phosphate dehydrogenase might be the main supplier of NADPH at the early stage of fermentation while malic enzyme might be the provider at the late stage. This information might explain the regulation mechanism of phosphate limitation for lipid production and be useful for further DHA production enhancement.

  9. Macroscopic and microscopic variation in recovered magnesium phosphate materials: implications for phosphorus removal processes and product re-use.

    PubMed

    Massey, Michael S; Ippolito, James A; Davis, Jessica G; Sheffield, Ron E

    2010-02-01

    Phosphorus (P) recovery and re-use will become increasingly important for water quality protection and sustainable nutrient cycling as environmental regulations become stricter and global P reserves decline. The objective of this study was to examine and characterize several magnesium phosphates recovered from actual wastewater under field conditions. Three types of particles were examined including crystalline magnesium ammonium phosphate hexahydrate (struvite) recovered from dairy wastewater, crystalline magnesium ammonium phosphate hydrate (dittmarite) recovered from a food processing facility, and a heterogeneous product also recovered from dairy wastewater. The particles were analyzed using "wet" chemical techniques, powder X-ray diffraction (XRD), and scanning electron microscopy in conjunction with energy dispersive X-ray spectroscopy (SEM-EDS). The struvite crystals had regular and consistent shape, size, and structure, and SEM-EDS analysis clearly showed the struvite crystals as a surface precipitate on calcium phosphate seed material. In contrast, the dittmarite crystals showed no evidence of seed material, and were not regular in size or shape. The XRD analysis identified no crystalline magnesium phosphates in the heterogeneous product and indicated the presence of sand particles. However, magnesium phosphate precipitates on calcium phosphate seed material were observed in this product under SEM-EDS examination. These substantial variations in the macroscopic and microscopic characteristics of magnesium phosphates recovered under field conditions could affect their potential for beneficial re-use and underscore the need to develop recovery processes that result in a uniform, consistent product.

  10. 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.

  11. Engineering the specificity of trehalose phosphorylase as a general strategy for the production of glycosyl phosphates.

    PubMed

    Chen, Chao; Van der Borght, Jef; De Vreese, Rob; D'hooghe, Matthias; Soetaert, Wim; Desmet, Tom

    2014-07-25

    A two-step process is reported for the anomeric phosphorylation of galactose, using trehalose phosphorylase as biocatalyst. The monosaccharide enters this process as acceptor but can subsequently be released from the donor side, thanks to the non-reducing nature of the disaccharide intermediate. A key development was the creation of an optimized enzyme variant that displays a strict specificity (99%) for β-galactose 1-phosphate as product.

  12. Inositol uptake in rat aorta

    SciTech Connect

    Rapoport, R.M.; Van Gorp, C.; Chang, Ki-Churl )

    1990-01-01

    {sup 3}H-inositol uptake into deendothelialized aorta was linear for at least 2 h and was composed of both a saturable, Na{sup +}-dependent, and a nonsaturable, Na{sup +}-independent component. The Na{sup +}-dependent component of inositol uptake had a K{sub m} of 50 {mu}M and a V{sub max} of 289 pmol/mg prot/h. Exposure to LiCl, ouabain, or Ca{sup 2+} - free Krebs-Ringer bicarbonate solution inhibited uptake. Metabolic poisoning with dinitrophenol, as well as incubation with phloretin, an inhibitor of carrier-mediated hexose transport, also inhibited uptake. Exposure to norepinephrine decreased inositol uptake, while phorbol myristate acetate was without effect. Isobutylmethylxanthine significantly increased inositol uptake, while the increased uptake due to dibutyryl cyclic AMP and forskolin were not statistically significant. Sodium nitroprusside, and activator of guanylate cyclase, and 8-bromo cyclic GMP, were without effect on uptake, as was methylene blue, an inhibitor of guanylate cyclase. Inositol uptake into the aorta was increased when the endothelium was allowed to remain intact, although this effect was likely due to uptake in both the endothelial and smooth muscle cells.

  13. 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.

  14. Metabolism of myo-Inositol by Legionella pneumophila Promotes Infection of Amoebae and Macrophages

    PubMed Central

    Manske, Christian; Schell, Ursula

    2016-01-01

    ABSTRACT Legionella pneumophila is a natural parasite of environmental amoebae and the causative agent of a severe pneumonia termed Legionnaires' disease. The facultative intracellular pathogen employs a bipartite metabolism, where the amino acid serine serves as the major energy supply, while glycerol and glucose are mainly utilized for anabolic processes. The L. pneumophila genome harbors the cluster lpg1653 to lpg1649 putatively involved in the metabolism of the abundant carbohydrate myo-inositol (here termed inositol). To assess inositol metabolism by L. pneumophila, we constructed defined mutant strains lacking lpg1653 or lpg1652, which are predicted to encode the inositol transporter IolT or the inositol-2-dehydrogenase IolG, respectively. The mutant strains were not impaired for growth in complex or defined minimal media, and inositol did not promote extracellular growth. However, upon coinfection of Acanthamoeba castellanii, the mutants were outcompeted by the parental strain, indicating that the intracellular inositol metabolism confers a fitness advantage to the pathogen. Indeed, inositol added to L. pneumophila-infected amoebae or macrophages promoted intracellular growth of the parental strain, but not of the ΔiolT or ΔiolG mutant, and growth stimulation by inositol was restored by complementation of the mutant strains. The expression of the Piol promoter and bacterial uptake of inositol required the alternative sigma factor RpoS, a key virulence regulator of L. pneumophila. Finally, the parental strain and ΔiolG mutant bacteria but not the ΔiolT mutant strain accumulated [U-14C6]inositol, indicating that IolT indeed functions as an inositol transporter. Taken together, intracellular L. pneumophila metabolizes inositol through the iol gene products, thus promoting the growth and virulence of the pathogen. IMPORTANCE The environmental bacterium Legionella pneumophila is the causative agent of a severe pneumonia termed Legionnaires' disease. The

  15. Ceramide and ceramide 1-phosphate are negative regulators of TNF-α production induced by lipopolysaccharide.

    PubMed

    Józefowski, Szczepan; Czerkies, Maciej; Łukasik, Anna; Bielawska, Alicja; Bielawski, Jacek; Kwiatkowska, Katarzyna; Sobota, Andrzej

    2010-12-01

    LPS is a constituent of cell walls of Gram-negative bacteria that, acting through the CD14/TLR4 receptor complex, causes strong proinflammatory activation of macrophages. In murine peritoneal macrophages and J774 cells, LPS at 1-2 ng/ml induced maximal TNF-α and MIP-2 release, and higher LPS concentrations were less effective, which suggested a negative control of LPS action. While studying the mechanism of this negative regulation, we found that in J774 cells, LPS activated both acid sphingomyelinase and neutral sphingomyelinase and moderately elevated ceramide, ceramide 1-phosphate, and sphingosine levels. Lowering of the acid sphingomyelinase and neutral sphingomyelinase activities using inhibitors or gene silencing upregulated TNF-α and MIP-2 production in J774 cells and macrophages. Accordingly, treatment of those cells with exogenous C8-ceramide diminished TNF-α and MIP-2 production after LPS stimulation. Exposure of J774 cells to bacterial sphingomyelinase or interference with ceramide hydrolysis using inhibitors of ceramidases also lowered the LPS-induced TNF-α production. The latter result indicates that ceramide rather than sphingosine suppresses TNF-α and MIP-2 production. Of these two cytokines, only TNF-α was negatively regulated by ceramide 1-phosphate as was indicated by upregulated TNF-α production after silencing of ceramide kinase gene expression. None of the above treatments diminished NO or RANTES production induced by LPS. Together the data indicate that ceramide negatively regulates production of TNF-α and MIP-2 in response to LPS with the former being sensitive to ceramide 1-phosphate as well. We hypothesize that the ceramide-mediated anti-inflammatory pathway may play a role in preventing endotoxic shock and in limiting inflammation.

  16. Detecting Adsorbed Sulfate and Phosphate on Nanophase Weathering Products on Mars

    NASA Astrophysics Data System (ADS)

    Rampe, E. B.; Morris, R. V.

    2012-12-01

    Characterizing the mineralogy and chemistry of aqueous alteration phases on the martian surface is essential for understanding past aqueous processes because the types of secondary phases present and their chemical compositions tell us about the environments in which they formed. Orbital mid-infrared data and in-situ mineralogical and chemical data from the martian surface indicate that Si/Al- and Fe-bearing nanophase weathering products are widespread, including allophane and nanophase ferric oxide (npOx), which includes any combination of superparamagnetic hematite and goethite, ferrihydrite, schwertmannite, akaganeite, iddingsite, and palagonite (altered basaltic glass) [Morris et al., 2006; Michalski et al., 2006; Rampe et al., in press]. These weathering products have larger surface areas and variable surface charge and can adsorb anions and cations onto their surfaces. Some anions, such as sulfate and phosphate, specifically chemically adsorb onto mineral/mineraloid surfaces so that these complexes are covalently bonded and form ligands. Nanophase weathering products on Earth can specifically adsorb up to a few weight percent to a few tens of weight percent phosphate and sulfate, respectively [Parfitt and Smart, 1978; Jara et al., 2006]. Phosphate and sulfate have been identified in martian rocks and soils in abundances of up to ~5 wt.% and ~30 wt.%, respectively [Gellert et al., 2006; Ming et al., 2006], and it has been suggested that phosphate and sulfate ions may be adsorbed on nanophase weathering products on the martian surface [Greenwood and Blake, 2006; Morris et al., 2006]. What is relatively unknown is how to use in-situ and orbital instruments on Mars to determine if these ions are present as discrete minerals or adsorbed onto the surfaces of weathering products. We adsorbed phosphate and sulfate onto allophane surfaces in the laboratory. Here, we present laboratory measurements of phosphate- and sulfate-adsorbed allophane to compare to in

  17. Prevention of Prostate Cancer by Inositol Hexaphosphate

    DTIC Science & Technology

    2007-02-01

    signaling pathways therby inhibit growth. A large number of studies have pointed out that inositol hexaphosphate ( IP6 ) could have beneficial effect on...years, a large number of studies have pointed out that inositol hexaphosphate ( IP6 ), the most abundant phosphorylated inositol present in beans, cereal... inositol hexaphosphate ( IP6 ) on the growth and development of prostate cancer in TRAMP mice. To test the efficacy of IP6 in preventing prostate cancer

  18. Prevention of Prostate Cancer by Inositol Hexaphosphate

    DTIC Science & Technology

    2005-02-01

    studies have pointed out that inositol hexaphosphate ( IP6 ), the most abundant phosphorylated inositol present in beans, cereal grains, lentils and legumes...under the "Statement of Work", I proposed that my first task would be to determine the in vivo effects of inositol hexaphosphate ( IP6 ) on the growth and...AD Award Number: DAMDl7-03-1-0080 TITLE: Prevention of Prostate Cancer by Inositol Hexaphosphate PRINCIPAL INVESTIGATOR: Partha P. Banerjee, Ph.D

  19. Plasma membrane inositol 1,4,5-trisphosphate receptor of lymphocytes: selective enrichment in sialic acid and unique binding specificity.

    PubMed Central

    Khan, A A; Steiner, J P; Snyder, S H

    1992-01-01

    The inositol 1,4,5-trisphosphate receptor (IP3R) associated with plasma membranes of lymphocytes differs in terminal sugar content and binding specificity from the cerebellar receptor, which is localized to endoplasmic reticulum. Lectin column chromatography reveals that 30% of IP3R in the thymus contains sialic acid, reflecting a plasma membrane association, in contrast to 5% of cerebellar IP3R. IP3R in thymus and plasma membrane fractions of Jurkat lymphocytes differs from IP3R of Jurkat microsomes and cerebellum in inositol phosphate specificity. The plasma membrane IP3R has lower affinity for IP3 but higher affinity for inositol 1,3,4,5-tetrakisphosphate, which may reflect a unique regulation of calcium at the plasma membrane by inositol phosphates. Images PMID:1313570

  20. Nutritional control of actinorhodin production by Streptomyces coelicolor A3(2): suppressive effects of nitrogen and phosphate.

    PubMed

    Doull, J L; Vining, L C

    1990-01-01

    Actinorhodin production in Streptomyces coelicolor A3(2) was relatively insensitive to the carbon source concentration but was elicited by nitrogen or phosphate depletion, or by a decline in the growth rate. In starch-glutamate media with nitrogen limitation, increasing the nitrogen supply delayed the onset of antibiotic synthesis and, at concentrations above 30 mM, decreased its rate. In a similar medium with phosphate limitation, increasing the initial phosphate concentration delayed actinorhodin formation and, above 2.5 mM, reduced the rate of synthesis. Experiments in which actinorhodin synthesis was elicited by phosphate depletion at various nitrogen concentrations demonstrated strong suppression by residual glutamate. Cultures in which actinorhodin biosynthesis was initiated by nitrogen depletion were not similarly suppressed by increasing amounts of residual phosphate. The results suggest that actinorhodin production in S. coelicolor A3(2) responds to interacting physiological controls, notable among which is nitrogen catabolite regulation.

  1. Rabbit synoviocyte inositol phospholipid metabolism is stimulated by hydroxyapatite crystals

    SciTech Connect

    Rothenberg, R.J.; Cheung, H.

    1988-04-01

    Inhibition of prostaglandin E2 synthesis partially ameliorates some aspects of synovitis, but joint destruction still progresses. Other aspects of phospholipid metabolism may play a role in synovial tissue pathophysiology. Products of phosphatidylinositol metabolism can activate intracellular processes in response to extracellular stimuli. We asked whether this pathway is activated in synoviocytes in monolayer tissue culture by the addition of hydroxyapatite (HA) crystals in medium. These crystals are found in pathological human synovial fluid. These crystals are associated with the secretion of degradative enzymes and with a destructive arthritis in humans. Rabbit synoviocyte cultures, previously incubated with (3H)inositol to label inositol phospholipids, were stimulated with the addition of hydroxyapatite (180 micrograms/ml) to the cultures. There was enhanced intracellular accumulation of (3H)inositol monophosphate (30-100%) after 4 h. This indicated an increased phospholipase C activity. The radioactivity in (3H)inositol bis- and trisphosphates was too low to reliably measure. The use of (32P)Pi allowed detection of these compounds. In the presence of HA, incorporation of (32P)Pi into phosphatidylinositol, phosphatidylinositol monophosphate, and phosphatidylinositol bisphosphate was increased. In addition, cultures exposed to (32P)Pi during stimulation with HA had an increased content of (32P)inositol monophosphate, bisphosphate, and trisphosphate.

  2. Dihydroxyacetone production in an engineered Escherichia coli through expression of Corynebacterium glutamicum dihydroxyacetone phosphate dephosphorylase.

    PubMed

    Jain, Vishist Kumar; Tear, Crystal Jing Ying; Lim, Chan Yuen

    2016-05-01

    Dihydroxyacetone (DHA) has several industrial applications such as a tanning agent in tanning lotions in the cosmetic industry; its production via microbial fermentation would present a more sustainable option for the future. Here we genetically engineered Escherichia coli (E. coli) for DHA production from glucose. Deletion of E. coli triose phosphate isomerase (tpiA) gene was carried out to accumulate dihydroxyacetone phosphate (DHAP), for use as the main intermediate or precursor for DHA production. The accumulated DHAP was then converted to DHA through the heterologous expression of Corynebacterium glutamicum DHAP dephosphorylase (cghdpA) gene. To conserve DHAP exclusively for DHA production we removed methylglyoxal synthase (mgsA) gene in the ΔtpiA strain. This drastically improved DHA production from 0.83g/l (0.06g DHA/g glucose) in the ΔtpiA strain bearing cghdpA to 5.84g/l (0.41g DHA/g glucose) in the ΔtpiAΔmgsA double mutant containing the same gene. To limit the conversion of intracellular DHA to glycerol, glycerol dehydrogenase (gldA) gene was further knocked out resulting in a ΔtpiAΔmgsAΔgldA triple mutant. This triple mutant expressing the cghdpA gene produced 6.60g/l of DHA at 87% of the maximum theoretical yield. In summary, we demonstrated an efficient system for DHA production in genetically engineered E. coli strain.

  3. Assaying inositol and phosphoinositide phosphatase enzymes.

    PubMed

    Donahue, Janet L; Ercetin, Mustafa; Gillaspy, Glenda E

    2013-01-01

    One critical aspect of phosphoinositide signaling is the turnover of signaling molecules in the pathway. These signaling molecules include the phosphatidylinositol phosphates (PtdInsPs) and inositol phosphates (InsPs). The enzymes that catalyze the breakdown of these molecules are thus important potential regulators of signaling, and in many cases the activity of such enzymes needs to be measured and compared to other enzymes. PtdInsPs and InsPs are broken down by sequential dephosphorylation reactions which are catalyzed by a set of specific phosphatases. Many of the phosphatases can act on both PtdInsP and InsP substrates. The protocols described in this chapter detail activity assays that allow for the measurement of PtdInsP and InsP phosphatase activities in vitro starting with native or recombinant enzymes. Three different assays are described that have different equipment requirements and allow one to test a range of PtdInsP and InsP phosphatases that act on different substrates.

  4. Dextransucrase production using cashew apple juice as substrate: effect of phosphate and yeast extract addition.

    PubMed

    Chagas, Clarice M A; Honorato, Talita L; Pinto, Gustavo A S; Maia, Geraldo A; Rodrigues, Sueli

    2007-05-01

    Cashew apples are considered agriculture excess in the Brazilian Northeast because cashew trees are cultivated primarily with the aim of cashew nut production. In this work, the use of cashew apple juice as a substrate for Leuconostoc mesenteroides cultivation was investigated. The effect of yeast extract and phosphate addition was evaluated using factorial planning tools. Both phosphate and yeast extract addition were significant factors for biomass growth, but had no significant effect on maximum enzyme activity. The enzyme activities found in cashew apple juice assays were at least 3.5 times higher than the activity found in the synthetic medium. Assays with pH control (pH = 6.5) were also carried out. The pH-controlled fermentation enhanced biomass growth, but decreased the enzyme activity. Crude enzyme free of cells produced using cashew apple juice was stable for 16 h at 30 degrees C at a pH of 5.0.

  5. Occupational exposures in two industrial plants devoted to the production of ammonium phosphate fertilisers.

    PubMed

    Bolívar, J P; García-Tenorio, R; Mosqueda, F; Gázquez, M J; López-Coto, I; Adame, J A; Vaca, F

    2013-03-01

    In order to fill a gap in the open literature, occupational exposures and activity concentrations have been assessed in two NORM industrial plants, located in the south-west of Spain, devoted to the production of mono-ammonium phosphate (MAP) and di-ammonium phosphate (DAP) fertilisers. The annual effective doses received by the workers from these plants are clearly below 1 mSv yr(-1) and the contribution due to external radiation is similar to that due to inhalation. The contribution to the maximum effective doses due to inhalation of particulate matter has been estimated to be about 0.12 mSv yr(-1), while the (222)Rn concentrations inside the plants are of no concern. Consequently, no additional actions or radiological protection measures need to be taken to decrease the natural radiation received by the workers in these facilities.

  6. Phosphates at the Surface of Mars: Primary Deposits and Alteration Products

    NASA Astrophysics Data System (ADS)

    Yen, A. S.; Gellert, R.; Clark, B. C.; Ming, D. W.; Mittlefehldt, D. W.; Arvidson, R. E.; McSween, H. Y., Jr.; Schröder, C.

    2014-12-01

    Phosphorus is an essential element in terrestrial organisms and thus characterizing the occurrences of phosphate phases at the martian surface is crucial in the assessment of habitability. The Alpha Particle X-Ray Spectrometers onboard Spirit, Opportunity and Curiosity discovered a variety of primary and secondary phosphate phases allowing direct comparisons across the three landing sites. The Spirit rover at Gusev Crater encountered the "Wishstone/Watchtower" class of P-rich (up to 5.2 wt% P2O5) rocks interpreted to be alkaline volcanic rocks with a physical admixture of ~10 to 20% merrillite [Usui et al 2008]. These rocks are characterized by elevated Ti and Y and anomalously low Cr and Ni, which could largely reflect the nature of the protoliths: Evolved magmatic rocks. Many of these chemical signatures are also found in pyroclastic deposits at nearby "Home Plate" and in phosphate precipitates derived from fluid interactions with these rocks ("Paso Robles" soils). The Opportunity rover at Meridiani Planum recently analyzed a ~4 cm clast in a fine-grained matrix, one of numerous rocks of similar appearance at the rim of Endeavour Crater. This clast, "Sarcobatus," has minor enrichments in Ca and P relative to the matrix, and like the P-rich rocks at Gusev, Sarcobatus also shows elevated Al and Ti. On the same segment of the Endeavour rim, subsurface samples were found with exceptional levels of Mn (~3.5 wt% MnO). These secondary and likely aqueous deposits contain strong evidence for associated Mg-sulfate and Ca-phosphate phases. Finally, the Curiosity traverse at Gale crater encountered P-rich rocks compositionally comparable to Wishstone at Gusev, including elevated Y. Phosphorous-rich rocks with similar chemical characteristics are prevalent on Mars, and the trace and minor element signatures provide constraints on whether these are primary deposits, secondary products of physical weathering, or secondary products of chemical weathering.

  7. Dried plum products as a substitute for phosphate in chicken marinade.

    PubMed

    Jarvis, Nathan; Clement, Ashley R; O'Bryan, Corliss A; Babu, Dinesh; Crandall, Philip G; Owens, Casey M; Meullenet, Jean-Francois; Ricke, Steven C

    2012-06-01

    In order to address the growing demand for more natural poultry products, alkaline phosphates commonly used in chicken breast meat marinades were replaced with plum ingredients and evaluated. For initial sensory evaluation, 200 consumers of chicken were served a small portion of the chicken breast on a plate and were asked to evaluate the product for overall impression, flavor, and texture on a 9-point hedonic scale with 1 = "dislike extremely" and 9 = "like extremely." Also, a 5-point just-about-right (JAR) scale was used on questions about tenderness, juiciness, overall flavor, and saltiness. Both hedonic and JAR demonstrated that the marinades of plum concentrate and the blend of plum fiber and powder were not distinguishable from the control (P > 0.05). Using two different percentages of fiber/powder blend, two different percentages of concentrate, sodium tripolyphosphate (STPP), and no marinade, measurements were made for marinade per cent pickup, thaw loss, and cook loss. Plum concentrate at 1.1% was most similar to STPP in marinade per cent pickup, thaw loss, drip loss, and cook loss. These results show that plum ingredients can potentially be used as a substitute in standard phosphate marinades. Consumers are increasingly demanding more natural foods with less artificial additives. This research presents the results of experiments using dried plum ingredients as a substitute for phosphates commonly used in marinades for chicken. Results indicate that dried plum ingredients may be a suitable substitute for phosphates in marinades. Journal of Food Science © 2012 Institute of Food Technologists® No claim to original US government works.

  8. Phosphates at the Surface of Mars: Primary Deposits and Alteration Products

    NASA Technical Reports Server (NTRS)

    Yen, Albert S.; Gellert, Ralf; Clark, Benton C.; Ming, Douglas W.; Mittlefehldt, David W.; Arvidson, Raymond E.; McSween, Harry Y., Jr.; Schroder, Christian

    2014-01-01

    Phosphorus is an essential element in terrestrial organisms and thus characterizing the occurrences of phosphate phases at the martian surface is crucial in the assessment of habitability. The Alpha Particle X-Ray Spectrometers onboard Spirit, Opportunity and Curiosity discovered a variety of primary and secondary phosphate phases allowing direct comparisons across the three landing sites. The Spirit rover at Gusev Crater encountered the "Wishstone/Watchtower" class of P-rich (up to 5.2 wt% P2O5) rocks interpreted to be alkaline volcanic rocks with a physical admixture of approximately 10 to 20% merrillite [Usui et al 2008]. These rocks are characterized by elevated Ti and Y and anomalously low Cr and Ni, which could largely reflect the nature of the protoliths: Evolved magmatic rocks. Many of these chemical signatures are also found in pyroclastic deposits at nearby "Home Plate" and in phosphate precipitates derived from fluid interactions with these rocks ("Paso Robles" soils). The Opportunity rover at Meridiani Planum recently analyzed approximately 4 cm clast in a fine-grained matrix, one of numerous rocks of similar appearance at the rim of Endeavour Crater. This clast, "Sarcobatus," has minor enrichments in Ca and P relative to the matrix, and like the P-rich rocks at Gusev, Sarcobatus also shows elevated Al and Ti. On the same segment of the Endeavour rim, subsurface samples were found with exceptional levels of Mn (approximately 3.5 wt% MnO). These secondary and likely aqueous deposits contain strong evidence for associated Mg-sulfate and Ca-phosphate phases. Finally, the Curiosity traverse at Gale crater encountered P-rich rocks compositionally comparable to Wishstone at Gusev, including elevated Y. Phosphorous-rich rocks with similar chemical characteristics are prevalent on Mars, and the trace and minor element signatures provide constraints on whether these are primary deposits, secondary products of physical weathering or secondary products of chemical

  9. [Production of timolol containing calcium-phosphate nanoparticles and evaluation of their effect on intraocular pressure in experiment].

    PubMed

    Shimanovskaia, E V; Beznos, O V; Kliachko, N L; Kost, O A; Nikol'skaia, I I; Pavlenko, T A; Chesnokova, N B; Kabanov, A V

    2012-01-01

    Methodology for production of calcium-phosphate nanoparticles is developed and its efficacy as a drug carrier system is estimated by example of timolol. Conditions for production of particles with optimal size and resistance are determined, methodology of loading of particles with timolol is developed. Physical parameters of particles (form, size, relief), kinetics of saturation with drug and its release are studied. Packaging of timolol into calcium phosphate nanoparticles was showed to enhance and prolong its hypotensive effect in experiment on healthy rabbits.

  10. Insulin activation of vacuolar protein sorting 34 mediates localized phosphatidylinositol 3-phosphate production at lamellipodia and activation of mTOR/S6K1.

    PubMed

    Hirsch, Dianne S; Shen, Yi; Dokmanovic, Milos; Yu, Joyce; Mohan, Nishant; Elzarrad, Mohammed Khair; Wu, Wen Jin

    2014-06-01

    The class III phosphatidylinositol 3-kinase, VPS34, phosphorylates the D3 hydroxyl of inositol generating phosphatidylinositol 3-phosphate (ptdins(3)p). Initial studies suggested that ptdins(3)p solely functioned as a component of vesicular and endosomal membranes and that VPS34 did not function in signal transduction. However, VPS34 has recently been shown to be required for insulin-mediated activation of S6 kinase 1 (S6K1). Whether VPS34 activity is directly regulated by insulin is unclear. It is also not known whether VPS34 activity can be spatially restricted in response to extracellular stimuli. Data presented here demonstrate that in response to insulin, VPS34 is activated and translocated to lamellipodia where it produces ptdins(3)p. The localized production of ptdins(3)p is dependent on Src phosphorylation of VPS34. In cells expressing VPS34 with mutations at Y231 or Y310, which are Src-phosphorylation sites, insulin-stimulated VPS34 translocation to the plasma membrane and lamellipodia formation are blocked. mTOR also colocalizes with VPS34 and ptdins(3)p at lamellipodia following insulin-stimulation. In cells expressing the VPS34-Y231F mutant, which blocks lamellipodia formation, mTOR localization at the plasma membrane and insulin-mediated S6K1 activation are reduced. This suggests that mTOR localization at lamellipodia is important for full activation of S6K1 induced by insulin. These data demonstrate that insulin can spatially regulate VPS34 activity through Src-mediated tyrosine phosphorylation and that this membrane localized activity contributes to lamellipodia formation and activation of mTOR/S6K1signaling.

  11. Development of Vanadium-Phosphate Catalysts for Methanol Production by Selective Oxidation of Methane.

    SciTech Connect

    McCormick, R.L.; Alptekin, G.O.

    1997-07-30

    This document is the seventeenth quarterly technical progress report under Contract No. DE-AC22-92PC92110 `Development of Vanadium- Phosphate Catalysts for Methanol Production by Selective Oxidation of Methane` and covers the period April-June, 1997. Vanadium phosphate, vanadyl pyrophosphate specifically, is used commercially to oxidize butane to maleic anhydride and is one of the few examples of an active and selective oxidation catalyst for alkanes. In this project we are examining this catalyst for the methane oxidation reaction. Initial process variable and kinetic studies indicated that vanadyl pyrophosphate is a reasonably active catalyst below 500{degrees}C but produces CO as the primary product, no formaldehyde or methanol were observed. A number of approaches for modification of the catalyst to improve selectivity have been tried. Results obtained earlier in this project are summarized under Project Description in the body of this report. Iron phosphate and iron phosphate supported on silica catalysts have been shown in our previous work to produce much higher yields of partial oxidation products from methane than VPO. During this quarter we have expanded these studies dramatically by detailed testing of a new silica support, by performance of detailed kinetic and product selectivity studies on the quartz form of FePO{sub 4}, both unsupported and supported on silica, by testing of a mixed valence iron phosphate Fe{sub 4}(P{sub 2}0{sub 7}), and by detailed characterization of and other materials by a number of methods including Moessbauer spectroscopy. The most selective catalyst examined to date is FePO{sub 4} supported on silica. This material has produced formaldehyde with space time yields of nearly 500 g/kg-h. Methanol yields are low but quantifiable at roughly 10 g/kg-h. Interestingly, addition of water to the feed gas produces large improvements in the formaldehyde yield by suppression of the parallel reaction to form carbon dioxide. Increasing

  12. Effect of fermented wastewaters from butter production on phosphates removal in a sequencing batch reactor.

    PubMed

    Janczukowicz, Wojciech; Rodziewicz, Joanna; Thornton, Arthur; Czaplicka, Kamila

    2012-09-01

    This study determined the potential for fermented wastewaters from butter production plant to act as a carbon source to facilitate phosphates removal. Synthetic dairy wastewaters were treated using SBR, with doses of fermented wastewaters. An increase in the fermented wastewater doses were found to improve the effluent quality in respect of phosphates and nitrates. The lowest concentrations of phosphate and nitrates, respectively 0.10 ± 0.04 mg PO(4)-PL(-1) and 1.03 ± 0.22 mg NO(3)-NL(-1), were noted in the effluent from the reactor fed with fermented wastewaters in a dose of 0.25 L d(-1) per 0.45 L d(-1) of wastewaters fed to the reactor. In the case of the two highest doses, an increase in effluent COD was stated. The higher effectiveness resulted from the fact that the introduction of fermented wastewaters caused an increase in the easily-available carbon compounds content and the predominance of acetic acid amongst VFAs available to dephosphatating and denitrifying bacteria.

  13. 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.

  14. myo-Inositol Oxygenase is Required for Responses to Low Energy Conditions in Arabidopsis thaliana.

    PubMed

    Alford, Shannon R; Rangarajan, Padma; Williams, Phoebe; Gillaspy, Glenda E

    2012-01-01

    myo-Inositol is a precursor for cell wall components, is used as a backbone of myo-inositol trisphosphate (Ins(1,4,5)P(3)) and phosphatidylinositol phosphate signaling molecules, and is debated about whether it is also a precursor in an alternate ascorbic acid synthesis pathway. Plants control inositol homeostasis by regulation of key enzymes involved in myo-inositol synthesis and catabolism. Recent transcriptional profiling data indicate up-regulation of the myo-inositol oxygenase (MIOX) genes under conditions in which energy or nutrients are limited. To test whether the MIOX genes are required for responses to low energy, we first examined MIOX2 and MIOX4 gene expression regulation by energy/nutrient conditions. We found that both MIOX2 and MIOX4 expression are suppressed by exogenous glucose addition in the shoot, but not in the root. Both genes were abundantly expressed during low energy/nutrient conditions. Loss-of-function mutants in MIOX genes contain alterations in myo-inositol levels and growth changes in the root. Miox2 mutants can be complemented with a MIOX2:green fluorescent protein fusion. Further we show here that MIOX2 is a cytoplasmic protein, while MIOX4 is present mostly in the cytoplasm, but also occasionally in the nucleus. Together, these data suggest that MIOX catabolism in the shoot may influence root growth responses during low energy/nutrient conditions.

  15. Recognizing Sulfate and Phosphate Complexes Adsorbed onto Nanophase Weathering Products on Mars

    NASA Technical Reports Server (NTRS)

    Rampe, E. B.; Morris, R. V.; Archer, P. D., Jr.

    2015-01-01

    Nanophase weathering products (i.e., secondary phases that lack long-range atomic order) have been recognized on the martian surface via orbital observations and in-situ measurements from landed missions. Allophane, a poorly crystalline, hydrated aluminosilicate, has been identified at the regional scale in models of thermal-infrared (TIR) data from the Thermal Emission Spectrometer (TES) and at the local scale from visible/near-IR (VNIR) data from the Compact Reconnaissance Impact Spectrometer for Mars (CRISM) instrument and phase calculations of Alpha Particle X-ray Spectrometer (APXS) data of rocks encountered by the Mars Exploration Rovers (MER) Spirit and Opportunity. Nanophase iron oxides (npOx) have been recognized in rocks and soils measured by the Mössbauer Spectrometer on Spirit and Opportunity. Furthermore, analyses of X-ray diffraction data measured by the CheMin instrument onboard the Mars Science Laboratory rover Curiosity indicate rock and soil samples are comprised of approx. 20-50 wt.% X-ray amorphous materials. Chemical measurements by landed missions indicate the presence of sulfur and phosphorus in martian rocks in soils, and APXS data from Gusev crater demonstrate abundances of up to approx. 5 wt.% P2O5 and approx. 30 wt.% SO3. However, the speciation of phosphorus and sulfur is not always evident. On Earth, phosphate and sulfate anions can be chemisorbed onto the surfaces of nanophase weathering products. This process may also occur on Mars, and calculations of the composition of the amorphous component at Gale crater using CheMin mineral models and APXS data show that amorphous material is enriched in volatiles, including S. Here, we examine the ability to detect chemisorbed sulfate and phosphate complexes by analyzing sulfate- and phosphate-adsorbed nanophase weathering products using instruments similar to those on landed and orbital missions.

  16. 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.

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

    PubMed Central

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

    2013-01-01

    SUMMARY 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. PMID:24072790

  18. Characterization of a Mannose-6-Phosphate Isomerase from Bacillus amyloliquefaciens and Its Application in Fructose-6-Phosphate Production

    PubMed Central

    Sigdel, Sujan; Singh, Ranjitha; Kim, Tae-Su; Li, Jinglin; Kim, Sang-Yong; Kim, In-Won; Jung, Woo-Suk; Pan, Cheol-Ho; Kang, Yun Chan; Lee, Jung-Kul

    2015-01-01

    The BaM6PI gene encoding a mannose-6-phosphate isomerase (M6PI, EC 5.3.1.8) was cloned from Bacillus amyloliquefaciens DSM7 and overexpressed in Escherichia coli. The enzyme activity of BaM6PI was optimal at pH and temperature of 7.5 and 70°C, respectively, with a kcat/Km of 13,900 s-1 mM-1 for mannose-6-phosphate (M6P). The purified BaM6PI demonstrated the highest catalytic efficiency of all characterized M6PIs. Although M6PIs have been characterized from several other sources, BaM6PI is distinguished from other M6PIs by its wide pH range and high catalytic efficiency for M6P. The binding orientation of the substrate M6P in the active site of BaM6PI shed light on the molecular basis of its unusually high activity. BaM6PI showed 97% substrate conversion from M6P to fructose-6-phosphate demonstrating the potential for using BaM6PI in industrial applications. PMID:26171785

  19. Mercury contamination of soil as the result of long-term phosphate fertilizer production.

    PubMed

    Mirlean, Nicolai; Baisch, Paulo; Machado, Isabel; Shumilin, Evgueni

    2008-09-01

    This study was aimed at determining the range and spatial distribution of mercury in a geographical area influenced by the emissions of phosphate fertilizers industries in Rio Grande, Brazil. The case study demonstrated that mercury concentrations in a fine fraction of the surface soil close to the fertilizer factory reached levels as high as 800 microg kg(-1). Increased mercury concentrations were detected up to 60 cm below the soil surface. Further, a significant impact of the elevated mercury levels was manifested in a 1-km zone around the factory. Technical grade sulfuric acid employed in superphosphate production is considered the principal source of this mercury contamination.

  20. Effects of Various Calcium Powders as Replacers for Synthetic Phosphate on the Quality Properties of Ground Pork Meat Products

    PubMed Central

    2017-01-01

    The aim of this study was to identify the optimal and superior type of natural calcium for replacing phosphate in cooked ground pork products. To achieve this, 0.5% eggshell calcium (ESC), oyster shell calcium (OSC), marine algae calcium (MAC), or milk calcium (MC) was added to ground pork meat products. The effect of this substitution was studied by comparing the substituted products with products containing 0.3% phosphate blend (control). ESC was considered an ideal phosphate replacer for minimizing the cooking loss, which likely resulted from the increase in the pH of the product. Among the other natural calcium types, OSC treatment did not cause a significant increase in pH, but it lowered the cooking loss. CIE L* values were higher (p<0.05) in products treated with OSC or MC than the control, and lowest (p<0.05) in the products with ESC. However, products with ESC had higher (p<0.05) CIE a* and CIE b* values than the control and products treated with other powders. Compared to the control, products treated with ESC and OSC had similar substitution effects on the textural properties of the products. Therefore, the results of this study suggested that the combined use of ESC and OSC could be a potentially effective method for replacing synthetic phosphate in ground pork products. PMID:28747832

  1. High bovine blastocyst development in a static in vitro production system using SOFaa medium supplemented with sodium citrate and myo-inositol with or without serum-proteins.

    PubMed

    Holm, P; Booth, P J; Schmidt, M H; Greve, T; Callesen, H

    1999-09-01

    We describe a bovine embryo culture system that supports repeatable high development in the presence of serum or BSA as well as under defined conditions in the absence of those components. In the first experiment, embryo development in SOF with amino acids (SOFaa), sodium citrate (SOFaac) and myo-inositol (SOFaaci) and with BSA or polyvinyl alcohol (PVA) was compared with that in a M199 granulosa cell co-culture (M199 co-culture). Subsequently, development and cell numbers of blastocysts cultured under defined conditions in SOFaaci with PVA (SOFaaci-PVA), or under undefined conditions in SOFaaci with 5% cow serum (SOFaaci-CS) or M199 co-culture were compared. The repeatability of culture results in SOFaaci-CS was checked by weekly replicates (n = 30) spread over 11 months. The viability of embryos developed in SOFaaci-PVA was estimated by transfer of morphologically good blastocysts (n = 10) to synchronized recipients. In the second experiment, the effect of omitting CS or BSA from IVM and IVM-IVF on subsequent embryo development in SOFaaci-PVA or in SOFaaci-CS was investigated. Blastocyst development in SOFaa-PVA, SOFaac-PVA, SOFaa-BSA and M199 was 16 +/- 3b, 23 +/- 2ab, 30 +/- 8a and 36 +/- 7a%, respectively (Pab < 0.05). Additional inclusion of myoinositol resulted in 42 +/- 1a% blastocysts in SOFaaci-PVA vs 19 +/- 3b% in SOFaac-PVA, 47 +/- 7a% in SOFaac-BSA, and 36 +/- 7a% in M199 co-culture, respectively (Pab < 0.01). In 30 replicates, the average cleavage and blastocyst rates of oocytes in SOFaaci-CS were 87 +/- 4 and 49 +/- 5%, respectively. Five normal calves were produced after transfer of 10 blastocysts developed in defined culture medium (i.e., SOFaaci-PVA). Defined IVM or IVM-IVF (i.e., in absence of CS and BSA) reduced cleavage rates (83 +/- 3 and 55 +/- 3% vs 90 +/- 1% in presence of CS; P < 0.01). Subsequent embryo development in SOFaaci-CS was not affected in either of these defined conditions. However, cleavage and blastocyst rates under completely

  2. Streptococcus pneumoniae PstS production is phosphate responsive and enhanced during growth in the murine peritoneal cavity

    NASA Technical Reports Server (NTRS)

    Orihuela, C. J.; Mills, J.; Robb, C. W.; Wilson, C. J.; Watson, D. A.; Niesel, D. W.

    2001-01-01

    Differential display-PCR (DDPCR) was used to identify a Streptococcus pneumoniae gene with enhanced transcription during growth in the murine peritoneal cavity. Northern dot blot analysis and comparative densitometry confirmed a 1.8-fold increase in expression of the encoded sequence following murine peritoneal culture (MPC) versus laboratory culture or control culture (CC). Sequencing and basic local alignment search tool analysis identified the DDPCR fragment as pstS, the phosphate-binding protein of a high-affinity phosphate uptake system. PCR amplification of the complete pstS gene followed by restriction analysis and sequencing suggests a high level of conservation between strains and serotypes. Quantitative immunodot blotting using antiserum to recombinant PstS (rPstS) demonstrated an approximately twofold increase in PstS production during MPC from that during CCs, a finding consistent with the low levels of phosphate observed in the peritoneum. Moreover, immunodot blot and Northern analysis demonstrated phosphate-dependent production of PstS in six of seven strains examined. These results identify pstS expression as responsive to the MPC environment and extracellular phosphate concentrations. Presently, it remains unclear if phosphate concentrations in vivo contribute to the regulation of pstS. Finally, polyclonal antiserum to rPstS did not inhibit growth of the pneumococcus in vitro, suggesting that antibodies do not block phosphate uptake; moreover, vaccination of mice with rPstS did not protect against intraperitoneal challenge as assessed by the 50% lethal dose.

  3. Streptococcus pneumoniae PstS production is phosphate responsive and enhanced during growth in the murine peritoneal cavity

    NASA Technical Reports Server (NTRS)

    Orihuela, C. J.; Mills, J.; Robb, C. W.; Wilson, C. J.; Watson, D. A.; Niesel, D. W.

    2001-01-01

    Differential display-PCR (DDPCR) was used to identify a Streptococcus pneumoniae gene with enhanced transcription during growth in the murine peritoneal cavity. Northern dot blot analysis and comparative densitometry confirmed a 1.8-fold increase in expression of the encoded sequence following murine peritoneal culture (MPC) versus laboratory culture or control culture (CC). Sequencing and basic local alignment search tool analysis identified the DDPCR fragment as pstS, the phosphate-binding protein of a high-affinity phosphate uptake system. PCR amplification of the complete pstS gene followed by restriction analysis and sequencing suggests a high level of conservation between strains and serotypes. Quantitative immunodot blotting using antiserum to recombinant PstS (rPstS) demonstrated an approximately twofold increase in PstS production during MPC from that during CCs, a finding consistent with the low levels of phosphate observed in the peritoneum. Moreover, immunodot blot and Northern analysis demonstrated phosphate-dependent production of PstS in six of seven strains examined. These results identify pstS expression as responsive to the MPC environment and extracellular phosphate concentrations. Presently, it remains unclear if phosphate concentrations in vivo contribute to the regulation of pstS. Finally, polyclonal antiserum to rPstS did not inhibit growth of the pneumococcus in vitro, suggesting that antibodies do not block phosphate uptake; moreover, vaccination of mice with rPstS did not protect against intraperitoneal challenge as assessed by the 50% lethal dose.

  4. 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.

  5. Expression of striatal D sub 1 dopamine receptors coupled to inositol phosphate production and Ca sup 2+ mobilization in Xenopus oocytes

    SciTech Connect

    Mahan, L.C.; Monsma, F.J. Jr.; Sibley, D.R. ); Burch, R.M. )

    1990-03-01

    Expression of central nervous system receptors for dopamine was examined by injection of poly(A){sup +} RNA (mRNA) from rat striatum into oocytes from Xenopus laevis. Electrophysiological measurements in mRNA-injected oocytes indicated that addition of 100 {mu}M dopamine induced an inward current that was consistent with the activation of endogenous Ca{sup 2+}-dependent Cl{sup {minus}} channels. This current was also elicited by addition of the selective D{sub 1} agonist SKF 38393 but not by the selective D{sub 2} agonist quinpirole. Using {sup 45}Ca{sup 2+} efflux assays, addition of 100 {mu}M dopamine to injected oocytes stimulated efflux 2- to 3-fold. This increase was mimicked by SKF 38393 and was blocked by the D{sub 1}-selective antagonist (+)SCH 23390 but not by the D{sub 2}-selective antagonist domperidone. No increase in {sup 45}Ca{sup 2+} efflux was seen with 100 {mu}M quinpirole. Size fractionation of striatal mRNA yielded a single peak (2.5-3.0 kilobases) of D{sub 1} receptor-mediated {sup 45}Ca{sup 2+} efflux activity in injected oocytes. Thus, in addition to D{sub 1} receptors that stimulate adenylyl cyclase, rat striatum contains D{sub 1} receptors that can couple to InsP{sub 3} formation and mobilization of intracellular Ca{sup 2+}.

  6. Src homology 2-containing 5-inositol phosphatase (SHIP) suppresses an early stage of lymphoid cell development through elevated interleukin-6 production by myeloid cells in bone marrow.

    PubMed

    Nakamura, Koji; Kouro, Taku; Kincade, Paul W; Malykhin, Alexander; Maeda, Kazuhiko; Coggeshall, K Mark

    2004-01-19

    The Src homology (SH)2-containing inositol 5-phosphatase (SHIP) negatively regulates a variety of immune responses through inhibitory immune receptors. In SHIP(-/-) animals, we found that the number of early lymphoid progenitors in the bone marrow was significantly reduced and accompanied by expansion of myeloid cells. We exploited an in vitro system using hematopoietic progenitors that reproduced the in vivo phenotype of SHIP(-/-) mice. Lineage-negative marrow (Lin(-)) cells isolated from wild-type mice failed to differentiate into B cells when cocultured with those of SHIP(-/-) mice. Furthermore, culture supernatants of SHIP(-/-) Lin(-) cells suppressed the B lineage expansion of wild-type lineage-negative cells, suggesting the presence of a suppressive cytokine. SHIP(-/-) Lin(-) cells contained more IL-6 transcripts than wild-type Lin(-) cells, and neutralizing anti-IL-6 antibody rescued the B lineage expansion suppressed by the supernatants of SHIP(-/-) Lin(-) cells. Finally, we found that addition of recombinant IL-6 to cultures of wild-type Lin(-) bone marrow cells reproduced the phenotype of SHIP(-/-) bone marrow cultures: suppression of B cell development and expansion of myeloid cells. The results identify IL-6 as an important regulatory cytokine that can suppress B lineage differentiation and drive excessive myeloid development in bone marrow.

  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. Determination of pre-cecal phosphorus digestibility of inorganic phosphates and bone meal products in broilers.

    PubMed

    van Harn, J; Spek, J W; van Vuure, C A; van Krimpen, M M

    2017-05-01

    A broiler study was performed to determine the pre-cecal phosphorus (P) digestibility of 5 P sources, 3 from animal (Delfos, Calfos, and porcine bone meal) and 2 of inorganic (monocalcium phosphate [MCP] and dicalcium phosphate [DCP]) origin. Delfos is processed from bones resulting in a dicalcium phosphate product, and Calfos is processed from bones in which part of the gelatin is removed but in which the hydroxy-apatite matrix is preserved. During the first 14 d, birds were housed in floor pens bedded with wood shavings and received a commercial starter diet. At d 14, broilers were randomly assigned to pens (0.9 m2, 10 birds/pen) with a slatted floor. From d 14 onwards, one of the 6 experimental diets (a basal diet, and 5 diets containing the P sources) was provided. Test diets were replicated 6 times, and the basal diet 8 times. Electron microscopy images of test products were made in order to verify whether the spatial structure of the test products could be related to the pre-cecal P digestibility of the same products. Diets met or exceeded CVB (2011) requirements for all nutrients except for P and were formulated to contain a calcium to total P ratio of between 1.4 and 1.6 and a minimal amount of phytate P. Diets contained 5 g/kg titanium oxide as a marker to determine digestibility of P. At d 24 all birds were euthanized, after which the content of the terminal part of the ileum was sampled. The P digestibility was calculated by linear regression according to World's Poultry Science Association (WPSA) protocol for determination of pre-cecal P digestibility. Pre-cecal P digestibility of MCP, DCP, Delfos, Calfos, and porcine bone meal was 88.5, 82.4, 94.5, 86.9, and 78.2%, respectively. Based on visual inspection of electron microscopy images of test products, the spatial structure of the test products might be related to P digestibility. It is concluded that processing of bone meal increases the pre-cecal P digestibility in broilers. © The Author 2017

  9. Calcium Phosphate Product Is Associated with Subclinical Carotid Atherosclerosis in Type 2 Diabetes

    PubMed Central

    Ramírez-Morros, Anna; Granado-Casas, Minerva; Martinez-Alonso, Montserrat; Real, Jordi; Castelblanco, Esmeralda; Esquerda, Aureli; Cao, Gonzalo; Alonso, Núria

    2017-01-01

    Aims To assess whether circulating 25-hydroxyvitamin D3 (25OHD) and mineral metabolism-related factors (serum phosphate, calcium, and parathormone) are associated with subclinical carotid atherosclerosis (SCA), defined as the presence of carotid atherosclerotic plaques (main study outcome), in patients with type 2 diabetes mellitus (T2DM) without kidney disease or previous cardiovascular disease. Methods We undertook a post hoc analysis of a cross-sectional study in adults with T2DM in whom we evaluated SCA. A total of 303 subjects with T2DM were included. Clinical variables and carotid ultrasound imaging were obtained. Results We found no association of 25OHD with the presence of SCA. However, calcium phosphate (CaP; mg2/dL2) product was positively associated with the presence of carotid plaques (ORadj = 1.078; 95% CI: 1.017–1.142). An inverse association was observed between higher levels of 25OHD (≥30 ng/mL versus <20 ng/mL concentrations) and common carotid intima-media thickness (cIMT; mm) (βadj ± SE = −0.055 ± 0.024). We conclude that the CaP product is independently associated with the presence of established subclinical carotid atherosclerosis in patients with T2DM. PMID:28840128

  10. A Study Update of Mortality in Workers at a Phosphate Fertilizer Production Facility

    PubMed Central

    Yiin, James H.; Daniels, Robert D.; Kubale, Travis L.; Dunn, Kevin L.; Stayner, Leslie T.

    2016-01-01

    Objective To evaluate the mortality experience among 3,199 workers employed 1951–1976 at a phosphate fertilizer production plant in central Florida with follow-up through2011. Methods Cause-specific standardized mortality ratios (SMRs) for the full cohort were calculated with the U.S. population as referent. Lung cancer and leukemia risks were further analyzed using conditional logistic regression. Results The mortality due to all-causes (SMR = 1.07, 95% confidence interval [CI] 1.02–1.13, observed deaths [n] = 1,473), all-cancers (SMR = 1.16, 95%CI 1.06–1.28, n = 431), and a priori outcomes of interests including lung cancer (SMR = 1.32, 95%CI = 1.13–1.53, n = 168) and leukemia (SMR = 1.74, 95%CI = 1.11–2.62, n = 23) were statistically significantly elevated. Regression modeling on employment duration or estimated radiation scores did not show exposure–response relation with lung cancer or leukemia mortality. Conclusion SMR results showed increased lung cancer and leukemia mortality in a full cohort of the phosphate fertilizer production facility. There was, however, no exposure–response relation observed among cases and matched controls. PMID:26523937

  11. A study update of mortality in workers at a phosphate fertilizer production facility.

    PubMed

    Yiin, James H; Daniels, Robert D; Kubale, Travis L; Dunn, Kevin L; Stayner, Leslie T

    2016-01-01

    To evaluate the mortality experience among 3,199 workers employed 1951-1976 at a phosphate fertilizer production plant in central Florida with follow-up through 2011. Cause-specific standardized mortality ratios (SMRs) for the full cohort were calculated with the U.S. population as referent. Lung cancer and leukemia risks were further analyzed using conditional logistic regression. The mortality due to all-causes (SMR = 1.07, 95% confidence interval [CI] 1.02-1.13, observed deaths [n] = 1,473), all-cancers (SMR = 1.16, 95%CI 1.06-1.28, n = 431), and a priori outcomes of interests including lung cancer (SMR = 1.32, 95%CI = 1.13-1.53, n = 168) and leukemia (SMR = 1.74, 95%CI = 1.11-2.62, n = 23) were statistically significantly elevated. Regression modeling on employment duration or estimated radiation scores did not show exposure-response relation with lung cancer or leukemia mortality. SMR results showed increased lung cancer and leukemia mortality in a full cohort of the phosphate fertilizer production facility. There was, however, no exposure-response relation observed among cases and matched controls. © 2015 Wiley Periodicals, Inc.

  12. Biochar enhances Aspergillus niger rock phosphate solubilization by increasing organic acid production and alleviating fluoride toxicity.

    PubMed

    Mendes, Gilberto de Oliveira; Zafra, David Lopez; Vassilev, Nikolay Bojkov; Silva, Ivo Ribeiro; Ribeiro, José Ivo; Costa, Maurício Dutra

    2014-05-01

    During fungal rock phosphate (RP) solubilization, a significant quantity of fluoride (F(-)) is released together with phosphorus (P), strongly inhibiting the process. In the present study, the effect of two F(-) adsorbents [activated alumina (Al2O3) and biochar] on RP solubilization by Aspergillus niger was examined. Al2O3 adsorbed part of the F(-) released but also adsorbed soluble P, which makes it inappropriate for microbial RP solubilization systems. In contrast, biochar adsorbed only F(-) while enhancing phosphate solubilization 3-fold, leading to the accumulation of up to 160 mg of P per liter. By comparing the values of F(-) measured in solution at the end of incubation and those from a predictive model, it was estimated that up to 19 mg of F(-) per liter can be removed from solution by biochar when added at 3 g liter(-1) to the culture medium. Thus, biochar acted as an F(-) sink during RP solubilization and led to an F(-) concentration in solution that was less inhibitory to the process. In the presence of biochar, A. niger produced larger amounts of citric, gluconic, and oxalic acids, whether RP was present or not. Our results show that biochar enhances RP solubilization through two interrelated processes: partial removal of the released F(-) and increased organic acid production. Given the importance of organic acids for P solubilization and that most of the RPs contain high concentrations of F(-), the proposed solubilization system offers an important technological improvement for the microbial production of soluble P fertilizers from RP.

  13. Phosphoinositol 3-phosphate acts as a timer for reactive oxygen species production in the phagosome.

    PubMed

    Song, Zhi Min; Bouchab, Leïla; Hudik, Elodie; Le Bars, Romain; Nüsse, Oliver; Dupré-Crochet, Sophie

    2017-01-17

    Production of reactive oxygen species (ROS) in the phagosome by the NADPH oxidase is critical for mammalian immune defense against microbial infections and phosphoinositides are important regulators in this process. Phosphoinositol 3-phosphate (PI(3)P) regulates ROS production at the phagosome via p40(phox) by an unknown mechanism. This study tested the hypothesis that PI(3)P controls ROS production by regulating the presence of p40(phox) and p67(phox) at the phagosomal membrane. Pharmacologic inhibition of PI(3)P synthesis at the phagosome decreased the ROS production both in differentiated PLB-985 cells and human neutrophils. It also releases p67(phox), the key cytosolic subunit of the oxidase, and p40(phox) from the phagosome. The knockdown of the PI(3)P phosphatase MTM1 or Rubicon or both increases the level of PI(3)P at the phagosome. That increase enhances ROS production inside the phagosome and triggers an extended accumulation of p67(phox) at the phagosome. Furthermore, the overexpression of MTM1 at the phagosomal membrane induces the disappearance of PI(3)P from the phagosome and prevents sustained ROS production. In conclusion, PI(3)P, indeed, regulates ROS production by maintaining p40(phox) and p67(phox) at the phagosomal membrane.

  14. Structural basis for phosphoinositide substrate recognition, catalysis, and membrane interactions in human inositol polyphosphate 5-phosphatases.

    PubMed

    Trésaugues, Lionel; Silvander, Camilla; Flodin, Susanne; Welin, Martin; Nyman, Tomas; Gräslund, Susanne; Hammarström, Martin; Berglund, Helena; Nordlund, Pär

    2014-05-06

    SHIP2, OCRL, and INPP5B belong to inositol polyphosphate 5-phophatase subfamilies involved in insulin regulation and Lowes syndrome. The structural basis for membrane recognition, substrate specificity, and regulation of inositol polyphosphate 5-phophatases is still poorly understood. We determined the crystal structures of human SHIP2, OCRL, and INPP5B, the latter in complex with phosphoinositide substrate analogs, which revealed a membrane interaction patch likely to assist in sequestering substrates from the lipid bilayer. Residues recognizing the 1-phosphate of the substrates are highly conserved among human family members, suggesting similar substrate binding modes. However, 3- and 4-phosphate recognition varies and determines individual substrate specificity profiles. The high conservation of the environment of the scissile 5-phosphate suggests a common reaction geometry for all members of the human 5-phosphatase family.

  15. Protection against cancer by dietary IP6 and inositol.

    PubMed

    Vucenik, Ivana; Shamsuddin, AbulKalam M

    2006-01-01

    Inositol hexaphosphate (IP(6)) is a naturally occurring polyphosphorylated carbohydrate, abundantly present in many plant sources and in certain high-fiber diets, such as cereals and legumes. In addition to being found in plants, IP(6) is contained in almost all mammalian cells, although in much smaller amounts, where it is important in regulating vital cellular functions such as signal transduction, cell proliferation, and differentiation. For a long time IP(6) has been recognized as a natural antioxidant. Recently IP(6) has received much attention for its role in cancer prevention and control of experimental tumor growth, progression, and metastasis. In addition, IP(6) possesses other significant benefits for human health, such as the ability to enhance immune system, prevent pathological calcification and kidney stone formation, lower elevated serum cholesterol, and reduce pathological platelet activity. In this review we show the efficacy and discuss some of the molecular mechanisms that govern the action of this dietary agent. Exogenously administered IP(6) is rapidly taken up into cells and dephosphorylated to lower inositol phosphates, which further affect signal transduction pathways resulting in cell cycle arrest. A striking anticancer action of IP(6) was demonstrated in different experimental models. In addition to reducing cell proliferation, IP(6) also induces differentiation of malignant cells. Enhanced immunity and antioxidant properties also contribute to tumor cell destruction. Preliminary studies in humans show that IP(6) and inositol, the precursor molecule of IP(6), appear to enhance the anticancer effect of conventional chemotherapy, control cancer metastases, and improve quality of life. Because it is abundantly present in regular diet, efficiently absorbed from the gastrointestinal tract, and safe, IP(6) + inositol holds great promise in our strategies for cancer prevention and therapy. There is clearly enough evidence to justify the

  16. Determination of myo-inositol (free and bound as phosphatidylinositol) in infant formula and adult nutritionals by liquid chromatography/pulsed amperometry with column switching: first action 2011.18.

    PubMed

    Schimpf, Karen; Thompson, Linda; Baugh, Steve

    2012-01-01

    Myo-inositol is a 6-carbon cyclic polyalcohol also known as meso-inositol, meat sugar, inosite, and i-inositol. It occurs in nature in both free (myo-inositol) and bound (inositol phosphates and phosphatidylinositol) forms. For the determination of free myo-inositol, samples are mixed with dilute hydrochloric acid to extract myo-inositol and precipitate proteins, diluted with water, and filtered. For the determination of myo-inositol bound as phosphatidylinositol, samples are extracted with chloroform, isolated from other fats with silica SPE cartridges, and hydrolyzed with concentrated acid to free myo-inositol. Prepared samples are first injected onto a Dionex CarboPac PA1 column, which separates myo-inositol from other late-eluting carbohydrates. After column switching, myo-inositol is further separated on a CarboPac MA1 column using a 0.12% sodium hydroxide mobile phase; strongly retained carbohydrates are eluted from the PA1 column with a 3% sodium hydroxide mobile phase. Eluant from the CarboPac MA1 analytical column passes through an electrochemical detector cell where myo-inositol is detected by pulsed amperometry using a gold electrode. The method showed appropriate performance characteristics versus selected established standard method performance requirement parameters for the determination of myo-inositol: linear response; repeatability (RSDr) of 2%; and intermediate precision (RSDir) of 2.5%. Instrument LOD and LOQ were 0.0004 and 0.0013 mg/100 mL, respectively, and correspond to a free myo-inositol quantitation limit of 0.026 mg/100 g and a phosphatidylinositol quantitation limit of 0.016 mg/100 g. Correlation with the reference microbiological assay was good. The proposed method has been accepted by the Expert Review Panel as an AOAC First Action Method, suitable for the routine determination of myo-inositol in infant formula and adult nutritionals.

  17. Stimulation of acid secretion and phosphoinositol production by rat parietal cell muscarinic M sub 2 receptors

    SciTech Connect

    Pfeiffer, A.; Rochlitz, H.; Herz, A.; Paumgartner, G. )

    1988-04-01

    The muscarinic receptor system involved in hydrogen production by enriched rat gastric parietal cells was investigated. Muscarinic receptor density determined by (N-methyl-{sup 3}H)scopolamine binding was 8,100/cell. The receptor appeared to be of the M{sub 2} muscarinic receptor subtype, since it had a low affinity (K{sub d} 189 nM) for the M{sub 1} receptor antagonist pirenzepine compared with atropine. Receptor activation by carbachol rapidly augmented levels of polyphosphoinositides, indicating an activation of phospholipase C. The dose-response relations for the increase in inositol phosphates closely paralleled the binding of carbachol to muscarinic receptors. The inositol phosphate response was antagonized by pirenzepine with a K{sub i} of 177 nM. the stimulation of inositol phosphate levels by carbachol correlated well with the stimulation of ({sup 14}C)aminopyrine uptake, determine as an index of acid secretion. The muscarinic agonists oxotremorine, pilocarpine, and bethanechol elicited partial increases in inositol phosphates at maximal drug concentrations, and these partial increases correlated with their ability to stimulate ({sup 14}C)aminopyrine uptake. These data indicate that inositolpolyphosphates may be a second messenger of M{sub 2} receptors stimulating acid secretion.

  18. 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

  19. Impact of Phosphate, Potassium, Yeast Extract, and Trace Metals on Chitosan and Metabolite Production by Mucor indicus.

    PubMed

    Safaei, Zahra; Karimi, Keikhosro; Zamani, Akram

    2016-08-30

    In this study the effects of phosphate, potassium, yeast extract, and trace metals on the growth of Mucor indicus and chitosan, chitin, and metabolite production by the fungus were investigated. Maximum yield of chitosan (0.32 g/g cell wall) was obtained in a phosphate-free medium. Reversely, cell growth and ethanol formation by the fungus were positively affected in the presence of phosphate. In a phosphate-free medium, the highest chitosan content (0.42 g/g cell wall) and cell growth (0.66 g/g sugar) were obtained at 2.5 g/L of KOH. Potassium concentration had no significant effect on ethanol and glycerol yields. The presence of trace metals significantly increased the chitosan yield at an optimal phosphate and potassium concentration (0.50 g/g cell wall). By contrast, production of ethanol by the fungus was negatively affected (0.33 g/g sugars). A remarkable increase in chitin and decrease in chitosan were observed in the absence of yeast extract and concentrations lower than 2 g/L. The maximum chitosan yield of 51% cell wall was obtained at 5 g/L of yeast extract when the medium contained no phosphate, 2.5 g/L KOH, and 1 mL/L trace metal solution.

  20. Impact of Phosphate, Potassium, Yeast Extract, and Trace Metals on Chitosan and Metabolite Production by Mucor indicus

    PubMed Central

    Safaei, Zahra; Karimi, Keikhosro; Zamani, Akram

    2016-01-01

    In this study the effects of phosphate, potassium, yeast extract, and trace metals on the growth of Mucor indicus and chitosan, chitin, and metabolite production by the fungus were investigated. Maximum yield of chitosan (0.32 g/g cell wall) was obtained in a phosphate-free medium. Reversely, cell growth and ethanol formation by the fungus were positively affected in the presence of phosphate. In a phosphate-free medium, the highest chitosan content (0.42 g/g cell wall) and cell growth (0.66 g/g sugar) were obtained at 2.5 g/L of KOH. Potassium concentration had no significant effect on ethanol and glycerol yields. The presence of trace metals significantly increased the chitosan yield at an optimal phosphate and potassium concentration (0.50 g/g cell wall). By contrast, production of ethanol by the fungus was negatively affected (0.33 g/g sugars). A remarkable increase in chitin and decrease in chitosan were observed in the absence of yeast extract and concentrations lower than 2 g/L. The maximum chitosan yield of 51% cell wall was obtained at 5 g/L of yeast extract when the medium contained no phosphate, 2.5 g/L KOH, and 1 mL/L trace metal solution. PMID:27589726

  1. 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

  2. Polymer coating for immobilizing soluble ions in a phosphate ceramic product

    DOEpatents

    Singh, Dileep; Wagh, Arun S.; Patel, Kartikey D.

    2000-01-01

    A polymer coating is applied to the surface of a phosphate ceramic composite to effectively immobilize soluble salt anions encapsulated within the phosphate ceramic composite. The polymer coating is made from ceramic materials, including at least one inorganic metal compound, that wet and adhere to the surface structure of the phosphate ceramic composite, thereby isolating the soluble salt anions from the environment and ensuring long-term integrity of the phosphate ceramic composite.

  3. Polymer Coating for Immobilizing Soluble Ions in a Phosphate Ceramic Product

    SciTech Connect

    Singh, Dileep; Wagh, Arun S.; Patel, Kartikey D.

    1999-05-05

    A polymer coating is applied to the surface of a phosphate ceramic composite to effectively immobilize soluble salt anions encapsulated within the phosphate ceramic composite. The polymer coating is made from ceramic materials, including at least one inorganic metal compound, that wet and adhere to the surface structure of the phosphate ceramic composite, thereby isolating the soluble salt anions from the environment and ensuring long-term integrity of the phosphate ceramic composite.

  4. The impact of phosphate scarcity on pharmaceutical protein production in S. cerevisiae: linking transcriptomic insights to phenotypic responses

    PubMed Central

    2011-01-01

    Background The adaptation of unicellular organisms like Saccharomyces cerevisiae to alternating nutrient availability is of great fundamental and applied interest, as understanding how eukaryotic cells respond to variations in their nutrient supply has implications spanning from physiological insights to biotechnological applications. Results The impact of a step-wise restricted supply of phosphate on the physiological state of S. cerevisiae cells producing human Insulin was studied. The focus was to determine the changes within the global gene expression of cells being cultured to an industrially relevant high cell density of 33 g/l cell dry weight and under six distinct phosphate concentrations, ranging from 33 mM (unlimited) to 2.6 mM (limited). An increased flux through the secretory pathway, being induced by the PHO circuit during low Pi supplementation, proved to enhance the secretory production of the heterologous protein. The re-distribution of the carbon flux from biomass formation towards increased glycerol production under low phosphate led to increased transcript levels of the insulin gene, which was under the regulation of the TPI1 promoter. Conclusions Our study underlines the dynamic character of adaptive responses of cells towards a change in their nutrient access. The gradual decrease of the phosphate supply resulted in a step-wise modulated phenotypic response, thereby alternating the specific productivity and the secretory flux. Our work emphasizes the importance of reduced phosphate supply for improved secretory production of heterologous proteins. PMID:22151908

  5. Production of cellulose phosphate from oil palm empty fruit bunch: Effect of chemical ratio

    NASA Astrophysics Data System (ADS)

    Rohaizu, R.; Wanrosli, W. D.

    2015-06-01

    Cellulose phosphate was synthesized from oil palm biomass residue that has the potential to represent a considerable added value product for the oil palm biomass utilization. Cellulose phosphate (CP) is prepared viaa phosphorylation process using the H3PO4/P2O5/Et3PO4/hexanol sequence using oil palm empty fruit bunch microcrystalline cellulose (OPEFB-MCC) as the starting material. Various factors affect its synthesis; one of them which is the subject of this investigation is the orthophosphoric acid (H3PO4) to triethylphosphate(Et3PO4) ratio which have the capability to increase the phosphorus content of CP. It is believed that during this reaction, the esterification of the free hydroxyl groups of the cellulose occurred. The H3PO4/Et3PO4 ratios applied were 0.16, 1.00, and 1.84. The effect of the H3PO4/Et3PO4 ratio on phosphorus content, yield, water swelling and molecular structure of CP are discussed.

  6. Initial pH of medium affects organic acids production but do not affect phosphate solubilization.

    PubMed

    Marra, Leandro M; de Oliveira-Longatti, Silvia M; Soares, Cláudio R F S; de Lima, José M; Olivares, Fabio L; Moreira, Fatima M S

    2015-06-01

    The pH of the culture medium directly influences the growth of microorganisms and the chemical processes that they perform. The aim of this study was to assess the influence of the initial pH of the culture medium on the production of 11 low-molecular-weight organic acids and on the solubilization of calcium phosphate by bacteria in growth medium (NBRIP). The following strains isolated from cowpea nodules were studied: UFLA03-08 (Rhizobium tropici), UFLA03-09 (Acinetobacter sp.), UFLA03-10 (Paenibacillus kribbensis), UFLA03-106 (Paenibacillus kribbensis) and UFLA03-116 (Paenibacillus sp.). The strains UFLA03-08, UFLA03-09, UFLA03-10 and UFLA03-106 solubilized Ca3(PO4)2 in liquid medium regardless of the initial pH, although without a significant difference between the treatments. The production of organic acids by these strains was assessed for all of the initial pH values investigated, and differences between the treatments were observed. Strains UFLA03-09 and UFLA03-10 produced the same acids at different initial pH values in the culture medium. There was no correlation between phosphorus solubilized from Ca3(PO4)2 in NBRIP liquid medium and the concentration of total organic acids at the different initial pH values. Therefore, the initial pH of the culture medium influences the production of organic acids by the strains UFLA03-08, UFLA03-09, UFLA03-10 and UFLA03-106 but it does not affect calcium phosphate solubilization.

  7. Initial pH of medium affects organic acids production but do not affect phosphate solubilization

    PubMed Central

    Marra, Leandro M.; de Oliveira-Longatti, Silvia M.; Soares, Cláudio R.F.S.; de Lima, José M.; Olivares, Fabio L.; Moreira, Fatima M.S.

    2015-01-01

    The pH of the culture medium directly influences the growth of microorganisms and the chemical processes that they perform. The aim of this study was to assess the influence of the initial pH of the culture medium on the production of 11 low-molecular-weight organic acids and on the solubilization of calcium phosphate by bacteria in growth medium (NBRIP). The following strains isolated from cowpea nodules were studied: UFLA03-08 (Rhizobium tropici), UFLA03-09 (Acinetobacter sp.), UFLA03-10 (Paenibacillus kribbensis), UFLA03-106 (Paenibacillus kribbensis) and UFLA03-116 (Paenibacillus sp.). The strains UFLA03-08, UFLA03-09, UFLA03-10 and UFLA03-106 solubilized Ca3(PO4)2 in liquid medium regardless of the initial pH, although without a significant difference between the treatments. The production of organic acids by these strains was assessed for all of the initial pH values investigated, and differences between the treatments were observed. Strains UFLA03-09 and UFLA03-10 produced the same acids at different initial pH values in the culture medium. There was no correlation between phosphorus solubilized from Ca3(PO4)2 in NBRIP liquid medium and the concentration of total organic acids at the different initial pH values. Therefore, the initial pH of the culture medium influences the production of organic acids by the strains UFLA03-08, UFLA03-09, UFLA03-10 and UFLA03-106 but it does not affect calcium phosphate solubilization. PMID:26273251

  8. Inositol trisphosphate stimulates calcium release from peeled skeletal muscle fibers.

    PubMed

    Donaldson, S K; Goldberg, N D; Walseth, T F; Huetteman, D A

    1987-01-19

    The effects of inositol phosphates (tris (InsP3), bis (InsP2), mono (InsP)) on rabbit adductor magnus and soleus muscles were determined using mechanically peeled fibers (sarcolemma removed). Isometric force generation of each fiber was continuously monitored and was used along with 45Ca to detect calcium release from internal fiber stores. All experiments were conducted at a physiological Mg2+ concentration (10(-3) M) of the bathing solutions. The inositol phosphates did not directly activate the contractile apparatus. At bath concentrations of 100-300 microM, only InsP3 was capable of stimulating Ca2+ release. In contrast, 1 microM InsP3 maximally and selectively stimulated Ca2+ release when microinjected into the myofilament lattice. Calcium releasing effects of InsP2 and InsP were manifested at 10 microM when they were microinjected. The end-to-end internal Ca2+ release and subsequent fiber force generation stimulated by the locally applied microinjected InsP3 suggests that the InsP3-induced Ca2+ release mechanism may involve propagation, but not via the Ca2+-induced Ca2+ release, since procaine did not inhibit this response. These findings support the possibility that InsP3 plays a role in skeletal muscle excitation-contraction coupling.

  9. 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

  10. Inositol 1,3,4,5,6-pentakisphosphate 2-kinase is a distant IPK member with a singular inositide binding site for axial 2-OH recognition

    PubMed Central

    González, Beatriz; Baños-Sanz, Jose Ignacio; Villate, Maider; Brearley, Charles Alistair; Sanz-Aparicio, Julia

    2010-01-01

    Inositol phosphates (InsPs) are signaling molecules with multiple roles in cells. In particular (InsP6) is involved in mRNA export and editing or chromatin remodeling among other events. InsP6 accumulates as mixed salts (phytate) in storage tissues of plants and plays a key role in their physiology. Human diets that are exclusively grain-based provide an excess of InsP6 that, through chelation of metal ions, may have a detrimental effect on human health. Ins(1,3,4,5,6)P5 2-kinase (InsP5 2-kinase or Ipk1) catalyses the synthesis of InsP6 from InsP5 and ATP, and is the only enzyme that transfers a phosphate group to the axial 2-OH of the myo-inositide. We present the first structure for an InsP5 2-kinase in complex with both substrates and products. This enzyme presents a singular structural region for inositide binding that encompasses almost half of the protein. The key residues in substrate binding are identified, with Asp368 being responsible for recognition of the axial 2-OH. This study sheds light on the unique molecular mechanism for the synthesis of the precursor of inositol pyrophosphates. PMID:20453199

  11. Regioselective Opening of myo-Inositol Orthoesters: Mechanism and Synthetic Utility

    PubMed Central

    2013-01-01

    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. PMID:23438216

  12. Potential Application of Biohydrogen Production Liquid Waste as Phosphate Solubilizing Agent-A Study Using Soybean Plants.

    PubMed

    Sarma, Saurabh Jyoti; Brar, Satinder Kaur; LeBihan, Yann; Buelna, Gerardo

    2016-03-01

    With CO2 free emission and a gravimetric energy density higher than gasoline, diesel, biodiesel, and bioethanol, biohydrogen is a promising green renewable energy carrier. During fermentative hydrogen production, 60-70 % of the feedstock is converted to different by-products, dominated by organic acids. In the present investigation, a simple approach for value addition of hydrogen production liquid waste (HPLW) containing these compounds has been demonstrated. In soil, organic acids produced by phosphate solubilizing bacteria chelate the cations of insoluble inorganic phosphates (e.g., Ca3 (PO4)2) and make the phosphorus available to the plants. Organic acid-rich HPLW, therefore, has been evaluated as soil phosphate solubilizer. Application of HPLW as soil phosphate solubilizer was found to improve the phosphorus uptake of soybean plants by 2.18- to 2.74-folds. Additionally, 33-100 % increase in seed germination rate was also observed. Therefore, HPLW has the potential to be an alternative for phosphate solubilizing biofertilizers available in the market. Moreover, the strategy can be useful for phytoremediation of phosphorus-rich soil.

  13. Stereospecificity of myo-inositol hexakisphosphate hydrolysis by a protein tyrosine phosphatase-like inositol polyphosphatase from Megasphaera elsdenii.

    PubMed

    Puhl, Aaron A; Greiner, Ralf; Selinger, L Brent

    2009-02-01

    Inositol polyphosphatases (IPPases), particularly those that can hydrolyze myo-inositol hexakisphosphate (Ins P(6)), are of biotechnological interest for their ability to reduce the metabolically unavailable organic phosphate content of feedstuffs and to produce lower inositol polyphosphates (IPPs) for research and pharmaceutical applications. Here, the gene coding for a new protein tyrosine phosphatase (PTP)-like IPPase was cloned from Megasphaera elsdenii (phyAme), and the biochemical properties of the recombinant protein were determined. The deduced amino acid sequence of PhyAme is similar to known PTP-like IPPases (29-44% identity), and the recombinant enzyme displayed strict specificity for IPP substrates. Optimal IPPase activity was displayed at an ionic strength of 250 mM, a pH of 5.0, and a temperature of 60 degrees C. In order to elucidate its stereospecificity of Ins P(6) dephosphorylation, a combination of high-performance ion-pair chromatography and kinetic studies was conducted. PhyAme displayed a stereospecificity that is unique among enzymes belonging to this class in that it preferentially cleaved Ins P(6) at one of two phosphate positions, 1D-3 or 1D-4. PhyAme followed two distinct and specific routes of hydrolysis, predominantly degrading Ins P(6) to Ins(2)P via: (a) 1D-Ins(1,2,4,5,6)P(5), 1D-Ins(1,2,5,6)P(4), 1D-Ins(1,2,6)P(3), and 1D-Ins(1,2)P(2) (60%) and (b) 1D-Ins(1,2,3,5,6)P(5), 1D-Ins(1,2,3,6)P(4), Ins(1,2,3)P(3), and D/L-Ins(1,2)P(2)(35%).

  14. Effects of inositol, inositol-generating phytase B applied alone, and in combination with 6-phytase A to phosphorus-deficient diets on laying performance, eggshell quality, yolk cholesterol, and fatty acid deposition in laying hens.

    PubMed

    Zyla, K; Mika, M; Duliński, R; Swiatkiewicz, S; Koreleski, J; Pustkowiak, H; Piironen, J

    2012-08-01

    Phytase B, a product of Aspergillus niger phyB gene expressed in Trichoderma reesei, which increased myo-inositol concentrations in 20 mM sodium phytate solution 7.5-fold during 120-min incubation, a combination of phytase B with 6-phytase A, and pure myo-inositol were tested as feed supplements in Bovans Brown laying hens. In the 2-factorial experiment (2×5), birds from wk 50 to 62 were fed 2 basal diets, corn-soybean (CSM) or wheat-soybean (WSM), using 12 one-hen cages per treatment. For both basal diets, the dietary treatments included negative control (0.08% nonphytate P in CSM, 0.13% nonphytate P in WSM; NC); internal control groups, NC+0.04% nonphytate P from monocalcium phosphate, MCP (IC); NC+0.1% of myo-inositol (Inos), NC+phytase B at 1,300 units of phytase B-acid phosphatase activity (AcPU)/kg (PhyB), NC+phytase B at 1,300 AcPU/kg+6-phytase A at 300 FTU/kg (PhyA+B). Feed intake, laying performance, and eggshell quality were determined. The total lipid and cholesterol contents as well as fatty acid profile were assessed in egg yolks collected from hens fed CSM diets, as was fatty acid profile. The hens fed the WSM diet consumed significantly more feed, laid a higher mass of eggs daily with higher mean weights, and had a higher hen-day egg production than the birds receiving the CSM diets. Similarly, higher values for yolk weights, shell weights, shell thickness, shell density, and breaking strengths were determined in the eggs laid by the hens fed the WSM diets. In hens fed either the CSM diets with phytase B alone, or in combination with 6-phytase A, enhanced feed intakes, egg mass, and hen-day egg production were recorded. Phytases also enhanced the eggshell quality parameters in the hens fed both variants of the diets. Phytase B alone, or in combination with 6-phytase A, reduced the total lipid and cholesterol concentrations in egg yolks collected from the hens fed the CSM diets, whereas the combination of both phytases improved the n-6:n-3

  15. The effects of inorganic phosphate and creatine phosphate on force production in skinned muscles from rat ventricle.

    PubMed Central

    Kentish, J C

    1986-01-01

    Thin ventricular trabeculae from rat hearts were skinned with the non-ionic detergent Triton X-100. The isometric force development of these muscles was investigated over a range of Ca2+ concentrations (0.2-200 microM) in the presence of various concentrations of creatine phosphate (CP), creatine and inorganic phosphate (Pi). The addition of Pi (1-30 microM) at constant ionic strength reduced the maximum Ca2+-regulated force (obtained at 200 microM-Ca2+). At 20 mM-Pi force was 31 +/- 5% (mean +/- S.E. of mean, n = 7) of that in Pi-free solution. This inhibitory action of Pi was not due to an inhibition of creatine kinase activity or to a reduction in the amount of free energy available for work from ATP hydrolysis. Increases in Pi concentration over the range 0-20 mM shifted the sigmoid relationship between force and [Ca2+] to higher Ca2+ concentrations. The [Ca2+] required for 50% activation rose from 8.3 +/- 1.5 microM (mean +/- S.E. of mean, n = 7) in Pi-free solutions to 19.8 +/- 4.2 microM in 20 mM-Pi. An increase in CP concentration in the range 10-30 mM had a small inhibitory effect on maximum Ca2+-regulated force but did not alter the force--[Ca2+] relationship. Creatine (0-30 mM) was without effect on the skinned muscles. The inhibitory effects of Pi suggest that a net hydrolysis of CP to Pi and creatine in the myoplasm of intact cardiac cells could reduce force development at a given myoplasmic [Ca2+], especially if the latter was below the level needed to fully activate the myofibrils. This suggestion is discussed in relation to the CP hydrolysis and decrease in force development that are observed in cardiac muscle during hypoxia or ischaemia. PMID:3958986

  16. Shear effects on aluminum phosphate adjuvant particle properties in vaccine drug products.

    PubMed

    Kolade, Olatomirin O; Jin, Wenbin; Tengroth, Charbel; Green, Kenneth D; Bracewell, Daniel G

    2015-02-01

    Adjuvant-containing drug products can be exposed to high levels of interfacial shear during manufacture. This may affect the integrity of the adjuvant, alter its interaction with the drug substance or change the physical characteristics of the drug product. In this study, a solid-liquid interfacial shear device was used to investigate the shear response of aluminum phosphate adjuvant alone and two adjuvant containing vaccine drug products (DP1 and DP2). The relationship between the shear sensitivity of each and its resuspension properties was determined. Changes in the particle dimensions of the bulk adjuvant were minimal at shear strain rates of 10,900 s(-1) . However, at 25,500 s(-1) , the median particle diameter was reduced from 6.2 to 3.5 μm and was marked by the presence of sub-micron fines. A formulation without drug substance and DP2 produced similar shear responses but with less impact on particle diameter. The behavior of DP1 was less predictable. Sheared DP1 was characterized by prolonged sedimentation because of the presence of fine particulates and required in excess of 300 rotations to resuspend after extended storage. The study confirms that the solid-liquid interfacial shear device may be applied to understand product shear sensitivity associated with vaccine manufacturing. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

  17. Biochar Enhances Aspergillus niger Rock Phosphate Solubilization by Increasing Organic Acid Production and Alleviating Fluoride Toxicity

    PubMed Central

    Mendes, Gilberto de Oliveira; Zafra, David Lopez; Vassilev, Nikolay Bojkov; Silva, Ivo Ribeiro; Ribeiro, José Ivo

    2014-01-01

    During fungal rock phosphate (RP) solubilization, a significant quantity of fluoride (F−) is released together with phosphorus (P), strongly inhibiting the process. In the present study, the effect of two F− adsorbents [activated alumina (Al2O3) and biochar] on RP solubilization by Aspergillus niger was examined. Al2O3 adsorbed part of the F− released but also adsorbed soluble P, which makes it inappropriate for microbial RP solubilization systems. In contrast, biochar adsorbed only F− while enhancing phosphate solubilization 3-fold, leading to the accumulation of up to 160 mg of P per liter. By comparing the values of F− measured in solution at the end of incubation and those from a predictive model, it was estimated that up to 19 mg of F− per liter can be removed from solution by biochar when added at 3 g liter−1 to the culture medium. Thus, biochar acted as an F− sink during RP solubilization and led to an F− concentration in solution that was less inhibitory to the process. In the presence of biochar, A. niger produced larger amounts of citric, gluconic, and oxalic acids, whether RP was present or not. Our results show that biochar enhances RP solubilization through two interrelated processes: partial removal of the released F− and increased organic acid production. Given the importance of organic acids for P solubilization and that most of the RPs contain high concentrations of F−, the proposed solubilization system offers an important technological improvement for the microbial production of soluble P fertilizers from RP. PMID:24610849

  18. Inositol, neural tube closure and the prevention of neural tube defects

    PubMed Central

    Leung, Kit‐Yi; Copp, Andrew J.

    2017-01-01

    Susceptibility to neural tube defects (NTDs), such as anencephaly and spina bifida is influenced by genetic and environmental factors including maternal nutrition. Maternal periconceptional supplementation with folic acid significantly reduces the risk of an NTD‐affected pregnancy, but does not prevent all NTDs, and “folic acid non‐responsive” NTDs continue to occur. Similarly, among mouse models of NTDs, some are responsive to folic acid but others are not. Among nutritional factors, inositol deficiency causes cranial NTDs in mice while supplemental inositol prevents spinal and cranial NTDs in the curly tail (Grhl3 hypomorph) mouse, rodent models of hyperglycemia or induced diabetes, and in a folate‐deficiency induced NTD model. NTDs also occur in mice lacking expression of certain inositol kinases. Inositol‐containing phospholipids (phosphoinositides) and soluble inositol phosphates mediate a range of functions, including intracellular signaling, interaction with cytoskeletal proteins, and regulation of membrane identity in trafficking and cell division. Myo‐inositol has been trialed in humans for a range of conditions and appears safe for use in human pregnancy. In pilot studies in Italy and the United Kingdom, women took inositol together with folic acid preconceptionally, after one or more previous NTD‐affected pregnancies. In nonrandomized cohorts and a randomized double‐blind study in the United Kingdom, no recurrent NTDs were observed among 52 pregnancies reported to date. Larger‐scale fully powered trials are needed to determine whether supplementation with inositol and folic acid would more effectively prevent NTDs than folic acid alone. Birth Defects Research 109:68–80, 2017. © 2016 The Authors Birth Defects Research Published by Wiley Periodicals, Inc. PMID:27324558

  19. Effects of combined inositol hexakisphosphate and inositol supplement on antioxidant activity and metabolic enzymes in the liver of streptozotocin-induced type 2 diabetic rats.

    PubMed

    Foster, Shadae R; Dilworth, Lowell L; Thompson, Rory K; Alexander-Lindo, Ruby L; Omoruyi, Felix O

    2017-09-25

    Diabetes mellitus is associated with elevated reactive oxygen species, lipid abnormalities, reduced antioxidant activity and organ damage. This study examines the effects of combined inositol hexakisphosphate (IP6) and inositol supplement on antioxidant levels and other biochemical parameters in the liver of type 2 diabetic rats. Five groups of Sprague-Dawley rats were studied. Six rats were fed normal diet (non-diabetic control), while 24 rats were fed high-fat diet (HFD) for 4 weeks. Diabetes was induced in 18 of the rats fed HFD by intraperitoneal administration of streptozotocin. The diabetic rats were separated into three groups namely: combined IP6 and inositol, glibenclamide and diabetic control. The non-diabetic group fed high-fat diet was classified as a high-fat control group. For the final four weeks of the experiment, all rats were fed normal diet and given their respective treatment regimes. Hepatic antioxidant status, metabolic enzyme activity, lipid profile, peroxidative damage and liver histology, as well as, serum aminotransferase and alkaline phosphatase activities, and total bilirubin concentration were assessed. Treatment with combined IP6 and inositol supplement significantly increased liver reduced glutathione and high-density lipoprotein levels while liver triglyceride levels and serum alkaline phosphatase activity were significantly reduced by 27%, 50%, 38.5%, and 69.2% respectively compared to the diabetic control. Hepatic superoxide dismutase, catalase, glucose-6-phosphate dehydrogenase activities were significantly upregulated by 55%, 26% and 53% respectively in the diabetic rats treated with combined IP6 and inositol compared to the diabetic control. Combined IP6 and inositol treatment resulted in the preservation of liver cell integrity and improved antioxidant status in type 2 diabetic rats. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Carbamoylcholine and gastrin induce inositol lipid turnover in canine gastric parietal cells

    SciTech Connect

    Chiba, T.; Fisher, S.K.; Park, J.; Seguin, E.B.; Agranoff, B.W.; Yamada, Tadataka )

    1988-07-01

    The potential role of inositol phospholipid turnover in mediating acid secretion was examined in a preparation enriched for isolated canine gastric parietal cells. The stimulatory effects of carbamoylcholine (carbachol) and gastrin on parietal cell uptake of ({sup 14}C)aminopyrine were linked to dose- and time-dependent selective reduction in cellular phosphatidylinositol content, although the specific fatty acid composition of the phosphoinositides was not altered. Analysis of ({sup 3}H)inositol phosphates accumulated in cells prelabeled with ({sup 3}H)inositol revealed an increase in labeled inositol trisphosphate by 5 min of incubation with either carbachol or gastrin. Furthermore, after preincubation of parietal cells in medium containing ({sup 32}P)orthophosphate, the two secretagogues elicited a time-dependent decrease in {sup 32}P labeling of phosphatidylinositol 4,5-bisphosphate and concomitant increase in labeling of phosphatidic acid. These data demonstrate that the acid secretagogue actions of carbachol and gastrin are correlated with turnover of cellular inositol phospholipids in a preparation consisting predominantly of parietal cells.

  1. Dephosphorylation of 1D-myo-inositol 1,4-bisphosphate in rat liver.

    PubMed Central

    Morris, A J; Storey, D J; Downes, C P; Michell, R H

    1988-01-01

    Dephosphorylation of 1D-myo-inositol 1,4-bisphosphate [Ins(1,4)P2] in rat liver is catalysed by a cytosolic phosphatase that removes the 1-phosphate group. The Km for Ins(1,4)P2 is approx. 17 microM. Li+ (100 mM) causes 50% inhibition of Ins(1,4)P2 phosphatase activity when activity is measured at the very low substrate concentration of 10 nM, but on raising the substrate concentration to 100 microM there is a greater than 10-fold increase in sensitivity to Li+, suggesting that Li+ acts mainly, but not entirely, as an uncompetitive inhibitor of Ins(1,4)P2 phosphatase. In addition, rat liver cytosol shows Li+-sensitive phosphatase activity against 1D-myo-inositol 1-,3- and 4-monophosphates. The Ins(1,4)P2 1-phosphatase and inositol monophosphatase activities all share an apparent Mr of 47 x 10(3), as determined by gel-filtration chromatography. However, the Ins(1,4)P2 1-phosphatase is more sensitive to inactivation by heat, and can be separated from inositol monophosphatase activity by anion-exchange chromatography. We conclude that rat liver cytosol contains an Ins(1,4)P2 1-phosphatase that is distinct from, but in many ways similar to, inositol monophosphatase. PMID:2848493

  2. Inositol trisphosphate and calcium signalling

    NASA Astrophysics Data System (ADS)

    Berridge, Michael J.

    1993-01-01

    Inositol trisphosphate is a second messenger that controls many cellular processes by generating internal calcium signals. It operates through receptors whose molecular and physiological properties closely resemble the calcium-mobilizing ryanodine receptors of muscle. This family of intracellular calcium channels displays the regenerative process of calcium-induced calcium release responsible for the complex spatiotemporal patterns of calcium waves and oscillations. Such a dynamic signalling pathway controls many cellular processes, including fertilization, cell growth, transformation, secretion, smooth muscle contraction, sensory perception and neuronal signalling.

  3. Synthesis of inositol 1,2-(cyclic)-4,5-trisphosphate.

    PubMed Central

    Auchus, R J; Kaiser, S L; Majerus, P W

    1987-01-01

    We have developed a method for synthesis of inositol 1,2-(cyclic)-4,5-trisphosphate from inositol 1,4,5-trisphosphate using a water-soluble carbodiimide. We obtained 1-1.5 mumol of the inositol cyclic trisphosphate starting with 5 mumol of inositol 1,4,5-trisphosphate. The cyclized product was isolated by HPLC on Partisil SAX. The identity of the cyclic product was verified by its hydrolysis to inositol 1,4,5-trisphosphate in acid and by its conversion to 1,2-(cyclic)-4-bisphosphate by a specific 5-phosphomonoesterase from platelets. We also identified the product by 31P NMR spectroscopy, which showed a peak at 17.2 ppm, characteristic of a five-membered cyclic phosphodiester ring, and peaks at 4.1 ppm and 0.8 ppm, indicative of phosphomonoesters. This relatively simple method for producing inositol 1,2-(cyclic)-4,5-trisphosphate will facilitate studies of the physiology of this compound in signal transduction. PMID:3469663

  4. Influence of Calcium Phosphate and Apatite Containing Products on Enamel Erosion

    PubMed Central

    Kensche, A.; Pötschke, S.; Hannig, C.; Richter, G.; Hoth-Hannig, W.; Hannig, M.

    2016-01-01

    For the purpose of erosion prevention the present study aimed to compare the efficacy of two biomimetic products and a fluoride solution to optimize the protective properties of the pellicle. After 1 min of in situ pellicle formation on bovine enamel slabs, 8 subjects adopted CPP-ACP (GC Tooth Mousse), a mouthwash with hydroxyapatite microclusters (Biorepair), or a fluoride based mouthwash (elmex Kariesschutz) for 1 min each. Afterwards, samples were exposed in the oral cavity for 28 min. Native enamel slabs and slabs exposed to the oral cavity for 30 min without any rinse served as controls. After oral exposure, slabs were incubated in HCl (pH values 2, 2.3, and 3) for 120 s and kinetics of calcium and phosphate release were measured photometrically; representative samples were evaluated by SEM and TEM. The physiological pellicle reduced demineralization at all pH values; the protective effect was enhanced by fluoride. The biomimetic materials also reduced ion release but their effect was less pronounced. SEM indicated no layer formation after use of the different products. However, TEM confirmed the potential accumulation of mineral components at the pellicle surface. The tested products improve the protective properties of the in situ pellicle but not as effectively as fluorides. PMID:27430013

  5. Enhancing solubility of deoxyxylulose phosphate pathway enzymes for microbial isoprenoid production

    PubMed Central

    2012-01-01

    Background Recombinant proteins are routinely overexpressed in metabolic engineering. It is well known that some over-expressed heterologous recombinant enzymes are insoluble with little or no enzymatic activity. This study examined the solubility of over-expressed homologous enzymes of the deoxyxylulose phosphate pathway (DXP) and the impact of inclusion body formation on metabolic engineering of microbes. Results Four enzymes of this pathway (DXS, ISPG, ISPH and ISPA), but not all, were highly insoluble, regardless of the expression systems used. Insoluble dxs (the committed enzyme of DXP pathway) was found to be inactive. Expressions of fusion tags did not significantly improve the solubility of dxs. However, hypertonic media containing sorbitol, an osmolyte, successfully doubled the solubility of dxs, with the concomitant improvement in microbial production of the metabolite, DXP. Similarly, sorbitol significantly improved the production of soluble and functional ERG12, the committed enzyme in the mevalonate pathway. Conclusion This study demonstrated the unanticipated findings that some over-expressed homologous enzymes of the DXP pathway were highly insoluble, forming inclusion bodies, which affected metabolite formation. Sorbitol was found to increase both the solubility and function of some of these over-expressed enzymes, a strategy to increase the production of secondary metabolites. PMID:23148661

  6. Studies on the properties of myo-inositol-1,4,5-trisphosphate 5-phosphatase and myo-inositol monophosphatase in bovine iris sphincter smooth muscle: effects of okadaic acid and protein phosphorylation.

    PubMed

    Wang, X L; Akhtar, R A; Abdel-Latif, A A

    1994-05-26

    In bovine iris sphincter, myo-inositol 1,4,5-trisphosphate (IP3) 5-phosphatase and myo-inositol 1-phosphate (IP1) monophosphatase are mainly localized in the microsomal and soluble fractions, respectively. Studies on the properties of these enzymes can be summarized as follows. (1) The microsomal IP3 5-phosphatase hydrolyzed IP3 to myo-inositol 1,4-bisphosphate with an apparent Km of 28 microM and Vmax of 32 nmol/min per mg protein. The IP1 monophosphatase in the soluble fraction hydrolyzed IP1 into free inositol with an apparent Km of 89 microM and Vmax of 7 nmol/min per mg protein. (2) IP3 5-phosphatase and IP1 monophosphatase had optimal pH values at 8.0 and 7.0, respectively. (3) Both enzymes required Mg2+ and their highest specific activities were at a cation concentration of 2 mM. (4) Ca2+ (> 0.5 microM) exerted an inhibitory effect on IP3 5-phosphatase activity, and marked inhibition (47%) was observed at a concentration of 10 microM. Higher concentrations of the cation (> 100 microM) were required to inhibit IP1 monophosphatase. (5) IP1 monophosphatase, but not IP3 5-phosphatase, was inhibited by Li+. Li+ had no effect on the contractile response in this smooth muscle. (6) Both enzymes were inhibited by ATP and by the thiol-blocking agent, disulfiram. In addition, thimerosal, a thiol reagent, also inhibited the IP3 5-phosphatase activity. (7) Protein phosphorylation of the microsomal and soluble fractions with PKA or PKC had no effect on the activities of these enzymes. (8) Okadaic acid, a protein phosphatase inhibitor, had no effect on the activity of IP3 5-phosphatase. However, in the intact iris sphincter the toxin significantly reduced the carbachol-induced IP3 production, 1,2-diacylglycerol formation, measured as phosphatidic acid, and caused muscle relaxation.

  7. Phosphatidylinositol(4,5)bisphosphate and phosphatidylinositol(4)phosphate in plant tissues. [Pisum sativum

    SciTech Connect

    Irvine, R.F.; Letcher, A.J.; Lander, D.J. ); Dawson, A.P. ); Musgrave, A. ); Drobak, B.K. )

    1989-03-01

    Pea (Pisum sativum) leaf discs or swimming suspensions of Chlamydomonas eugametos were radiolabeled with ({sup 3}H)myo-inositol or ({sup 32}P)Pi and the lipids were extracted, deacylated, and their glycerol moieties removed. The resulting inositol trisphosphate and bisphosphate fractions were examined by periodate degradation, reduction and dephosphorylation, or by incubation with human red cell membranes. Their likely structures were identified as D-myo-inositol(1,4,5)trisphosphate and D-myo-inositol(1,4,)-bisphosphate. It is concluded that plants contain phosphatidylinositol(4)phosphate and phosphatidylinositol(4,5)bisphosphate; no other polyphosphoinositides were detected.

  8. Regulation of serum phosphate

    PubMed Central

    Lederer, Eleanor

    2014-01-01

    The regulation of serum phosphate, an acknowledged risk factor for chronic kidney disease and cardiovascular mortality, is poorly understood. The discovery of fibroblast growth factor 23 (FGF23) as a key regulator of renal phosphate handling and activation of vitamin D has revolutionized our comprehension of phosphate homeostasis. Through as yet undetermined mechanisms, circulating and dietary phosphate appear to have a direct effect on FGF23 release by bone cells that, in turn, causes renal phosphate excretion and decreases intestinal phosphate absorption through a decrease in vitamin D production. Thus, the two major phosphaturic hormones, PTH and FGF23, have opposing effects on vitamin D production, placing vitamin D at the nexus of phosphate homeostasis. While our understanding of phosphate homeostasis has advanced, the factors determining regulation of serum phosphate level remain enigmatic. Diet, time of day, season, gender, age and genetics have all been identified as significant contributors to serum phosphate level. The effects of these factors on serum phosphate have major implications for what is understood as ‘normal’ and for studies of phosphate homeostasis and metabolism. Moreover, other hormonal mediators such as dopamine, insulin-like growth factor, and angiotensin II also affect renal handling of phosphate. How the major hormone effects on phosphate handling are regulated and how the effect of these other factors are integrated to yield the measurable serum phosphate are only now beginning to be studied. PMID:24973411

  9. Inositol pyrophosphates mediate the effects of aging on bone marrow mesenchymal stem cells by inhibiting Akt signaling

    PubMed Central

    2014-01-01

    Introduction Bone marrow-derived mesenchymal stem cells (BM-MSCs) have been proposed as an ideal autologous stem cell source for cell-based therapy for myocardial infarction (MI). However, decreased viability and impaired function of aged MSCs hampered the therapeutic efficacy of engrafted MSCs, and the underlying mechanisms remain unclarified. Here, we investigated the role of inositol phosphates 6 kinase (IP6Ks) inhibition on the therapeutic efficacy of BM-MSCs and its underlying mechanism. Methods BM-MSCs isolated from young (8-week-old) or aged (18-month-old) donor male C57BL/6 mice, were subjected to hypoxia and serum deprivation (H/SD) injury with or without administration of inositol phosphates 6 kinase (IP6Ks) inhibitor TNP (10 μM). MSC apoptosis induced by H/SD was determined by flow cytometry and TUNEL assays. Protein expressions were evaluated by Western blot assay. Furthermore, the paracrine effects of MSCs were measured by reverse transcriptase–polymerized chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) analyses. Results Aged BM-MSCs exhibited more Inositol pyrophosphate 7 (IP7) production, compared with young BM-MSCs. Meanwhile, the expression of phospho-Akt (Thr308) was significantly decreased in the aged MSCs, resulting in enhanced Bad activation and decreased Bax/Bcl-2 ratio. Moreover, the apoptosis in aged BM-MSCs was increased, compared with young BM-MSCs. Furthermore, TNP administration significantly inhibited IP7 production and increased the phosphorylation of Akt under both normoxic and hypoxic conditions. Meanwhile, IP6Ks inhibition reduced apoptotic index of aged MSCs, associated with decreased expressions of pro-apoptotic proteins Bax and Bad and increased anti-apoptotic protein Bcl-2. The expressions of angiogenic factors, including VEGF, bFGF, IGF-1 and HGF, were decreased in MSCs from aged mice. In addition, TNP administration enhanced the paracrine efficiency of aged BM-MSCs under normoxic and hypoxic conditions

  10. Transient and sustained increases in inositol 1,4,5-trisphosphate precede the differential growth response in gravistimulated maize pulvini

    NASA Technical Reports Server (NTRS)

    Perera, I. Y.; Heilmann, I.; Boss, W. F.; Davies, E. (Principal Investigator)

    1999-01-01

    The internodal maize pulvinus responds to gravistimulation with differential cell elongation on the lower side. As the site of both graviperception and response, the pulvinus is an ideal system to study how organisms sense changes in orientation. We observed a transient 5-fold increase in inositol 1,4,5-trisphosphate (IP3) within 10 s of gravistimulation in the lower half of the pulvinus, indicating that the positional change was sensed immediately. Over the first 30 min, rapid IP3 fluctuations were observed between the upper and lower halves. Maize plants require a presentation time of between 2 and 4 h before the cells on the lower side of the pulvinus are committed to elongation. After 2 h of gravistimulation, the lower half consistently had higher IP3, and IP3 levels on the lower side continued to increase up to approximately 5-fold over basal levels before visible growth. As bending became visible after 8-10 h, IP3 levels returned to basal values. Additionally, phosphatidylinositol 4-phosphate 5-kinase activity in the lower pulvinus half increased transiently within 10 min of gravistimulation, suggesting that the increased IP3 production was accompanied by an up-regulation of phosphatidylinositol 4, 5-bisphosphate biosynthesis. Neither IP3 levels nor phosphatidylinositol 4-phosphate 5-kinase activity changed in pulvini halves from vertical control plants. Our data indicate the involvement of IP3 and inositol phospholipids in both short- and long-term responses to gravistimulation. As a diffusible second messenger, IP3 provides a mechanism to transmit and amplify the signal from the perceiving to the responding cells in the pulvinus, coordinating a synchronized growth response.

  11. Transient and sustained increases in inositol 1,4,5-trisphosphate precede the differential growth response in gravistimulated maize pulvini

    NASA Technical Reports Server (NTRS)

    Perera, I. Y.; Heilmann, I.; Boss, W. F.; Davies, E. (Principal Investigator)

    1999-01-01

    The internodal maize pulvinus responds to gravistimulation with differential cell elongation on the lower side. As the site of both graviperception and response, the pulvinus is an ideal system to study how organisms sense changes in orientation. We observed a transient 5-fold increase in inositol 1,4,5-trisphosphate (IP3) within 10 s of gravistimulation in the lower half of the pulvinus, indicating that the positional change was sensed immediately. Over the first 30 min, rapid IP3 fluctuations were observed between the upper and lower halves. Maize plants require a presentation time of between 2 and 4 h before the cells on the lower side of the pulvinus are committed to elongation. After 2 h of gravistimulation, the lower half consistently had higher IP3, and IP3 levels on the lower side continued to increase up to approximately 5-fold over basal levels before visible growth. As bending became visible after 8-10 h, IP3 levels returned to basal values. Additionally, phosphatidylinositol 4-phosphate 5-kinase activity in the lower pulvinus half increased transiently within 10 min of gravistimulation, suggesting that the increased IP3 production was accompanied by an up-regulation of phosphatidylinositol 4, 5-bisphosphate biosynthesis. Neither IP3 levels nor phosphatidylinositol 4-phosphate 5-kinase activity changed in pulvini halves from vertical control plants. Our data indicate the involvement of IP3 and inositol phospholipids in both short- and long-term responses to gravistimulation. As a diffusible second messenger, IP3 provides a mechanism to transmit and amplify the signal from the perceiving to the responding cells in the pulvinus, coordinating a synchronized growth response.

  12. 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.

  13. Conversion of cassava wastes for biofertilizer production using phosphate solubilizing fungi.

    PubMed

    Ogbo, Frank C

    2010-06-01

    Two fungi characterized as Aspergillus fumigatus and Aspergillus niger, isolated from decaying cassava peels were used to convert cassava wastes by the semi-solid fermentation technique to phosphate biofertilizer. The isolates solubilized Ca(3)(PO(4))(2), AlPO(4) and FePO(4) in liquid Pikovskaya medium, a process that was accompanied by acid production. Medium for the SSF fermentation was composed of 1% raw cassava starch and 3% poultry droppings as nutrients and 96% ground (0.5-1.5mm) dried cassava peels as carrier material. During the 14days fermentation, both test organisms increased in biomass in this medium as indicated by increases in phosphatase activity and drop in pH. Ground cassava peels satisfied many properties required of carrier material particularly in respect of the organisms under study. Biofertilizer produced using A. niger significantly (p<.05) improved the growth of pigeon pea [Cajanus cajan (L.) Millsp.] in pot experiments but product made with A. fumigatus did not. Copyright 2009 Elsevier Ltd. All rights reserved.

  14. Phosphate laser glass for NIF: production status, slab selection, and recent technical advances

    NASA Astrophysics Data System (ADS)

    Suratwala, Tayyab I.; Campbell, John H.; Miller, Philip E.; Thorsness, Charles B.; Riley, Michael O.; Ehrmann, Paul R.; Steele, Rusty A.

    2004-05-01

    The National Ignition Facility (NIF) at the Lawrence Livermore National Laboratory is a stadium-sized high-energy (1.8 megajoule) / high-peak power (500 terawatt) laser system, which will utilize over 3000 meter-size Nd-doped metaphosphate glasses as its gain media. The current production status, the selection criteria of individual slabs for specific beam line locations, and some recent technical advances are reviewed. The glass blanks are manufactured by a novel continuous glass melting process, and the finished slabs are then prepared by epoxy bonding a Cu-doped phosphate glass edge cladding and by advanced finishing techniques. To date, nearly 3400 slab equivalents have been melted, 2600 have been rough-cut to blanks, 1200 have been finished, and 144 have been installed in NIF. A set of selection rules, which are designed to optimize laser performance (e.g., maintain gain balance between beam lines and minimize beam walkoff) and to maximize glass lifetime with respect to Pt damage site growth, have been established for assigning individual slabs to specific beam line locations. Recent technical advances for amplifier slab production, which include: 1) minimizing surface pitting (hazing) after final finishing; 2) minimizing humidity-induced surface degradation (weathering) upon storage and use; and 3) preventing mounting-induced surface fractures upon installation, have contributed in improving the laser glass quality.

  15. Transport and homeostasis of potassium and phosphate: limiting factors for sustainable crop production.

    PubMed

    Luan, Mingda; Tang, Ren-Jie; Tang, Yumei; Tian, Wang; Hou, Congong; Zhao, Fugeng; Lan, Wenzhi; Luan, Sheng

    2017-06-01

    Potassium (K) and phosphate (Pi) are both macronutrients essential for plant growth and crop production, but the unrenewable resources of phosphorus rock and potash have become limiting factors for food security. One critical measure to help solve this problem is to improve nutrient use efficiency (NUE) in plants by understanding and engineering genetic networks for ion uptake, translocation, and storage. Plants have evolved multiple systems to adapt to various nutrient conditions for growth and production. Within the NUE networks, transport proteins and their regulators are the primary players for maintaining nutrient homeostasis and could be utilized to engineer high NUE traits in crop plants. A large number of publications have detailed K+ and Pi transport proteins in plants over the past three decades. Meanwhile, the discovery and validation of their regulatory mechanisms are fast-track topics for research. Here, we provide an overview of K+ and Pi transport proteins and their regulatory mechanisms, which participate in the uptake, translocation, storage, and recycling of these nutrients in plants. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  16. Efficient recovery of nitrate and phosphate from wastewater by an amine-grafted adsorbent for cyanobacterial biomass production.

    PubMed

    Kim, Jungmin; Hwang, Min-Jin; Lee, Sang-Jun; Noh, Won; Kwon, Jung Min; Choi, Jin Soo; Kang, Chang-Min

    2016-04-01

    Various types of wastewater have been widely utilized in microalgae and cyanobacteria cultivation for environmental and economic reasons. However, the problems of low cell growth and biomass contamination due to direct use of wastewater remain unresolved. In the present study, nitrate and phosphate were separated from wastewater by adsorption and subsequently used for cyanobacterial biomass production. To this end, an amine-grafted magnetic absorbent was synthesized. The synthesized absorbent recovered ca. 78% nitrate and 93% phosphate from wastewater. Regenerated medium was prepared using recovered nutrients as nitrogen and phosphate sources, which were efficiently assimilated by cyanobacterial culture. Compared to synthetic medium, there was no difference in growth and nutrient removal using regenerated medium. The proposed indirect method of wastewater utilization would prevent contamination of the produced biomass by unfavorable substances, which will broaden its potential applications.

  17. Glucose-6-phosphate dehydrogenase-derived NADPH fuels superoxide production in the failing heart

    USDA-ARS?s Scientific Manuscript database

    In the failing heart, NADPH oxidase and uncoupled NO synthase utilize cytosolic NADPH to form superoxide. NADPH is supplied principally by the pentose phosphate pathway, whose rate-limiting enzyme is glucose 6-phosphate dehydrogenase (G6PD). Therefore, we hypothesized that cardiac G6PD activation dr...

  18. 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.

  19. Enhancing pentose phosphate pathway in Corynebacterium glutamicum to improve l-isoleucine production.

    PubMed

    Ma, Wenjian; Wang, Jianli; Li, Ye; Hu, Xiaoqing; Shi, Feng; Wang, Xiaoyuan

    2016-11-01

    Three genes, gnd, pgl, and fbp, relevant to the pentose phosphate pathway (PPP) were overexpressed in Corynebacterium glutamicum IWJ001, leading to increase of l-isoleucine production. The transcriptional levels of gnd, pgl, and fbp significantly increased in IWJ001/pDXW-8-gnd-fbp-pgl. Compared with the control strain IWJ001/pDXW-8, intracellular NADPH/NADP(+) ratios in IWJ001/pDXW-8-gnd and IWJ001/pDXW-8-gnd-fbp cells grown for 36 H increased threefold and fourfold, respectively, indicating that overexpression of gnd and fbp redirected the carbon flux to PPP. Intracellular NADPH/NADP(+) ratio in IWJ001/pDXW-8-gnd-fbp-pgl grown for 36 H was similar to IWJ001/pDXW-8, suggesting that the NADPH produced by PPP could be quickly consumed for l-isoleucine production. 10.9 and 28.96 g/L of l-isoleucine was produced in IWJ001/pDXW-8-gnd-fbp-pgl in shake flask cultivation and fed-batch fermentation, respectively. In addition, IWJ001/pDXW-8-gnd-fbp-pgl grew fast, its dry cell weight reached 49 g/L after 48 H, whereas the start strain IWJ001/pDXW-8 reached only 40 g/L. After 96 H fermentation, l-isoleucine yield on glucose in IWJ001/pDXW-8-gnd-fbp-pgl reached 0.138 g/g. The results demonstrate that carbon flux redirection to PPP is an efficient approach to enhance l-isoleucine production in C. glutamicum.

  20. Performance of microbial phytases for gastric inositol phosphate degradation.

    PubMed

    Nielsen, Anne Veller Friis; Nyffenegger, Christian; Meyer, Anne S

    2015-01-28

    Microbial phytases catalyze dephosphorylation of phytic acid, thereby potentially releasing chelated iron and improving human iron absorption from cereal-based diets. For this catalysis to take place in vivo, the phytase must be robust to low pH and proteolysis in the gastric ventricle. This study compares the robustness of five different microbial phytases, evaluating thermal stability, activity retention, and extent of dephosphorylation of phytic acid in a simulated low-pH/pepsin gastric environment and examines secondary protein structural changes at low pH via circular dichroism. The Peniophora lycii phytase was found to be the most thermostable, but the least robust enzyme in gastric conditions, whereas the Aspergillus niger and Escherichia coli phytases proved to be most resistant to gastric conditions. The phytase from Citrobacter braakii showed intermediate robustness. The extent of loss of secondary structure at low pH correlated positively with the extent of activity loss at low pH.

  1. Large scale production of yolk-shell β-tricalcium phosphate powders, and their bioactivities as novel bone substitutes.

    PubMed

    Cho, Jung Sang; Lee, Jong-Heun; Kang, Yun Chan

    2014-08-28

    This paper proposes the production of yolk-shell structured β-tricalcium phosphate (β-TCP) powders using a spray-drying method, suitable for commercial scale production. Spray-dried precursor powders, consisting of calcium-phosphate salts and each of the various carbon source materials, are combusted in an oxygen atmosphere to obtain a yolk-shell structure. Only dextrin among the carbon source materials investigated shows promise in the production of β-TCP yolk-shell powders. By evaluating their apatite-forming capacity in simulated body fluid, the outstanding bioactivity of β-TCP yolk-shell powders is confirmed: numerous acicular and newly formed hydroxyl carbonate apatite crystals cover the entire β-TCP surface after a single day of soaking. These crystals are observed on both the outer and inner surfaces of the shells, and on the outer surface of the core, which is encouraging for its potential use as a bone grafting material.

  2. Complete hydrolysis of myo-inositol hexakisphosphate by a novel phytase from Debaryomyces castellii CBS 2923.

    PubMed

    Ragon, Mélanie; Aumelas, André; Chemardin, Patrick; Galvez, Santiago; Moulin, Guy; Boze, Hélène

    2008-02-01

    Debaryomyces castellii phytase was purified to homogeneity in a single step by hydrophobic interaction chromatography. Its molecular mass is 74 kDa with 28.8% glycosylation. Its activity was optimal at 60 degrees C and pH 4.0. The K (m) value for sodium phytate was 0.532 mM. The enzyme exhibited a low specificity and hydrolyzed many phosphate esters. The phytase fully hydrolyzed myo-inositol hexakisphosphate (or phytic acid, Ins P(6)) to inositol and inorganic phosphate. The sequence of Ins P(6) hydrolysis was determined by combining results from high-performance ionic chromatography and nuclear magnetic resonance. D. castellii phytase is a 3-phytase that sequentially releases phosphate groups through Ins (1,2,4,5,6) P(5), Ins (1,2,5,6) P(4), Ins (1,2,6) P(3), Ins (1,2) P(2), Ins (1 or 2) P(1), and inositol (notation 3/4/5/6/1 or 2).

  3. Role for ionotropic and metabotropic receptors in quisqualate-stimulated inositol polyphosphate accumulation in rat cerebral cortex.

    PubMed

    Baird, J G; Challiss, R A; Nahorski, S R

    1991-06-01

    The actions of the excitatory amino acid quisqualate (QA) on inositol polyphosphate accumulation in cerebral cortex slices have been assessed using both [3H]inositol prelabeling and mass measurements over relatively short incubation periods. QA stimulated accumulation of all the inositol polyphosphates, with similar EC50 values (2.8 +/- 0.7 microM). High performance liquid chromatography analysis of isomeric forms of inositol polyphosphates and specific mass assays revealed that both phosphorylation and dephosphorylation products of inositol-1,4,5-trisphosphate accumulate. A large component of the QA-stimulated inositol polyphosphate accumulation was inhibited by the ionotropic antagonist 6,7-dinitroquinoxaline-2,3-dione in a competitive manner. This implied that the QA response may be due to entry of Ca2+ via voltage-sensitive calcium channels as a consequence of an ionotropic receptor-induced depolarization. In support of this mechanism, the QA-induced response was dependent on the presence of extracellular calcium, whereas the well characterized muscarinic receptor agonist response to carbachol showed only a slight reduction under the same conditions. The concentration-dependent (EC50 8.8 +/- 3 microM) response to the selective ionotropic agonist amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA) differed from that to QA or carbachol, in that accumulation of only [3H]inositol mono- and bisphosphates was stimulated, with no increase in the [3H]inositol tris- or tetrakisphosphates. Use of the metabotropic agonist (trans)-(+/-)-1-aminocyclopentyl-1,3-dicarboxylate (ACPD), however, produced concentration-dependent increases in all [3H]inositol polyphosphates. Although both AMPA and ACPD responses alone were smaller in magnitude than that to QA, when present together AMPA and ACPD produced additive responses on [3H]inositol mono- and bisphosphate and a marked synergistic increase in [3H]inositol tetrakisphosphate accumulation, resulting in a response similar to

  4. Ablation of Succinate Production from Glucose Metabolism in the Procyclic Trypanosomes Induces Metabolic Switches to the Glycerol 3-Phosphate/Dihydroxyacetone Phosphate Shuttle and to Proline Metabolism*

    PubMed Central

    Ebikeme, Charles; Hubert, Jane; Biran, Marc; Gouspillou, Gilles; Morand, Pauline; Plazolles, Nicolas; Guegan, Fabien; Diolez, Philippe; Franconi, Jean-Michel; Portais, Jean-Charles; Bringaud, Frédéric

    2010-01-01

    Trypanosoma brucei is a parasitic protist that undergoes a complex life cycle during transmission from its mammalian host (bloodstream forms) to the midgut of its insect vector (procyclic form). In both parasitic forms, most glycolytic steps take place within specialized peroxisomes, called glycosomes. Here, we studied metabolic adaptations in procyclic trypanosome mutants affected in their maintenance of the glycosomal redox balance. T. brucei can theoretically use three strategies to maintain the glycosomal NAD+/NADH balance as follows: (i) the glycosomal succinic fermentation branch; (ii) the glycerol 3-phosphate (Gly-3-P)/dihydroxyacetone phosphate (DHAP) shuttle that transfers reducing equivalents to the mitochondrion; and (iii) the glycosomal glycerol production pathway. We showed a hierarchy in the use of these glycosomal NADH-consuming pathways by determining metabolic perturbations and adaptations in single and double mutant cell lines using a combination of NMR, ion chromatography-MS/MS, and HPLC approaches. Although functional, the Gly-3-P/DHAP shuttle is primarily used when the preferred succinate fermentation pathway is abolished in the Δpepck knock-out mutant cell line. In the absence of these two pathways (Δpepck/RNAiFAD-GPDH.i mutant), glycerol production is used but with a 16-fold reduced glycolytic flux. In addition, the Δpepck mutant cell line shows a 3.3-fold reduced glycolytic flux compensated by an increase of proline metabolism. The inability of the Δpepck mutant to maintain a high glycolytic flux demonstrates that the Gly-3-P/DHAP shuttle is not adapted to the procyclic trypanosome context. In contrast, this shuttle was shown earlier to be the only way used by the bloodstream forms of T. brucei to sustain their high glycolytic flux. PMID:20702405

  5. Pseudomonas-Derived Ceramidase Induces Production of Inflammatory Mediators from Human Keratinocytes via Sphingosine-1-Phosphate

    PubMed Central

    Oizumi, Ami; Nakayama, Hitoshi; Okino, Nozomu; Iwahara, Chihiro; Kina, Katsunari; Matsumoto, Ryo; Ogawa, Hideoki; Takamori, Kenji; Ito, Makoto; Suga, Yasushi; Iwabuchi, Kazuhisa

    2014-01-01

    Ceramide is important for water retention and permeability barrier functions in the stratum corneum, and plays a key role in the pathogenesis of atopic dermatitis (AD). A Pseudomonas aeruginosa-derived neutral ceramidase (PaCDase) isolated from a patient with AD was shown to effectively degrade ceramide in the presence of Staphylococcus aureus-derived lipids or neutral detergents. However, the effect of ceramide metabolites on the functions of differentiating keratinocytes is poorly understood. We found that the ceramide metabolite sphingosine-1-phosphate (S1P) stimulated the production of inflammatory mediators such as TNF-α and IL-8 from three-dimensionally cultured human primary keratinocytes (termed “3D keratinocytes”), which form a stratum corneum. PaCDase alone did not affect TNF-α gene expression in 3D keratinocytes. In the presence of the detergent Triton X-100, which damages stratum corneum structure, PaCDase, but not heat-inactivated PaCDase or PaCDase-inactive mutant, induced the production of TNF-α, endothelin-1, and IL-8, indicating that this production was dependent on ceramidase activity. Among various ceramide metabolites, sphingosine and S1P enhanced the gene expression of TNF-α, endothelin-1, and IL-8. The PaCDase-enhanced expression of these genes was inhibited by a sphingosine kinase inhibitor and by an S1P receptor antagonist VPC 23019. The TNF-α-binding antibody infliximab suppressed the PaCDase-induced upregulation of IL-8, but not TNF-α, mRNA. PaCDase induced NF-κB p65 phosphorylation. The NF-κB inhibitor curcumin significantly inhibited PaCDase-induced expression of IL-8 and endothelin-1. VPC 23019 and infliximab inhibited PaCDase-induced NF-κB p65 phosphorylation and reduction in the protein level of the NF-κB inhibitor IκBα. Collectively, these findings suggest that (i) 3D keratinocytes produce S1P from sphingosine, which is produced through the hydrolysis of ceramide by PaCDase, (ii) S1P induces the production of TNF

  6. Structure-based identification of inositol polyphosphate 1-phosphatase from Entamoeba histolytica.

    PubMed

    Faisal Tarique, Khaja; Arif Abdul Rehman, Syed; Betzel, Christian; Gourinath, Samudrala

    2014-11-01

    Inositol polyphosphate 1-phosphatase from Entamoeba histolytica (EhIPPase) is an Mg(2+)-dependent and Li(+)-sensitive enzyme that catalyzes the hydrolysis of inositol 1,4-bisphosphate [Ins(1,4)P2] into myo-inositol 1-monophosphate and PO4(3-). In the present work, EhIPPase has been biochemically identified and its crystal structure has been determined in the presence of Mg(2+) and PO4(3-) at 2.5 Å resolution. This enzyme was previously classified as a 3'(2'),5'-bisphosphate nucleotidase in the NCBI, but its biochemical activity and structural analysis suggest that this enzyme behaves more like an inositol polyphosphate 1-phosphatase. The ability of EhIPPase to hydrolyze the smaller Ins(1,4)P2 better than the bulkier 3'-phosphoadenosine 5'-phosphate (PAP) is explained on the basis of the orientations of amino-acid residues in the binding site. This structure is the first of its class to be determined from any protozoan parasite, and is the third to determined among all organisms, following its rat and bovine homologues. The three-dimensional fold of EhIPPase is similar to those of other members of the inositol monophosphatase superfamily, which also includes inositol monophosphatase, 3'(2'),5'-bisphosphate nucleotidase and fructose-1,6-bisphosphate 1-phosphatase. They all share conserved residues essential for metal binding and substrate hydrolysis, with the motif D-Xn-EE-Xn-DP(I/L)DG(S/T)-Xn-WD-Xn-GG. The structure is divided into two domains, namely α+β and α/β, and the substrate and metal ions bind between them. However, the ability of each enzyme class to act specifically on its cognate substrate is governed by the class-specific amino-acid residues at the active site.

  7. Importance and regulation of inositol biosynthesis during growth and differentiation of Streptomyces.

    PubMed

    Zhang, Guohua; Tian, Yuqing; Hu, Kun; Zhu, Yu; Chater, Keith F; Feng, Chi; Liu, Gang; Tan, Huarong

    2012-03-01

    Unusually among bacteria, actinobacteria possess myo-inositol 1-phosphate synthase (mIPS). In the developmentally complex Streptomyces coelicolor, the mIPS-encoding gene (inoA) is cotranscribed with a putative regulatory gene (inoR). The inoRA transcript was more abundant in an inoR in-frame deletion mutant, and InoR formed different complexes in vitro with an extensive region around the inoRA promoter. Binding was relieved by adding glucose 6-phosphate. Thus, InoR is a metabolite-sensitive autorepressor that influences inoA expression, and hence the level of inositol, by controlling transcription from P(inoRA) . Disruption of inoA resulted in inositol-dependent growth and development, with full phenotypic correction at 0.1 mM inositol: at lower inositol concentrations differentiation was arrested at intermediate stages. This pattern may partly reflect increased demand for membrane phospholipids during sporulation septation. A corresponding sharp upregulation of inoRA transcription coincident with sporulation was dependent on a developmental regulator, WhiI. A truncated form of WhiI could bind two sites downstream of P(inoRA) , and one of the WhiI-binding sites overlapped the InoR-binding site. The combined action of a metabolic regulator and a developmental regulator at the simple P(inoRA) promoter is a previously undescribed strategy for the differential provision of developmentally appropriate levels of a substance required during the formation of spore chains.

  8. Whole-Genome Analysis Reveals that Mutations in Inositol Polyphosphate Phosphatase-like 1 Cause Opsismodysplasia

    PubMed Central

    Below, Jennifer E.; Earl, Dawn L.; Shively, Kathryn M.; McMillin, Margaret J.; Smith, Joshua D.; Turner, Emily H.; Stephan, Mark J.; Al-Gazali, Lihadh I.; Hertecant, Jozef L.; Chitayat, David; Unger, Sheila; Cohn, Daniel H.; Krakow, Deborah; Swanson, James M.; Faustman, Elaine M.; Shendure, Jay; Nickerson, Deborah A.; Bamshad, Michael J.

    2013-01-01

    Opsismodysplasia is a rare, autosomal-recessive skeletal dysplasia characterized by short stature, characteristic facial features, and in some cases severe renal phosphate wasting. We used linkage analysis and whole-genome sequencing of a consanguineous trio to discover that mutations in inositol polyphosphate phosphatase-like 1 (INPPL1) cause opsismodysplasia with or without renal phosphate wasting. Evaluation of 12 families with opsismodysplasia revealed that INPPL1 mutations explain ∼60% of cases overall, including both of the families in our cohort with more than one affected child and 50% of the simplex cases. PMID:23273567

  9. Structural basis for phosphatidylinositol-phosphate biosynthesis

    NASA Astrophysics Data System (ADS)

    Clarke, Oliver B.; Tomasek, David; Jorge, Carla D.; Dufrisne, Meagan Belcher; Kim, Minah; Banerjee, Surajit; Rajashankar, Kanagalaghatta R.; Shapiro, Lawrence; Hendrickson, Wayne A.; Santos, Helena; Mancia, Filippo

    2015-10-01

    Phosphatidylinositol is critical for intracellular signalling and anchoring of carbohydrates and proteins to outer cellular membranes. The defining step in phosphatidylinositol biosynthesis is catalysed by CDP-alcohol phosphotransferases, transmembrane enzymes that use CDP-diacylglycerol as donor substrate for this reaction, and either inositol in eukaryotes or inositol phosphate in prokaryotes as the acceptor alcohol. Here we report the structures of a related enzyme, the phosphatidylinositol-phosphate synthase from Renibacterium salmoninarum, with and without bound CDP-diacylglycerol to 3.6 and 2.5 Å resolution, respectively. These structures reveal the location of the acceptor site, and the molecular determinants of substrate specificity and catalysis. Functional characterization of the 40%-identical ortholog from Mycobacterium tuberculosis, a potential target for the development of novel anti-tuberculosis drugs, supports the proposed mechanism of substrate binding and catalysis. This work therefore provides a structural and functional framework to understand the mechanism of phosphatidylinositol-phosphate biosynthesis.

  10. Structural basis for phosphatidylinositol-phosphate biosynthesis

    PubMed Central

    Clarke, Oliver B.; Tomasek, David; Jorge, Carla D.; Dufrisne, Meagan Belcher; Kim, Minah; Banerjee, Surajit; Rajashankar, Kanagalaghatta R.; Shapiro, Lawrence; Hendrickson, Wayne A.; Santos, Helena; Mancia, Filippo

    2015-01-01

    Phosphatidylinositol is critical for intracellular signalling and anchoring of carbohydrates and proteins to outer cellular membranes. The defining step in phosphatidylinositol biosynthesis is catalysed by CDP-alcohol phosphotransferases, transmembrane enzymes that use CDP-diacylglycerol as donor substrate for this reaction, and either inositol in eukaryotes or inositol phosphate in prokaryotes as the acceptor alcohol. Here we report the structures of a related enzyme, the phosphatidylinositol-phosphate synthase from Renibacterium salmoninarum, with and without bound CDP-diacylglycerol to 3.6 and 2.5 Å resolution, respectively. These structures reveal the location of the acceptor site, and the molecular determinants of substrate specificity and catalysis. Functional characterization of the 40%-identical ortholog from Mycobacterium tuberculosis, a potential target for the development of novel anti-tuberculosis drugs, supports the proposed mechanism of substrate binding and catalysis. This work therefore provides a structural and functional framework to understand the mechanism of phosphatidylinositol-phosphate biosynthesis. PMID:26510127

  11. Metabolic engineering of enhanced glycerol-3-phosphate synthesis to increase lipid production in Synechocystis sp. PCC 6803.

    PubMed

    Wang, Xi; Xiong, Xiaochao; Sa, Na; Roje, Sanja; Chen, Shulin

    2016-07-01

    With the growing attention to global warming and energy sustainability, biosynthesis of lipids by photosynthetic microorganisms has attracted more interest for the production of renewable transportation fuels. Recently, the cyanobacterium Synechocystis sp. PCC 6803 has been widely used for biofuel production through metabolic engineering because of its efficient photosynthesis and well-developed genetic tools. In lipid biosynthesis, glycerol-3-phosphate (G3P) is a key node for both CO2 fixation and lipid metabolism in cyanobacteria. However, few studies have explored the use of G3P synthesis to improve photosynthetic lipid production. In this study, metabolic engineering combined with flux balance analysis (FBA) was conducted to reveal the effect of G3P synthesis on lipid production. Heterologous genes that encoded glycerol-3-phosphate dehydrogenase (GPD) and diacylglycerol acyltransferase (DGAT) were engineered into Synechocystis sp. PCC 6803 to enhance G3P supply and lipid production. The resultant recombinant Synechocystis produced higher levels of lipids without a significant reduction in cell growth. Compared with the wild-type strain, lipid content and productivity of the engineered cyanobacteria increased by up to 36 and 31 %, respectively, under autotrophic conditions. Lipid production under mixotrophic conditions of the engineered cyanobacteria was also investigated. This work demonstrated that enhanced G3P synthesis was an important factor in photosynthetic lipid production and that introducing heterologous GPD and DGAT genes was an effective strategy to increase lipid production in Synechocystis sp. PCC 6803.

  12. Egg Shell and Oyster Shell Powder as Alternatives for Synthetic Phosphate: Effects on the Quality of Cooked Ground Pork Products

    PubMed Central

    2017-01-01

    This study aimed to determine the optimal ratio of natural calcium powders (oyster shell and egg shell calcium) as synthetic phosphate replacers in pork products. Ground pork samples were subjected to six treatments, as follows: control (−) (no phosphate added), control (+) (0.3% phosphate blend added), treatment 1 (0.5% oyster shell calcium powder added), treatment 2 (0.3% oyster shell calcium powder and 0.2% egg shell calcium powder added), treatment 3 (0.2% oyster shell calcium powder and 0.3% egg shell calcium powder added), and treatment 4 (0.5% egg shell calcium powder added). The addition of natural calcium powders resulted in an increase in the pH values of meat products, regardless of whether they were used individually or mixed. The highest cooking loss was observed (p<0.05) in the negative control samples, whereas the cooking loss in samples with natural calcium powder added was similar (p>0.05) to that in the positive control samples. CIE L* values decreased as the amount of added egg shell calcium powder increased. CIE a* values were higher (p<0.05) in samples containing natural calcium powder (treatments 1, 2, 3, and 4) than in the positive control. The combination of oyster shell calcium powder and egg shell powder (treatment 2 or 3) was effective for the improvement of textural properties of the pork products. The findings show that the combined use of 0.2% oyster shell calcium and 0.3% egg shell calcium should enable the replacement of synthetic phosphate in the production of cooked pork products with desirable qualities. PMID:28943770

  13. Egg Shell and Oyster Shell Powder as Alternatives for Synthetic Phosphate: Effects on the Quality of Cooked Ground Pork Products.

    PubMed

    Cho, Min Guk; Bae, Su Min; Jeong, Jong Youn

    2017-01-01

    This study aimed to determine the optimal ratio of natural calcium powders (oyster shell and egg shell calcium) as synthetic phosphate replacers in pork products. Ground pork samples were subjected to six treatments, as follows: control (-) (no phosphate added), control (+) (0.3% phosphate blend added), treatment 1 (0.5% oyster shell calcium powder added), treatment 2 (0.3% oyster shell calcium powder and 0.2% egg shell calcium powder added), treatment 3 (0.2% oyster shell calcium powder and 0.3% egg shell calcium powder added), and treatment 4 (0.5% egg shell calcium powder added). The addition of natural calcium powders resulted in an increase in the pH values of meat products, regardless of whether they were used individually or mixed. The highest cooking loss was observed (p<0.05) in the negative control samples, whereas the cooking loss in samples with natural calcium powder added was similar (p>0.05) to that in the positive control samples. CIE L* values decreased as the amount of added egg shell calcium powder increased. CIE a* values were higher (p<0.05) in samples containing natural calcium powder (treatments 1, 2, 3, and 4) than in the positive control. The combination of oyster shell calcium powder and egg shell powder (treatment 2 or 3) was effective for the improvement of textural properties of the pork products. The findings show that the combined use of 0.2% oyster shell calcium and 0.3% egg shell calcium should enable the replacement of synthetic phosphate in the production of cooked pork products with desirable qualities.

  14. Improving isobutanol production in metabolically engineered Escherichia coli by co-producing ethanol and modulation of pentose phosphate pathway.

    PubMed

    Liu, Zichun; Liu, Pingping; Xiao, Dongguang; Zhang, Xueli

    2016-06-01

    Redox imbalance has been regarded as the key limitation for anaerobic isobutanol production in metabolically engineered Escherichia coli strains. In this work, the ethanol synthetic pathway was recruited to solve the NADH redundant problem while the pentose phosphate pathway was modulated to solve the NADPH deficient problem for anaerobic isobutanol production. Recruiting the ethanol synthetic pathway in strain AS108 decreased isobutanol yield from 0.66 to 0.29 mol/mol glucose. It was found that there was a negative correlation between aldehyde/alcohol dehydrogenase (AdhE) activity and isobutanol production. Decreasing AdhE activity increased isobutanol yield from 0.29 to 0.6 mol/mol. On the other hand, modulation of the glucose 6-phosphate dehydrogenase gene of the pentose phosphate pathway increased isobutanol yield from 0.29 to 0.41 mol/mol. Combination of these two strategies had a synergistic effect on improving isobutanol production. Isobutanol titer and yield of the best strain ZL021 were 53 mM and 0.74 mol/mol, which were 51 % and 12 % higher than the starting strain AS108, respectively. The total alcohol yield of strain ZL021 was 0.81 mol/mol, which was 23 % higher than strain AS108.

  15. Effects of phosphate limitation on soluble microbial products and microbial community structure in semi-continuous Synechocystis-based photobioreactors.

    PubMed

    Zevin, Alexander S; Nam, Taekgul; Rittmann, Bruce; Krajmalnik-Brown, Rosa

    2015-09-01

    All bacteria release organic compounds called soluble microbial products (SMP) as a part of their normal metabolism. In photobioreactor (PBR) settings, SMP produced by cyanobacteria represent a major pool of carbon and electrons available to heterotrophic bacteria. Thus, SMP in PBRs are a major driver for the growth of heterotrophic bacteria, and understanding the distribution of SMP in PBRs is an important step toward proper management of PBR microbial communities. Here, we analyzed the SMP and microbial communities in two Synechocystis sp. PCC6803-based PBRs. The first PBR (PBRP0) became phosphate limited after several days of operation, while the second PBR (PBRP+) did not have phosphate limitation. Heterotrophic bacteria were detected in both PBRs, but PBRP0 had a much higher proportion of heterotrophic bacteria than PBRP+. Furthermore, PBRP+ had greater biomass production and lower SMP production per unit biomass than PBRP0. Carbohydrates that were most likely derived from hydrolysis of extracellular polymeric substances (EPS) dominated the SMP in PBRP0, while products resulting from cell lysis or decay dominated the SMP in PBRP+. Together, our data support that maintaining phosphate availability in Synechocystis-based PBRs is important for managing SMP and, thus, the heterotrophic community.

  16. Fourier Transform Infrared Spectroscopy and Multivariate Analysis for Online Monitoring of Dibutyl Phosphate Degradation Product in Tributyl Phosphate /n-Dodecane/Nitric Acid Solvent

    SciTech Connect

    Levitskaia, Tatiana G.; Peterson, James M.; Campbell, Emily L.; Casella, Amanda J.; Peterman, Dean; Bryan, Samuel A.

    2013-11-05

    In liquid-liquid extraction separation processes, accumulation of organic solvent degradation products is detrimental to the process robustness and frequent solvent analysis is warranted. Our research explores feasibility of online monitoring of the organic solvents relevant to used nuclear fuel reprocessing. This paper describes the first phase of developing a system for monitoring the tributyl phosphate (TBP)/n-dodecane solvent commonly used to separate used nuclear fuel. In this investigation, the effect of extraction of nitric acid from aqueous solutions of variable concentrations on the quantification of TBP and its major degradation product dibutyl phosphoric acid (HDBP) was assessed. Fourier Transform Infrared Spectroscopy (FTIR) spectroscopy was used to discriminate between HDBP and TBP in the nitric acid-containing TBP/n-dodecane solvent. Multivariate analysis of the spectral data facilitated the development of regression models for HDBP and TBP quantification in real time, enabling online implementation of the monitoring system. The predictive regression models were validated using TBP/n-dodecane solvent samples subjected to the high dose external gamma irradiation. The predictive models were translated to flow conditions using a hollow fiber FTIR probe installed in a centrifugal contactor extraction apparatus demonstrating the applicability of the FTIR technique coupled with multivariate analysis for the online monitoring of the organic solvent degradation products.

  17. Fourier Transform Infrared Spectroscopy and Multivariate Analysis for Online Monitoring of Dibutyl Phosphate Degradation Product in Tributyl Phosphate/n-Dodecane/Nitric Acid Solvent

    SciTech Connect

    Tatiana G. Levitskaia; James M. Peterson; Emily L. Campbell; Amanda J. Casella; Dean R. Peterman; Samuel A. Bryan

    2013-12-01

    In liquid–liquid extraction separation processes, accumulation of organic solvent degradation products is detrimental to the process robustness, and frequent solvent analysis is warranted. Our research explores the feasibility of online monitoring of the organic solvents relevant to used nuclear fuel reprocessing. This paper describes the first phase of developing a system for monitoring the tributyl phosphate (TBP)/n-dodecane solvent commonly used to separate used nuclear fuel. In this investigation, the effect of extraction of nitric acid from aqueous solutions of variable concentrations on the quantification of TBP and its major degradation product dibutylphosphoric acid (HDBP) was assessed. Fourier transform infrared (FTIR) spectroscopy was used to discriminate between HDBP and TBP in the nitric acid-containing TBP/n-dodecane solvent. Multivariate analysis of the spectral data facilitated the development of regression models for HDBP and TBP quantification in real time, enabling online implementation of the monitoring system. The predictive regression models were validated using TBP/n-dodecane solvent samples subjected to high-dose external ?-irradiation. The predictive models were translated to flow conditions using a hollow fiber FTIR probe installed in a centrifugal contactor extraction apparatus, demonstrating the applicability of the FTIR technique coupled with multivariate analysis for the online monitoring of the organic solvent degradation products.

  18. Lipophosphonoglycan of the plasma membrane of A canthamoeba castellanii. Inositol and phytosphingosine content and general structural features.

    PubMed

    Dearborn, D G; Smith, S; Korn, E D

    1976-05-25

    Lipophosphonoglycan, a major component of the plasma membrane of Acanthamoeba castellanii, has now been shown to contain 8% inositol and 13% C25- and C24-phytosphingosines in addition to the previously identified content of neutral sugars (26%), amino sugars (3%), aminophosphonates (10%), acidhydrolyzable phosphate (3%), and long chain fatty acids (14%). The fatty acids and phytosphingosines are in ceramide groups. Lipophosphonoglycan can be separated by dodecyl sulfate-polyacrylamide electrophoresis into two major components that are similar in composition except for different oligosaccharide groups. A tentative structural model incorporating these features is proposed in which each of the two components of lipophosphonoglycan is conceived as an oligomeric inositol-containing glycosphingolipid.

  19. 1D-myo-inositol 1,4,5-trisphosphate dephosphorylation by rat enterocytes involves an intracellular 5-phosphatase and non-specific phosphatase activity at the cell surface.

    PubMed Central

    Rubiera, C; Velasco, G; Michell, R H; Lazo, P S; Shears, S B

    1988-01-01

    We studied the dephosphorylation of Ins(1,4,5)P3 (inositol 1,4,5-trisphosphate) by permeabilized rat intestinal epithelial cells incubated in a medium resembling intracellular ionic strength and pH. Saponin-permeabilized cells rapidly dephosphorylated Ins(1,4,5)P3 to a mixture of three InsP2 (inositol bisphosphate) isomers, namely Ins(1,4)P2, Ins(1,5)P2 and Ins(4,5)P2. These products were identified by h.p.l.c. analysis after dephosphorylation of both 3H- and 32P-labelled Ins(1,4,5)P3. Ins(1,4)P2 accumulated to about half of the concentration attained by Ins(1,5)P2 and Ins(4,5)P2. Ins(1,4,5)P3 dephosphorylation was inhibited, by up to 75%, by 10 mM-glucose 6-phosphate. In these conditions Ins(1,4)P2 became the predominant product, indicating that glucose 6-phosphate inhibited non-specific dephosphorylation of Ins(1,4,5)P3, at least at the 1- and 4-phosphate groups. Ins(1,4)P2 was further dephosphorylated, and the major InsP (inositol monophosphate) product was Ins4P. Most of the glucose 6-phosphate-inhibitable Ins(1,4,5)P3 phosphatase activity was exposed on the cell surface. The glucose 6-phosphate-insensitive Ins(1,4,5)P3 5-phosphatase activity was not detected until the cells were permeabilized with saponin. This intracellular 5-phosphatase activity was: (i) predominantly associated with the particulate portion of the cell; (ii) strongly inhibited by 10 mM-2,3-bisphosphoglycerate; (iii) insensitive to 50 mM-Li+. Therefore the Ins(1,4,5)P3 5-phosphatase activity in enterocytes appears similar to the 5-phosphatase that has been characterized in a number of cell types. PMID:2848503

  20. Efficient production of 2-deoxyribose 5-phosphate from glucose and acetaldehyde by coupling of the alcoholic fermentation system of Baker's yeast and deoxyriboaldolase-expressing Escherichia coli.

    PubMed

    Horinouchi, Nobuyuki; Ogawa, Jun; Kawano, Takako; Sakai, Takafumi; Saito, Kyota; Matsumoto, Seiichiro; Sasaki, Mie; Mikami, Yoichi; Shimizu, Sakayu

    2006-06-01

    2-Deoxyribose 5-phosphate production through coupling of the alcoholic fermentation system of baker's yeast and deoxyriboaldolase-expressing Escherichia coli was investigated. In this process, baker's yeast generates fructose 1,6-diphosphate from glucose and inorganic phosphate, and then the E. coli convert the fructose 1,6-diphosphate into 2-deoxyribose 5-phosphate via D-glyceraldehyde 3-phosphate. Under the optimized conditions with toluene-treated yeast cells, 356 mM (121 g/l) fructose 1,6-diphosphate was produced from 1,111 mM glucose and 750 mM potassium phosphate buffer (pH 6.4) with a catalytic amount of AMP, and the reaction supernatant containing the fructose 1,6-diphosphate was used directly as substrate for 2-deoxyribose 5-phosphate production with the E. coli cells. With 178 mM enzymatically prepared fructose 1,6-diphosphate and 400 mM acetaldehyde as substrates, 246 mM (52.6 g/l) 2-deoxyribose 5-phosphate was produced. The molar yield of 2-deoxyribose 5-phosphate as to glucose through the total two step reaction was 22.1%. The 2-deoxyribose 5-phosphate produced was converted to 2-deoxyribose with a molar yield of 85% through endogenous or exogenous phosphatase activity.

  1. Kinetic mechanism of the Zn-dependent aryl-phosphatase activity of myo-inositol-1-phosphatase.

    PubMed

    Caselli, Anna; Casolaro, Mario; Ranaldi, Francesco; Manao, Giampaolo; Camici, Guido; Giachetti, Eugenio

    2007-02-01

    Myo-inositol-1-phosphatase (EC 3.1.3.25) is able to hydrolyze myo-inositol-1-phosphate in the presence of Mg(2+) ions at neutral pH, and also p-nitrophenyl phosphate in the presence of Zn(2+)-ions at acidic pH. This enzyme plays a role in phosphatidylinositol cell signalling and is a putative target of lithium therapy in manic depression. We elucidate here the kinetic mechanism of the Zn-dependent activity of myo-inositol-1-phosphatase. As part of this analysis it was necessary to determine the basicity constants of p-nitrophenyl phosphate and the stability constant of its metal-complex in the presence of zinc chloride. We find that the Zn-dependent reaction may be described either by a rapid-equilibrium random mechanism or an ordered steady-state mechanism in which the substrate binds to the free enzyme prior to the metal ion. In both models the Zn-substrate complex acts as a high affinity inhibitor, yielding a dead-end species through its binding to the enzyme-Zn-substrate in rapid-equilibrium or to the enzyme-phosphate complexes in a steady-state model. Phosphate is a competitive inhibitor of the enzyme with respect to the substrate and an uncompetitive inhibitor with respect to zinc ions.

  2. Stereospecificity of myo-inositol hexakisphosphate dephosphorylation by a phytate-degrading enzyme of Escherichia coli.

    PubMed

    Greiner, R; Carlsson, N; Alminger, M L

    2001-11-17

    Using a combination of high-performance ion chromatography analysis and kinetic studies, the stereospecificity of myo-inositol hexakisphosphate dephosphorylation by the phytate-degrading enzyme P2 of Escherichia coli was established. High-performance ion chromatography revealed that the phytate-degrading enzyme P2 of E. coli degrades myo-inositol hexakisphosphate by stepwise dephosphorylation via D/L-Ins(1,2,3,4,5)P(5), D/L-Ins(2,3,4,5)P(4), D/L-Ins(2,4,5)P(3) or D/L-Ins(1,2,4)P(3), D/L-Ins(1,2)P(2) or Ins(2, 5)P(2) or D/L-Ins(4,5)P(2) to finally Ins(2)P or Ins(5)P. Kinetic parameters for myo-inositol pentakisphosphate hydrolysis by E. coli and wheat phytase, respectively, showed that the myo-inositol pentakisphosphate intermediate produced either by the phytate-degrading enzyme of wheat or E. coli are not identical. The absolute configuration of the myo-inositol pentakisphosphate isomer produced by the E. coli enzyme was determined by taking into consideration that wheat phytase produces predominantly the D-Ins(1, 2,3,5,6)P(5) isomer (Lim, P.E., Tate, M.E., 1973. The phytases: II. Properties of phytase fraction F(1) and F(2) from wheat bran and the myo-inositol phosphates produced by fraction F(2). Biochim. Biophys. Acta 302, 326-328). The data demonstrate that the phytate-degrading enzyme P2 of E. coli dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-Ins(1,2,3,4,5)P(5), D-Ins(2,3,4,5)P(4), D-Ins(2,4,5)P(3), Ins(2,5)P(2) to finally Ins(2)P (notation 6/1/3/4/5).

  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. Copyright 2010 Elsevier Inc. All rights reserved.

  4. The crystal structure of mammalian inositol 1,3,4,5,6-pentakisphosphate 2-kinase reveals a new zinc-binding site and key features for protein function.

    PubMed

    Franco-Echevarría, Elsa; Sanz-Aparicio, Julia; Brearley, Charles A; González-Rubio, Juana M; González, Beatriz

    2017-06-23

    Inositol 1,3,4,5,6-pentakisphosphate 2-kinases (IP5 2-Ks) are part of a family of enzymes in charge of synthesizing inositol hexakisphosphate (IP6) in eukaryotic cells. This protein and its product IP6 present many roles in cells, participating in mRNA export, embryonic development, and apoptosis. We reported previously that the full-length IP5 2-K from Arabidopsis thaliana is a zinc metallo-enzyme, including two separated lobes (the N- and C-lobes). We have also shown conformational changes in IP5 2-K and have identified the residues involved in substrate recognition and catalysis. However, the specific features of mammalian IP5 2-Ks remain unknown. To this end, we report here the first structure for a murine IP5 2-K in complex with ATP/IP5 or IP6 Our structural findings indicated that the general folding in N- and C-lobes is conserved with A. thaliana IP5 2-K. A helical scaffold in the C-lobe constitutes the inositol phosphate-binding site, which, along with the participation of the N-lobe, endows high specificity to this protein. However, we also noted large structural differences between the orthologues from these two eukaryotic kingdoms. These differences include a novel zinc-binding site and regions unique to the mammalian IP5 2-K, as an unexpected basic patch on the protein surface. In conclusion, our findings have uncovered distinct features of a mammalian IP5 2-K and set the stage for investigations into protein-protein or protein-RNA interactions important for IP5 2-K function and activity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Inositol pyrophosphates modulate hydrogen peroxide signalling.

    PubMed

    Onnebo, Sara Maria Nancy; Saiardi, Adolfo

    2009-09-14

    Inositol pyrophosphates are involved in a variety of cellular functions, but the specific pathways and/or downstream targets remain poorly characterized. In the present study we use Saccharomyces cerevisiae mutants to examine the potential roles of inositol pyrophosphates in responding to cell damage caused by ROS (reactive oxygen species). Yeast lacking kcs1 [the S. cerevisiae IP6K (inositol hexakisphosphate kinase)] have greatly reduced IP7 (diphosphoinositol pentakisphosphate) and IP8 (bisdiphosphoinositol tetrakisphosphate) levels, and display increased resistance to cell death caused by H2O2, consistent with a sustained activation of DNA repair mechanisms controlled by the Rad53 pathway. Other Rad53-controlled functions, such as actin polymerization, appear unaffected by inositol pyrophosphates. Yeast lacking vip1 [the S. cerevisiae PP-IP5K (also known as IP7K, IP7 kinase)] accumulate large amounts of the inositol pyrophosphate IP7, but have no detectable IP8, indicating that this enzyme represents the physiological IP7 kinase. Similar to kcs1Delta yeast, vip1Delta cells showed an increased resistance to cell death caused by H2O2, indicating that it is probably the double-pyrophosphorylated form of IP8 [(PP)2-IP4] which mediates the H2O2 response. However, these inositol pyrophosphates are not involved in directly sensing DNA damage, as kcs1Delta cells are more responsive to DNA damage caused by phleomycin. We observe in vivo a rapid decrease in cellular inositol pyrophosphate levels following exposure to H2O2, and an inhibitory effect of H2O2 on the enzymatic activity of Kcs1 in vitro. Furthermore, parallel cysteine mutagenesis studies performed on mammalian IP6K1 are suggestive that the ROS signal might be transduced by the direct modification of this evolutionarily conserved class of enzymes.

  6. 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.

  7. Polyols accumulated in ribose-5-phosphate isomerase deficiency increase mitochondrial superoxide production and improve antioxidant defenses in rats' prefrontal cortex.

    PubMed

    Stone, V; Kudo, K Y; August, P M; Marcelino, T B; Matté, C

    2014-10-01

    The ribose-5-phosphate isomerase deficiency is an inherited condition, which results in cerebral d-arabitol and ribitol accumulation. Patients present leukoencephalopathy, mental retardation, and psychomotor impairment. Considering that the pathophysiology of this disorder is still unclear, and literature are sparse and contradictory, reporting pro and antioxidant activities of polyols, the main objective of this study was to investigate some parameters of oxidative homeostasis of prefrontal cortex of rats incubated with d-arabitol and ribitol. We found evidences that ribitol promoted an increase in antioxidant enzymes activity (superoxide dismutase, catalase, and glutathione peroxidase), probably secondary to enhanced production of superoxide radical, measured by flow cytometry. Oxidation of proteins and lipids was not induced by polyols. Our data allow us to conclude that, at least in our methodological conditions, arabitol and ribitol probably have a secondary effect on the pathophysiology of ribose-5-phosphate isomerase deficiency. Copyright © 2014 ISDN. Published by Elsevier Ltd. All rights reserved.

  8. Myo-inositol inhibits intestinal glucose absorption and promotes muscle glucose uptake: a dual approach study.

    PubMed

    Chukwuma, Chika Ifeanyi; Ibrahim, Mohammed Auwal; Islam, Md Shahidul

    2016-12-01

    The present study investigated the effects of myo-inositol on muscle glucose uptake and intestinal glucose absorption ex vivo as well as in normal and type 2 diabetes model of rats. In ex vivo study, both intestinal glucose absorption and muscle glucose uptake were studied in isolated rat jejunum and psoas muscle respectively in the presence of increasing concentrations (2.5 % to 20 %) of myo-inositol. In the in vivo study, the effect of a single bolus dose (1 g/kg bw) of oral myo-inositol on intestinal glucose absorption, blood glucose, gastric emptying and digesta transit was investigated in normal and type 2 diabetic rats after 1 h of co-administration with 2 g/kg bw glucose, when phenol red was used as a recovery marker. Myo-inositol inhibited intestinal glucose absorption (IC50 = 28.23 ± 6.01 %) and increased muscle glucose uptake, with (GU50 = 2.68 ± 0.75 %) or without (GU50 = 8.61 ± 0.55 %) insulin. Additionally, oral myo-inositol not only inhibited duodenal glucose absorption and reduced blood glucose increase, but also delayed gastric emptying and accelerated digesta transit in both normal and diabetic animals. Results of this study suggest that dietary myo-inositol inhibits intestinal glucose absorption both in ex vivo and in normal or diabetic rats and also promotes muscle glucose uptake in ex vivo condition. Hence, myo-inositol may be further investigated as a possible anti-hyperglycaemic dietary supplement for diabetic foods and food products.

  9. Effect of Co-solutes on the Products and Solubility of Uranium(VI) Precipitated with Phosphate

    SciTech Connect

    Mehta, Vrajesh; Maillot, Fabien; Wang, Zheming; Catalano, Jeffrey G.; Giammar, Daniel E.

    2014-01-22

    Uranyl phosphate solids are often found with uranium ores, and their low solubility makes them promising target phases for in situ remediation of uranium-contaminated subsurface environments. The products and solubility of uranium(VI) precipitated with phosphate can be affected by the pH, dissolved inorganic carbon (DIC) concentration, and co-solute composition (e.g. Na+/Ca2+) of the groundwater. Batch experiments were performed to study the effect of these parameters on the products and extent of uranium precipitation induced by phosphate addition. In the absence of co-solute cations, chernikovite [H3O(UO2)(PO4)•3H2O] precipitated despite uranyl orthophosphate [(UO2)3(PO4)2•4H2O] being thermodynamically more favorable under certain conditions. As determined using X-ray diffraction, electron microscopy, and laser induced fluorescence spectroscopy, the presence of Na+ or Ca2+ as a co-solute led to the precipitation of sodium autunite ([Na2(UO2)2(PO4)2] and autunite [Ca(UO2)2(PO4)2]), which are structurally similar to chernikovite. In the presence of sodium, the dissolved U(VI) concentrations were generally in agreement with equilibrium predictions of sodium autunite solubility. However, in the calcium-containing systems, the observed concentrations were below the predicted solubility of autunite, suggesting the possibility of uranium adsorption to or incorporation in a calcium phosphate precipitate in addition to the precipitation of autunite.

  10. Structural profiling and quantitation of glycosyl inositol phosphoceramides in plants with Fourier transform mass spectrometry.

    PubMed

    Blaas, Nina; Humpf, Hans-Ulrich

    2013-05-08

    Glycosyl inositol phosphoceramides (GIPC) are the main sphingolipids in plants, and optimization of their extraction and detection is still in the focus of research. Mass spectrometry provides new options for the analysis and structural elucidation of this complex class of lipids. The coupling of linear ion trap and orbitrap (LTQ Orbitrap) enabled various fragmentation experiments (MS(2), MS(3)) by collision-induced dissociation (CID) and pulsed-Q dissociation (PQD). For structural analysis, GIPC-fragment ions were detected in the positive and negative ion mode with exact masses; therefore, fragmentation patterns were observed and finally structures have been characterized regarding polar head group, fatty acid, and sphingoid base. GIPC profiling was performed for spinach, white cabbage, sunflower seeds, and soybeans. The total GIPC concentration in these plants ranged from 1.1 to 88.4 μg/100 g dry weight with t18:1/h24:0 as the main ceramide structure and hexose-hexuronic acid-inositol phosphate and N-acetylhexosamine-hexuronic acid-inositol phosphate as polar head groups.

  11. Bradykinin-activated transmembrane signals are coupled via N/sub o/ or N/sub i/ to production of inositol 1,4,5-trisphosphate, a second messenger in NG108-15 neuroblastoma-glioma hybrid cells

    SciTech Connect

    Higashida, H.; Streaty, R.A.; Klee, W.; Nirenberg, M.

    1986-02-01

    The addition of bradykinin to NG108-15 cells results in a transient hyperpolarization followed by prolonged cell depolarization. Injection of inositol 1,4,5-trisphosphate or CaS into the cytoplasm of NG108-15 cells also elicits cell hyperpolarization followed by depolarization. Tetraethylammonium ions inhibit the hyperpolarizing response of cells to bradykinin or inositol 1,4,5-trisphosphate. Thus, the hyperpolarizing phase of the cell response may be due to inositol 1,4,5-trisphosphate-dependent release of stored UVCa-labelled CaS into the cytoplasm, which activates CaS -dependent K channels. The depolarizing phase of the cell response to bradykinin is due largely to inhibition of M channels, thereby decreasing the rate of K efflux from cells and, to a lesser extent, to activation of CaS -dependent ion channels and CaS channels. In contrast, injection of inositol 1,4,5-trisphosphate or CaS into the cytosol did not alter M channel activity. Incubation of NG108-15 cells with pertussis toxin inhibits bradykinin-dependent cell hyperpolarization and depolarization. Bradykinin stimulates low K/sub m/ GTPase activity and inhibits adenylate cyclase in NG108-15 membrane preparations but not in membranes prepared from cells treated with pertussis toxin. These results show that (bradykinin-receptor) complexes interact with N/sub o/ or N/sub i/ and suggest that N/sub o/ and/or N/sub i/ mediate the transduction of signals from bradykinin receptors to phospholipase C and adenylate cyclase.

  12. 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

  13. Biomineralization of U(VI) phosphate promoted by microbially-mediated phytate hydrolysis in contaminated soils

    NASA Astrophysics Data System (ADS)

    Salome, Kathleen R.; Beazley, Melanie J.; Webb, Samuel M.; Sobecky, Patricia A.; Taillefert, Martial

    2017-01-01

    The bioreduction of uranium may immobilize a significant fraction of this toxic contaminant in reduced environments at circumneutral pH. In oxic and low pH environments, however, the low solubility of U(VI)-phosphate minerals also makes them good candidates for the immobilization of U(VI) in the solid phase. As inorganic phosphate is generally scarce in soils, the biomineralization of U(VI)-phosphate minerals via microbially-mediated organophosphate hydrolysis may represent the main immobilization process of uranium in these environments. In this study, contaminated sediments were incubated aerobically in two pH conditions to examine whether phytate, a naturally-occurring and abundant organophosphate in soils, could represent a potential phosphorous source to promote U(VI)-phosphate biomineralization by natural microbial communities. While phytate hydrolysis was not evident at pH 7.0, nearly complete hydrolysis was observed both with and without electron donor at pH 5.5, suggesting indigenous microorganisms express acidic phytases in these sediments. While the rate of hydrolysis of phytate generally increased in the presence of uranium, the net rate of inorganic phosphate production in solution was decreased and inositol phosphate intermediates were generated in contrast to similar incubations conducted without uranium. These findings suggest uranium stress enhanced the phytate-metabolism of the microbial community, while simultaneously inhibiting phosphatase production and/or activity by the indigenous population. Finally, phytate hydrolysis drastically decreased uranium solubility, likely due to formation of ternary sorption complexes, U(VI)-phytate precipitates, and U(VI)-phosphate minerals. Overall, the results of this study provide evidence for the ability of natural microbial communities to liberate phosphate from phytate in acidic sediments, possibly as a detoxification mechanism, and demonstrate the potential utility of phytate-promoted uranium

  14. Effect of ACP-CPP Chewing Gum and Natural Chewable Products on Plaque pH, Calcium and Phosphate Concentration

    PubMed Central

    Sultan, Saima; Chaudhary, Seema; Manuja, Naveen; Kaur, Harsimran; Amit, Sinha Ashish; Lingesha, Ravishankar Telgi

    2016-01-01

    Introduction Numerous epidemiological studies have documented dental caries as the major public health problems throughout the world. It is gradually increasing in the underdeveloped and developing countries especially in children due to increasing popularity of refined sugars. Aim The aim of the study was to evaluate the effect of natural chewable products (Tulsi, sesame seeds, fennel seeds, coconut) and ACP-CPP chewing gum on plaque pH, calcium and phosphate concentration. Materials and Methods A randomized controlled trial, with a cross-over study design, was conducted. Ten subjects aged 15-17 years who agreed to refrain from oral hygiene practice for 48 hours prior to the sample collection were selected for the study. The baseline plaque pH, calcium and phosphate was measured and repeated after 5 and 30 minutes. It was ensured that each study participant was subjected to all the products making an effective sample of ten subjects per product. The data was statistically analysed. Results The mean pH in all the study groups increased after 5 minutes and 30 minutes compared to baseline, except for coconut group at 30 minutes and fennel group at 5 minutes. Highest increase in plaque calcium concentration was found in fennel group followed by recaldent and sesame, respectively. Whereas, the highest increase in plaque phosphate was found in recaldent group followed by sesame group and fennel group respectively. Conclusion Plant products can be effective, inexpensive, easily accessible methods of maintaining oral health. Further studies are recommended to confirm long term effects. PMID:27190943

  15. A Ralstonia solanacearum type III effector directs the production of the plant signal metabolite trehalose-6-phosphate.

    PubMed

    Poueymiro, M; Cazalé, A C; François, J M; Parrou, J L; Peeters, N; Genin, S

    2014-12-23

    The plant pathogen Ralstonia solanacearum possesses two genes encoding a trehalose-6-phosphate synthase (TPS), an enzyme of the trehalose biosynthetic pathway. One of these genes, named ripTPS, was found to encode a protein with an additional N-terminal domain which directs its translocation into host plant cells through the type 3 secretion system. RipTPS is a conserved effector in the R. solanacearum species complex, and homologues were also detected in other bacterial plant pathogens. Functional analysis of RipTPS demonstrated that this type 3 effector synthesizes trehalose-6-phosphate and identified residues essential for this enzymatic activity. Although trehalose-6-phosphate is a key signal molecule in plants that regulates sugar status and carbon assimilation, the disruption of ripTPS did not alter the virulence of R. solanacearum on plants. However, heterologous expression assays showed that this effector specifically elicits a hypersensitive-like response on tobacco that is independent of its enzymatic activity and is triggered by the C-terminal half of the protein. Recognition of this effector by the plant immune system is suggestive of a role during the infectious process. Ralstonia solanacearum, the causal agent of bacterial wilt disease, infects more than two hundred plant species, including economically important crops. The type III secretion system plays a major role in the pathogenicity of this bacterium, and approximately 70 effector proteins have been shown to be translocated into host plant cells. This study provides the first description of a type III effector endowed with a trehalose-6-phosphate synthase enzymatic activity and illustrates a new mechanism by which the bacteria may manipulate the plant metabolism upon infection. In recent years, trehalose-6-phosphate has emerged as an essential signal molecule in plants, connecting plant metabolism and development. The finding that a bacterial pathogen could induce the production of trehalose-6

  16. Kinetics, substrate specificity, and stereospecificity of two new protein tyrosine phosphatase-like inositol polyphosphatases from Selenomonas lacticifex.

    PubMed

    Puhl, Aaron A; Greiner, Ralf; Selinger, L Brent

    2008-08-01

    Inositol polyphosphatases (IPPases) play an important role in the metabolism of inositol polyphosphates, a class of molecules involved in signal transduction. Here we characterize 2 new protein tyrosine phosphatase-like IPPases (PhyAsl and PhyBsl) cloned from Selenomonas lacticifex that can hydrolyze myo-inositol hexakisphosphate (InsP6) in vitro. To determine their preferred substrates and stereospecificity of InsP6 dephosphorylation, a combination of kinetic and high-performance ion pair chromatography studies were conducted. Despite only 33% amino acid sequence identity between them, both enzymes display strict specificity for IPP substrates and cleave InsP6 primarily at the D-3-phosphate position (>90%). Furthermore, both enzymes predominantly degrade InsP6 to Ins(2)P via identical and very specific routes of dephosphorylation (3,4,5,6,1). Despite these similarities, PhylAsl is shown to have a slight kinetic preference for the major inositol pentakisphosphate intermediate in its InsP6 hydrolysis pathway, whereas PhyBsl displays a unique and substantial preference for an inositol tetrakisphosphate intermediate.

  17. Regulation of PtdIns(3,4,5)P3/Akt signalling by inositol polyphosphate 5-phosphatases.

    PubMed

    Eramo, Matthew J; Mitchell, Christina A

    2016-02-01

    The phosphoinositide 3-kinase (PI3K) generated lipid signals, PtdIns(3,4,5)P3 and PtdIns(3,4)P2, are both required for the maximal activation of the serine/threonine kinase proto-oncogene Akt. The inositol polyphosphate 5-phosphatases (5-phosphatases) hydrolyse the 5-position phosphate from the inositol head group of PtdIns(3,4,5)P3 to yield PtdIns(3,4)P2. Extensive work has revealed several 5-phosphatases inhibit PI3K-driven Akt signalling, by decreasing PtdIns(3,4,5)P3 despite increasing cellular levels of PtdIns(3,4)P2. The roles that 5-phosphatases play in suppressing cell proliferation and transformation are slow to emerge; however, the 5-phosphatase PIPP [proline-rich inositol polyphosphate 5-phosphatase; inositol polyphosphate 5-phosphatase (INPP5J)] has recently been identified as a putative tumour suppressor in melanoma and breast cancer and SHIP1 [SH2 (Src homology 2)-containing inositol phosphatase 1] inhibits haematopoietic cell proliferation. INPP5E regulates cilia stability and INPP5E mutations have been implicated ciliopathy syndromes. This review will examine 5-phosphatase regulation of PI3K/Akt signalling, focussing on the role PtdIns(3,4,5)P3 5-phosphatases play in developmental diseases and cancer.

  18. 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

  19. Phosphate Limitation Induces Drastic Physiological Changes, Virulence-Related Gene Expression, and Secondary Metabolite Production in Pseudovibrio sp. Strain FO-BEG1

    PubMed Central

    González, José M.; Bondarev, Vladimir

    2015-01-01

    Phosphorus is a vital nutrient for living organisms and is obtained by bacteria primarily via phosphate uptake. However, phosphate is often scarcely accessible in nature, and there is evidence that in many areas of the ocean, its concentration limits bacterial growth. Surprisingly, the phosphate starvation response has been extensively investigated in different model organisms (e.g., Escherichia coli), but there is a dearth of studies on heterotrophic marine bacteria. In this work, we describe the response of Pseudovibrio sp. strain FO-BEG1, a metabolically versatile alphaproteobacterium and potential symbiont of marine sponges, to phosphate limitation. We compared the physiology, protein expression, and secondary metabolite production under phosphate-limited conditions to those under phosphate surplus conditions. We observed that phosphate limitation had a pleiotropic effect on the physiology of the strain, triggering cell elongation, the accumulation of polyhydroxyalkanoate, the degradation of polyphosphate, and the exchange of membrane lipids in favor of phosphorus-free lipids such as sulfoquinovosyl diacylglycerols. Many proteins involved in the uptake and degradation of phospho-organic compounds were upregulated, together with subunits of the ABC transport system for phosphate. Under conditions of phosphate limitation, FO-BEG1 secreted compounds into the medium that conferred an intense yellow coloration to the cultures. Among these compounds, we identified the potent antibiotic tropodithietic acid. Finally, toxin-like proteins and other proteins likely involved in the interaction with the eukaryotic host were also upregulated. Altogether, our data suggest that phosphate limitation leads to a pronounced reorganization of FO-BEG1 physiology, involving phosphorus, carbon, and sulfur metabolism; cell morphology; secondary metabolite production; and the expression of virulence-related genes. PMID:25769826

  20. The product of microbial uranium reduction includes multiple species with U(IV)-phosphate coordination

    NASA Astrophysics Data System (ADS)

    Alessi, Daniel S.; Lezama-Pacheco, Juan S.; Stubbs, Joanne E.; Janousch, Markus; Bargar, John R.; Persson, Per; Bernier-Latmani, Rizlan

    2014-04-01

    Until recently, the reduction of U(VI) to U(IV) during bioremediation was assumed to produce solely the sparingly soluble mineral uraninite, UO2(s). However, results from several laboratories reveal other species of U(IV) characterized by the absence of an EXAFS U-U pair correlation (referred to here as noncrystalline U(IV)). Because it lacks the crystalline structure of uraninite, this species is likely to be more labile and susceptible to reoxidation. In the case of single species cultures, analyses of U extended X-ray fine structure (EXAFS) spectra have previously suggested U(IV) coordination to carboxyl, phosphoryl or carbonate groups. In spite of this evidence, little is understood about the species that make up noncrystalline U(IV), their structural chemistry and the nature of the U(IV)-ligand interactions. Here, we use infrared spectroscopy (IR), uranium LIII-edge X-ray absorption spectroscopy (XAS), and phosphorus K-edge XAS analyses to constrain the binding environments of phosphate and uranium associated with Shewanella oneidensis MR-1 bacterial cells. Systems tested as a function of pH included: cells under metal-reducing conditions without uranium, cells under reducing conditions that produced primarily uraninite, and cells under reducing conditions that produced primarily biomass-associated noncrystalline U(IV). P X-ray absorption near-edge structure (XANES) results provided clear and direct evidence of U(IV) coordination to phosphate. Infrared (IR) spectroscopy revealed a pronounced perturbation of phosphate functional groups in the presence of uranium. Analysis of these data provides evidence that U(IV) is coordinated to a range of phosphate species, including monomers and polymerized networks. U EXAFS analyses and a chemical extraction measurements support these conclusions. The results of this study provide new insights into the binding mechanisms of biomass-associated U(IV) species which in turn sheds light on the mechanisms of biological U

  1. 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

  2. Cyclic nucleotide regulation of inositol lipid metabolism in rat cerebral cortex

    SciTech Connect

    Nicchitta, C.V.; Williamson, J.R.

    1986-05-01

    The authors have investigated the effects of compounds known to elevate cAMP levels and/or activate the cAMP dependent protein kinase on resting and stimulated inositol lipid metabolism in rat cortical slices and synaptosomes. In (/sup 3/H)-myo-inositol-labeled brain slices, carbamylcholine-stimulated accumulation of (/sup 3/H)-myo-inositol 1-phosphate (IP/sub 1/) was decreased 50% by forskolin (150..mu..M) isobutylmethylxantihine (IBMX) (1mM), 8-bromo cAMP (5mM) or 8-(4-chlorophenylthio) cAMP (5mM). In studies with synaptosomes, the incorporation of (/sup 32/Pi) into phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP/sub 2/) and phosphatidic acid (PA) was also reduced in the presence of forskolin or the cAMP phosphodiesterase inhibitors IBMX and RO20-1724 (+/-)-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone. In the presence of maximally effective concentrations of these agents, steady state PIP levels were reduced by 50% and PIP/sub 2/ and PA levels by 40%. The effects of these compounds on (/sup 32/Pi) labeling of the phosphoinositides and PA are consistent with an inhibition of flux through the inositide cycle, presumably through the action of cAMP. Such an inhibition may account for the reduction in carbamylcholine sensitive IP/sub 1/ accumulation observed in brain slices. These studies indicate that in rat brain, ..beta..-adrenergic stimulation may be involved in modulating the activity of muscarinic cholinergic agonists. This regulation appears to be at the level of inositol lipid metabolism.

  3. 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.

  4. Production of clover broomrape seed germination stimulants by red clover root requires nitrate but is inhibited by phosphate and ammonium.

    PubMed

    Yoneyama, Koichi; Takeuchi, Yasutomo; Yokota, Takao

    2001-05-01

    The effect of nutrients (nitrate, ammonium, urea, phosphate and potassium) on the production and/or exudation of germination stimulants for clover broomrape (Orobanche minor Sm.), a root holoparasite, by its host red clover (Trifolium pratense L.) was examined using hydroponically grown material. Potassium (K2SO4) concentrations up to 100 mg l-1 (based on K) did not affect the production of germination stimulants by red clover while, in contrast, phosphate (NaH2PO4) was highly inhibitory even at concentrations as low as 1 mg l-1 (based on P). Nitrate (NaNO3) markedly promoted stimulant production in a dose-dependent manner from 2 to 50 mg l-1 (based on N). Ammonium [(NH4)2SO4] had no effect at 2 mg l-1 (based on N) but was inhibitory at higher concentrations. Ammonium is known to be a seed germination inhibitor of root parasites, indicating that ammonium has a dual inhibitory action. Urea had no effect at 2 mg l-1 (based on N) but was promotive at higher concentrations. These results provide a basis for the inhibitory effects of nitrogen fertilizer on infection by root parasitic weeds, broomrapes and witchweeds, and explain why these parasites prevail in areas where there is lower phosphorus availability in soils.

  5. Bioreduction of hydrogen uranyl phosphate: mechanisms and U(IV) products.

    PubMed

    Rui, Xue; Kwon, Man Jae; O'Loughlin, Edward J; Dunham-Cheatham, Sarrah; Fein, Jeremy B; Bunker, Bruce; Kemner, Kenneth M; Boyanov, Maxim I

    2013-06-04

    The mobility of uranium (U) in subsurface environments is controlled by interrelated adsorption, redox, and precipitation reactions. Previous work demonstrated the formation of nanometer-sized hydrogen uranyl phosphate (abbreviated as HUP) crystals on the cell walls of Bacillus subtilis, a non-U(VI)-reducing, Gram-positive bacterium. The current study examined the reduction of this biogenic, cell-associated HUP mineral by three dissimilatory metal-reducing bacteria, Anaeromyxobacter dehalogenans strain K, Geobacter sulfurreducens strain PCA, and Shewanella putrefaciens strain CN-32, and compared it to the bioreduction of abiotically formed and freely suspended HUP of larger particle size. Uranium speciation in the solid phase was followed over a 10- to 20-day reaction period by X-ray absorption fine structure spectroscopy (XANES and EXAFS) and showed varying extents of U(VI) reduction to U(IV). The reduction extent of the same mass of HUP to U(IV) was consistently greater with the biogenic than with the abiotic material under the same experimental conditions. A greater extent of HUP reduction was observed in the presence of bicarbonate in solution, whereas a decreased extent of HUP reduction was observed with the addition of dissolved phosphate. These results indicate that the extent of U(VI) reduction is controlled by dissolution of the HUP phase, suggesting that the metal-reducing bacteria transfer electrons to the dissolved or bacterially adsorbed U(VI) species formed after HUP dissolution, rather than to solid-phase U(VI) in the HUP mineral. Interestingly, the bioreduced U(IV) atoms were not immediately coordinated to other U(IV) atoms (as in uraninite, UO2) but were similar in structure to the phosphate-complexed U(IV) species found in ningyoite [CaU(PO4)2·H2O]. This indicates a strong control by phosphate on the speciation of bioreduced U(IV), expressed as inhibition of the typical formation of uraninite under phosphate-free conditions.

  6. Phosphate-binding pocket in Dicer-2 PAZ domain for high-fidelity siRNA production.

    PubMed

    Kandasamy, Suresh K; Fukunaga, Ryuya

    2016-12-06

    The enzyme Dicer produces small silencing RNAs such as micro-RNAs (miRNAs) and small interfering RNAs (siRNAs). In Drosophila, Dicer-1 produces ∼22-24-nt miRNAs from pre-miRNAs, whereas Dicer-2 makes 21-nt siRNAs from long double-stranded RNAs (dsRNAs). How Dicer-2 precisely makes 21-nt siRNAs with a remarkably high fidelity is unknown. Here we report that recognition of the 5'-monophosphate of a long dsRNA substrate by a phosphate-binding pocket in the Dicer-2 PAZ (Piwi, Argonaute, and Zwille/Pinhead) domain is crucial for the length fidelity, but not the efficiency, in 21-nt siRNA production. Loss of the length fidelity, meaning increased length heterogeneity of siRNAs, caused by point mutations in the phosphate-binding pocket of the Dicer-2 PAZ domain decreased RNA silencing activity in vivo, showing the importance of the high fidelity to make 21-nt siRNAs. We propose that the 5'-monophosphate of a long dsRNA substrate is anchored by the phosphate-binding pocket in the Dicer-2 PAZ domain and the distance between the pocket and the RNA cleavage active site in the RNaseIII domain corresponds to the 21-nt pitch in the A-form duplex of a long dsRNA substrate, resulting in high-fidelity 21-nt siRNA production. This study sheds light on the molecular mechanism by which Dicer-2 produces 21-nt siRNAs with a remarkably high fidelity for efficient RNA silencing.

  7. Development of vanadium-phosphate catalysts for methanol production by selective oxidation of methane. Quarterly report, July - September 1996

    SciTech Connect

    McCormick, R.L.; Alptekin, G.O.

    1996-12-01

    This document covers the period July-September, 1996. Activities included studies of the oxidation of dimethyl ether over vanadyl pyrophosphate and synthesis of all previously acquired kinetic data. This synthesis revealed the need for additional data on methane and methanol oxidation and these experiments were performed. A further series of methanol oxidation/dehydration experiments was conducted on samples with varying surface acidity that have been described in earlier reports. Oxidation of methane over Cr- promoted VPO was also reinvestigated. The kinetic studies performed to date allow us to determine optimum conditions for methanol and formaldehyde production from methane using VPO catalysts, and in particular determine the effect of lean conditions (excess oxygen), oxygen deficient conditions (used in most other methane oxidation studies), and the potential of using the catalyst as a stoichiometric oxidant or oxygen carrier. However, unpromoted VPO yields only CO as the primary oxidation product. Studies of promoters have shown improvements in the formaldehyde selectivity but no methanol has been observed. The best promoters tested have been Fe and Cr (results for Cr are described in this report). We have also examined the use of iron phosphate for the methane conversion reaction. FePO{sub 4}is a more selectivity catalyst than the promoted VPO materials. Support of this iron phosphate on silica results in further improvements in selectivity. Current work is directed at understanding the improved selectivity for promoted VPO and at obtaining a knowledge of the optimum conditions for methane conversion of iron phosphate. 15 refs., 2 figs., 1 tab.

  8. Barley (Hordeum vulgare L.) inositol monophosphatase: gene structure and enzyme characteristics

    USDA-ARS?s Scientific Manuscript database

    The de novo synthesis of myo-inositol (Ins) is catalyzed by two enzymatic activities; Ins(3)P1 synthase (MIPS; EC. 5.5.1.4) and Ins monophosphatase (IMPase; EC 3.1.3.25). The barley IMP-1 gene and gene products were studied to facilitate research into the regulation of Ins synthesis and supply. In m...

  9. Production of Glucaric Acid from a Synthetic Pathway in Recombinant Escherichia coli▿ †

    PubMed Central

    Moon, Tae Seok; Yoon, Sang-Hwal; Lanza, Amanda M.; Roy-Mayhew, Joseph D.; Prather, Kristala L. Jones

    2009-01-01

    A synthetic pathway has been constructed for the production of glucuronic and glucaric acids from glucose in Escherichia coli. Coexpression of the genes encoding myo-inositol-1-phosphate synthase (Ino1) from Saccharomyces cerevisiae and myo-inositol oxygenase (MIOX) from mice led to production of glucuronic acid through the intermediate myo-inositol. Glucuronic acid concentrations up to 0.3 g/liter were measured in the culture broth. The activity of MIOX was rate limiting, resulting in the accumulation of both myo-inositol and glucuronic acid as final products, in approximately equal concentrations. Inclusion of a third enzyme, uronate dehydrogenase (Udh) from Pseudomonas syringae, facilitated the conversion of glucuronic acid to glucaric acid. The activity of this recombinant enzyme was more than 2 orders of magnitude higher than that of Ino1 and MIOX and increased overall flux through the pathway such that glucaric acid concentrations in excess of 1 g/liter were observed. This represents a novel microbial system for the biological production of glucaric acid, a “top value-added chemical” from biomass. PMID:19060162

  10. 21 CFR 184.1370 - Inositol.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 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 FOOD... the Food Chemicals Codex, 3d Ed. (1981), p. 150, which is incorporated by reference. Copies are...

  11. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. D-chiro-inositol metabolism in diabetes mellitus.

    PubMed

    Ostlund, R E; McGill, J B; Herskowitz, I; Kipnis, D M; Santiago, J V; Sherman, W R

    1993-11-01

    D-chiro-inositol is a rare inositol isomer present in inositol phosphoglycans which are proposed mediators of insulin action. To study D-chiro-inositol metabolism in diabetes mellitus, a sensitive and specific assay was developed using negative-ion chemical ionization gas chromatography/mass spectrometry. Median urinary D-chiro-inositol excretion, which was 2.1 mumol/day in nondiabetics, was substantially increased to 12 mumol/day in non-insulin-dependent diabetes (P < 0.0001) and to 74 mumol/day in insulin-dependent diabetes (P < 0.0001). Urinary D-chiro-inositol was strongly correlated with fasting plasma glucose (r = 0.568, P < 0.0001), glycated hemoglobin (r = 0.529, P < 0.0001), and urinary glucose (r = 0.368, P = 0.01). The renal clearance of D-chiro-inositol was selectively elevated in both non-insulin-dependent and insulin-dependent diabetes when compared with the clearances of L-chiro-inositol or myo-inositol and exceeded the glomerular filtration rate in 71% of the diabetics but in none of the nondiabetics. In poorly controlled diabetic patients insulin treatment reduced urinary D-chiro-inositol losses by 63% and increased plasma levels by 8.8-fold. The metabolism of D-chiro-inositol is abnormal in diabetes and appears to be influenced by short- and long-term metabolic control.

  13. SGPL1 (sphingosine phosphate lyase 1) modulates neuronal autophagy via phosphatidylethanolamine production.

    PubMed

    Mitroi, Daniel N; Karunakaran, Indulekha; Gräler, Markus; Saba, Julie D; Ehninger, Dan; Ledesma, María Dolores; van Echten-Deckert, Gerhild

    2017-05-04

    Macroautophagy/autophagy defects have been identified as critical factors underlying the pathogenesis of neurodegenerative diseases. The roles of the bioactive signaling lipid sphingosine-1-phosphate (S1P) and its catabolic enzyme SGPL1/SPL (sphingosine phosphate lyase 1) in autophagy are increasingly recognized. Here we provide in vitro and in vivo evidence for a previously unidentified route through which SGPL1 modulates autophagy in neurons. SGPL1 cleaves S1P into ethanolamine phosphate, which is directed toward the synthesis of phosphatidylethanolamine (PE) that anchors LC3-I to phagophore membranes in the form of LC3-II. In the brains of SGPL1(fl/fl/Nes) mice with developmental neural specific SGPL1 ablation, we observed significantly reduced PE levels. Accordingly, alterations in basal and stimulated autophagy involving decreased conversion of LC3-I to LC3-II and increased BECN1/Beclin-1 and SQSTM1/p62 levels were apparent. Alterations were also noticed in downstream events of the autophagic-lysosomal pathway such as increased levels of lysosomal markers and aggregate-prone proteins such as APP (amyloid β [A4] precursor protein) and SNCA/α-synuclein. In vivo profound deficits in cognitive skills were observed. Genetic and pharmacological inhibition of SGPL1 in cultured neurons promoted these alterations, whereas addition of PE was sufficient to restore LC3-I to LC3-II conversion, and control levels of SQSTM1, APP and SNCA. Electron and immunofluorescence microscopy showed accumulation of unclosed phagophore-like structures, reduction of autolysosomes and altered distribution of LC3 in SGPL1(fl/fl/Nes) brains. Experiments using EGFP-mRFP-LC3 provided further support for blockage of the autophagic flux at initiation stages upon SGPL1 deficiency due to PE paucity. These results emphasize a formerly overlooked direct role of SGPL1 in neuronal autophagy and assume significance in the context that autophagy modulators hold an enormous therapeutic potential in the

  14. 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.

  15. PuPO4(cr, hyd.) Solubility Product and Pu3+ Complexes With Phosphate and Ethylenediaminetetraacetic Acid

    SciTech Connect

    Rai, Dhanpat; Moore, Dean A.; Felmy, Andrew R.; Rosso, Kevin M.; Bolton, Harvey

    2010-06-15

    To determine the solubility product of PuPO4(cr, hyd.) and the complexation constants of Pu(III) with phosphate and EDTA, the solubility of PuPO4(cr, hyd.) was investigated as a function of: 1) time and pH varying from 1.0 to 12.0 and at a fixed 0.00032 M phosphate concentration; 2) NaH2PO4 concentrations varying from 0.0001 M to 1.0 M and at a fixed pH value of 2.5; 3) time and pH varying from 1.3 to 13.0 at fixed concentrations of 0.00032 M phosphate and 0.0004 M or 0.002 M Na2H2EDTA; and 4) Na2H2EDTA concentrations varying from 0.00005 M to 0.0256 M at a fixed 0.00032 M phosphate concentration and at pH values of approximately 3.5, 10.6, and 12.6. A combination of solvent extraction and spectrophotometric techniques confirmed that the use of hydroquinone and Na2S2O4 helped maintain Pu as Pu(III). The solubility data were interpreted using Pitzer and SIT models, and both provided similar values for the solubility product of PuPO4(cr, hyd.) and for the formation constant of PuEDTA-. The log10 of the solubility product of PuPO4(cr, hyd.) (PuPO4(cr, hyd.) = Pu3+ + PO4 ) was determined to be –(24.42 ± 0.38). Pitzer modeling showed that phosphate interactions with Pu3+ were extremely weak and did not require any phosphate complexes (e.g., PuPO4(aq), PuH2PO42+, Pu(H2PO4)2+, Pu(H2PO4)3(aq), and Pu(H2PO4)4-), as proposed in existing literature, to explain the experimental data. SIT modeling, however, required the inclusion of PuH2PO42+ to explain the data in high NaH2PO4 concentrations; this illustrates the differences one can expect when using these two chemical models to interpret the data. As the Pu(III)-EDTA species, only PuEDTA- was needed to interpret the experimental data in a large range in pH values (1.3–12.9) and EDTA concentrations (0.00005–0.256 M). Calculations based on density functional theory support the existence of PuEDTA- (with prospective stoichiometry as Pu(OH2)3EDTA-) as the chemically and structurally stable species. The log10 of the

  16. Application of calcium phosphate flocculation in high-density cell culture fluid with high product titer of monoclonal antibody.

    PubMed

    Chen, Guoqiang; Su, Zhiguo; Li, Feng; Liu, Hui Fang

    2017-05-01

    The calcium phosphate [Ca3(PO4)2] precipitation was used for improving the clarification efficiency in harvest process of the monoclonal antibody (mAb) containing cell culture fluid (CCF) with high turbidity and product titer. The flocculation conditions (concentration, addition order of flocculants, pH, and operation time), and the effect of flocculants on the mAb physical chemical properties (such as distribution of charge variants and aggregates) and process-related impurities removal (such as DNA and CHOP) were evaluated in this study. The results showed that the turbidity of CCF supernatant was significantly reduced at pH 7, 120 min with addition of phosphate ions first, while a high mAb recovery yield was kept in the CCF supernatant after flocculation. Addition of calcium ions at 15-60 mM was sufficient for flocculation in this study. A relationship between turbidity/mAb recovery yield and the concentration of calcium ions was established. More than 85% DNA in the CCF were effectively removed by the addition of optimal concentration of flocculants. Flocculation process of Ca3(PO4)2 is an effective pretreatment method in purification processes of mAbs from the CCF with high turbidity and product titer.

  17. Determination of neo- and d-chiro-Inositol Hexakisphosphate in Soils by Solution 31P NMR Spectroscopy

    PubMed Central

    2012-01-01

    The inositol phosphates are an abundant but poorly understood group of organic phosphorus compounds found widely in the environment. Four stereoisomers of inositol hexakisphosphate (IP6) occur, although for three of these (scyllo, neo, and d-chiro) the origins, dynamics, and biological function remain unknown, due in large part to analytical limitations in their measurement in environmental samples. We synthesized authentic neo- and d-chiro-IP6 and used them to identify signals from these compounds in three soils from the Falkland Islands. Both compounds resisted hypobromite oxidation and gave quantifiable 31P NMR signals at δ = 6.67 ppm (equatorial phosphate groups of the 4-equatorial/2-axial conformer of neo-IP6) and δ = 6.48 ppm (equatorial phosphate groups of the 2-equatorial/4-axial conformer of d-chiro-IP6) in soil extracts. Inositol hexakisphosphate accounted for 46–54% of the soil organic phosphorus, of which the four stereoisomers constituted, on average, 55.9% (myo), 32.8% (scyllo), 6.1% (neo), and 5.2% (d-chiro). Reappraisal of the literature based on the new signal assignments revealed that neo- and d-chiro-IP6 occur widely in both terrestrial and aquatic ecosystems. These results confirm that the inositol phosphates can constitute a considerable fraction of the organic phosphorus in soils and reveal the prevalence of neo- and d-chiro-IP6 in the environment. The hypobromite oxidation and solution 31P NMR spectroscopy procedure allows the simultaneous quantification of all four IP6 stereoisomers in environmental samples and provides a platform for research into the origins and ecological significance of these enigmatic compounds. PMID:22489788

  18. Inositol pyrophosphates inhibit synaptotagmin-dependent exocytosis

    PubMed Central

    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

    2016-01-01

    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 Ca2+ 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 Ca2+ levels. Thus, 5-IP7 appears to act through Syt1 binding to interfere with the fusogenic activity of Ca2+. 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

  19. 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

  20. Phosphate salts

    MedlinePlus

    ... taken by mouth or used as enemas. Indigestion. Aluminum phosphate and calcium phosphate are FDA-permitted ingredients ... Phosphate salts containing sodium, potassium, aluminum, or calcium are LIKELY SAFE for most people when taken by mouth short-term, when sodium phosphate is inserted into the ...

  1. Conformational Stability of Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase (IPK1) Dictates Its Substrate Selectivity*

    PubMed Central

    Gosein, Varin; Miller, Gregory J.

    2013-01-01

    Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) converts inositol 1,3,4,5,6-pentakisphosphate(IP5) to inositol hexakisphosphate (IP6). IPK1 shares structural similarity with protein kinases and is suspected to employ a similar mechanism of activation. Previous studies revealed roles for the 1- and 3-phosphates of IP5 in IPK1 activation and revealed that the N-lobe of IPK1 is unstable in the absence of inositol phosphate (IP). Here, we demonstrate the link between IPK1 substrate specificity and the stability of its N-lobe. Limited proteolysis of IPK1 revealed that N-lobe stability is dependent on the presence of the 1-phosphate of the substrate, whereas overall stability of IPK1 was increased in ternary complexes with nucleotide and IPs possessing 1- and 3-phosphates that engage the N-lobe of IPK1. Thus, the 1- and 3-phosphates possess dual roles in both IPK1 activation and IPK1 stability. To test whether kinase stability directly contributed to substrate selectivity of the kinase, we engineered IPK1 mutants with disulfide bonds that artificially stabilized the N-lobe in an IP-independent manner thereby mimicking its substrate-bound state in the absence of IP. IPK1 E82C/S142C exhibited a DTT-sensitive 5-fold increase in kcat for 3,4,5,6-inositol tetrakisphosphate (3,4,5,6-IP4) as compared with wild-type IPK1. The crystal structure of the IPK1 E82C/S142C mutant confirmed the presence of the disulfide bond and revealed a small shift in the N-lobe. Finally, we determined that IPK1 E82C/S142C is substantially more stable than wild-type IPK1 under nonreducing conditions, revealing that increased stability of IPK1 E82C/S142C correlates with changes in substrate specificity by allowing IPs lacking the stabilizing 1-phosphate to be used. Taken together, our results show that IPK1 substrate selection is linked to the ability of each potential substrate to stabilize IPK1. PMID:24165122

  2. Enhanced photocatalytic H2 production on CdS nanorod using cobalt-phosphate as oxidation cocatalyst

    NASA Astrophysics Data System (ADS)

    Di, Tingmin; Zhu, Bicheng; Zhang, Jun; Cheng, Bei; Yu, Jiaguo

    2016-12-01

    Employing visible light responsive semiconductor for photocatalytic hydrogen production by water splitting is an efficient way for utilizing renewable solar energy to solve the depletion of fossil fuel and environmental contamination. Herein, we report enhanced photocatalytic H2-production performance over CdS nanorod using cobalt-phosphate (Co-Pi) as a water oxdation cocatalyst. The optimal Co-Pi modified CdS nanocomposite photocatalyst with the Co-Pi content of 8.4 mol% has a superior visible light H2-production rate of 13.3 mmol h-1 g-1 with an apparent quantum efficiency of 24.3% at 420 nm, which is even higher than that of 1 wt% Pt-CdS (11.3 mmol h-1 g-1) under the same conditions. The enhanced visible-light photocatalytic H2 production activity was attributed to the hole trapping and collecting ability of Co-Pi cocatalyst, which could effectively suppress the recombination of photogenerated electron-hole pairs and increase the electron density for hydrogen production. This work shows a possibility of using earth-abundant Co-Pi as cocatalyst for enhancing photocatalytic H2 production.

  3. SGPL1 (sphingosine phosphate lyase 1) modulates neuronal autophagy via phosphatidylethanolamine production

    PubMed Central

    Mitroi, Daniel N.; Karunakaran, Indulekha; Gräler, Markus; Saba, Julie D.; Ehninger, Dan; Ledesma, María Dolores; van Echten-Deckert, Gerhild

    2017-01-01

    ABSTRACT Macroautophagy/autophagy defects have been identified as critical factors underlying the pathogenesis of neurodegenerative diseases. The roles of the bioactive signaling lipid sphingosine-1-phosphate (S1P) and its catabolic enzyme SGPL1/SPL (sphingosine phosphate lyase 1) in autophagy are increasingly recognized. Here we provide in vitro and in vivo evidence for a previously unidentified route through which SGPL1 modulates autophagy in neurons. SGPL1 cleaves S1P into ethanolamine phosphate, which is directed toward the synthesis of phosphatidylethanolamine (PE) that anchors LC3-I to phagophore membranes in the form of LC3-II. In the brains of SGPL1fl/fl/Nes mice with developmental neural specific SGPL1 ablation, we observed significantly reduced PE levels. Accordingly, alterations in basal and stimulated autophagy involving decreased conversion of LC3-I to LC3-II and increased BECN1/Beclin-1 and SQSTM1/p62 levels were apparent. Alterations were also noticed in downstream events of the autophagic-lysosomal pathway such as increased levels of lysosomal markers and aggregate-prone proteins such as APP (amyloid β [A4] precursor protein) and SNCA/α-synuclein. In vivo profound deficits in cognitive skills were observed. Genetic and pharmacological inhibition of SGPL1 in cultured neurons promoted these alterations, whereas addition of PE was sufficient to restore LC3-I to LC3-II conversion, and control levels of SQSTM1, APP and SNCA. Electron and immunofluorescence microscopy showed accumulation of unclosed phagophore-like structures, reduction of autolysosomes and altered distribution of LC3 in SGPL1fl/fl/Nes brains. Experiments using EGFP-mRFP-LC3 provided further support for blockage of the autophagic flux at initiation stages upon SGPL1 deficiency due to PE paucity. These results emphasize a formerly overlooked direct role of SGPL1 in neuronal autophagy and assume significance in the context that autophagy modulators hold an enormous therapeutic potential

  4. Molecular definition of a novel inositol polyphosphate metabolic pathway initiated by inositol 1,4,5-trisphosphate 3-kinase activity in Saccharomyces cerevisiae.

    PubMed

    Seeds, Andrew M; Bastidas, Robert J; York, John D

    2005-07-29

    The production of inositol polyphosphate (IPs) and pyrophosphates (PP-IPs) from inositol 1,4,5-trisphosphate (I(1,4,5)P3) requires the 6-/3-/5-kinase activity of Ipk2 (also known as Arg82 and inositol polyphosphate multikinase). Here, we probed the distinct roles for I(1,4,5)P3 6- versus 3-kinase activities in IP metabolism and cellular functions reported for Ipk2. Expression of either I(1,4,5)P3 6- or 3-kinase activity rescued growth of ipk2-deficient yeast at high temperatures, whereas only 6-kinase activity enabled growth on ornithine as the sole nitrogen source. Analysis of IP metabolism revealed that the 3-kinase initiated the synthesis of novel pathway consisting of over eleven IPs and PP-IPs. This pathway was present in wild-type and ipk2 null cells, albeit at low levels as compared with inositol hexakisphosphate synthesis. The primary route of synthesis was: I(1,4,5)P3 --> I(1,3,4,5)P4 --> I(1,2,3,4,5)P5 --> PP-IP4 --> PP2-IP3 and required Kcs1 (or possibly Ipk2), Ipk1, a novel inositol pyrophosphate synthase, and then Kcs1 again, respectively. Mutation of kcs1 ablated this pathway in ipk2 null cells and overexpression of Kcs1 in ipk2 mutant cells phenocopied IP3K expression, confirming it harbors a novel 3-kinase activity. Our work provides a revised genetic map of IP metabolism in yeast and evidence for dosage compensation between IPs and PP-IPs downstream of I(1,4,5)P3 in the regulation of nucleocytoplasmic processes.

  5. Arabidopsis FHY3 and FAR1 Regulate Light-Induced myo-Inositol Biosynthesis and Oxidative Stress Responses by Transcriptional Activation of MIPS1.

    PubMed

    Ma, Lin; Tian, Tian; Lin, Rongcheng; Deng, Xing-Wang; Wang, Haiyang; Li, Gang

    2016-04-04

    myo-Inositol-1-phosphate synthase (MIPS) catalyzes the limiting step of inositol biosynthesis and has crucial roles in plant growth and development. In response to stress, the transcription of MIPS1 is induced and the biosynthesis of inositol or inositol derivatives is promoted by unknown mechanisms. Here, we found that the light signaling protein FAR-RED ELONGATED HYPOCOTYL3 (FHY3) and its homolog FAR-RED IMPAIRED RESPONSE1 (FAR1) regulate light-induced inositol biosynthesis and oxidative stress responses by activating the transcription of MIPS1. Disruption of FHY3 and FAR1 caused light-induced cell death after dark-light transition, precocious leaf senescence, and increased sensitivity to oxidative stress. Reduction of salicylic acid (SA) accumulation by overexpression of SALICYLIC ACID 3-HYDROXYLASE largely suppressed the cell death phenotype of fhy3 far1 mutant plants, suggesting that FHY3- and FAR1-mediated cell death is dependent on SA. Furthermore, comparative analysis of chromatin immunoprecipitation sequencing and microarray results revealed that FHY3 and FAR1 directly target both MIPS1 and MIPS2. The fhy3 far1 mutant plants showed severely decreased MIPS1/2 transcript levels and reduced inositol levels. Conversely, constitutive expression of MIPS1 partially rescued the inositol contents, caused reduced transcript levels of SA-biosynthesis genes, and prevented oxidative stress in fhy3 far1. Taken together, our results indicate that the light signaling proteins FHY3 and FAR1 directly bind the promoter of MIPS1 to activate its expression and thereby promote inositol biosynthesis to prevent light-induced oxidative stress and SA-dependent cell death.

  6. 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.

  7. Fed-Batch Production of Glucose 6-Phosphate Dehydrogenase Using Recombinant Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Das Neves, Luiz Carlos Martins; Pessoa, Adalberto; Vitolo, Michele

    The strain Saccharomyces cerevisiae W303-181, having the plasmid YEpPGK-G6P (built by coupling the vector YEPLAC 181 with the promoter phosphoglycerate kinase 1), was cultured by fed-batch process in order to evaluate its capability in the formation of glucose 6-phosphate dehydrogenase (EC.1.1.1.49). Two liters of culture medium (10.0 g/L glucose, 3.7 g/L yeast nitrogen broth (YNB), 0.02 g/L l-tryptophan, 0.02 g/L l-histidine, 0.02 g/L uracil, and 0.02 g/L adenine) were inoculated with 1.5 g dry cell/L and left fermenting in the batch mode at pH 5.7, aeration of 2.2 vvm, 30°C, and agitation of 400 rpm. After glucose concentration in the medium was lower than 1.0 g/L, the cell culture was fed with a solution of glucose (10.0 g/L) or micronutrients (l-tryptophan, l-histidine, uracil, and adenine each one at a concentration of 0.02 g/L) following the constant, linear, or exponential mode. The volume of the culture medium in the fed-batch process was varied from 2 L up to 3 L during 5 h. The highest glucose 6-phosphate dehydrogenase activity (350 U/L; 1 U=1 μmol of NADP/min) occurred when the glucose solution was fed into the fermenter through the decreasing linear mode.

  8. Mechanistic Insights into Validoxylamine A 7'-Phosphate Synthesis by VldE Using the Structure of the Entire Product Complex

    SciTech Connect

    Cavalier, Michael C.; Yim, Young-Sun; Asamizu, Shumpei; Neau, David; Almabruk, Khaled H.; Mahmud, Taifo; Lee, Yong-Hwan

    2013-09-09

    The pseudo-glycosyltransferase VldE catalyzes non-glycosidic C-N coupling between an unsaturated cyclitol and a saturated aminocyclitol with the conservation of the stereochemical configuration of the substrates to form validoxylamine A 7'-phosphate, the biosynthetic precursor of the antibiotic validamycin A. To study the molecular basis of its mechanism, the three-dimensional structures of VldE from Streptomyces hygroscopicus subsp. limoneus was determined in apo form, in complex with GDP, in complex with GDP and validoxylamine A 7'-phosphate, and in complex with GDP and trehalose. The structure of VldE with the catalytic site in both an “open” and “closed” conformation is also described. With these structures, the preferred binding of the guanine moiety by VldE, rather than the uracil moiety as seen in OtsA could be explained. The elucidation of the VldE structure in complex with the entirety of its products provides insight into the internal return mechanism by which catalysis occurs with a net retention of the stereochemical configuration of the donated cyclitol.

  9. Deoxyxylulose 5-phosphate reductoisomerase is not a rate-determining enzyme for essential oil production in spike lavender.

    PubMed

    Mendoza-Poudereux, Isabel; Muñoz-Bertomeu, Jesús; Arrillaga, Isabel; Segura, Juan

    2014-11-01

    Spike lavender (Lavandula latifolia) is an economically important aromatic plant producing essential oils, whose components (mostly monoterpenes) are mainly synthesized through the plastidial methylerythritol 4-phosphate (MEP) pathway. 1-Deoxy-D-xylulose-5-phosphate (DXP) synthase (DXS), that catalyzes the first step of the MEP pathway, plays a crucial role in monoterpene precursors biosynthesis in spike lavender. To date, however, it is not known whether the DXP reductoisomerase (DXR), that catalyzes the conversion of DXP into MEP, is also a rate-limiting enzyme for the biosynthesis of monoterpenes in spike lavender. To investigate it, we generated transgenic spike lavender plants constitutively expressing the Arabidopsis thaliana DXR gene. Although two out of the seven transgenic T0 plants analyzed accumulated more essential oils than the controls, this is hardly imputable to the DXR transgene effect since a clear correlation between transcript accumulation and monoterpene production could not be established. Furthermore, these increased essential oil phenotypes were not maintained in their respective T1 progenies. Similar results were obtained when total chlorophyll and carotenoid content in both T0 transgenic plants and their progenies were analyzed. Our results then demonstrate that DXR enzyme does not play a crucial role in the synthesis of plastidial monoterpene precursors, suggesting that the control flux of the MEP pathway in spike lavender is primarily exerted by the DXS enzyme.

  10. Sorbitol production from lactose by engineered Lactobacillus casei deficient in sorbitol transport system and mannitol-1-phosphate dehydrogenase.

    PubMed

    De Boeck, Reinout; Sarmiento-Rubiano, Luz Adriana; Nadal, Inmaculada; Monedero, Vicente; Pérez-Martínez, Gaspar; Yebra, María J

    2010-02-01

    Sorbitol is a sugar alcohol largely used in the food industry as a low-calorie sweetener. We have previously described a sorbitol-producing Lactobacillus casei (strain BL232) in which the gutF gene, encoding a sorbitol-6-phosphate dehydrogenase, was expressed from the lactose operon. Here, a complete deletion of the ldh1 gene, encoding the main L-lactate dehydrogenase, was performed in strain BL232. In a resting cell system with glucose, the new strain, named BL251, accumulated sorbitol in the medium that was rapidly metabolized after glucose exhaustion. Reutilization of produced sorbitol was prevented by deleting the gutB gene of the phosphoenolpyruvate: sorbitol phosphotransferase system (PTS(Gut)) in BL251. These results showed that the PTS(Gut) did not mediate sorbitol excretion from the cells, but it was responsible for uptake and reutilization of the synthesized sorbitol. A further improvement in sorbitol production was achieved by inactivation of the mtlD gene, encoding a mannitol-1-phosphate dehydrogenase. The new strain BL300 (lac::gutF Deltaldh1 DeltagutB mtlD) showed an increase in sorbitol production whereas no mannitol synthesis was detected, avoiding thus a polyol mixture. This strain was able to convert lactose, the main sugar from milk, into sorbitol, either using a resting cell system or in growing cells under pH control. A conversion rate of 9.4% of lactose into sorbitol was obtained using an optimized fed-batch system and whey permeate, a waste product of the dairy industry, as substrate.

  11. Effects of varied nitrate and phosphate supply on polysaccharidic and proteinaceous gel particle production during tropical phytoplankton bloom experiments

    NASA Astrophysics Data System (ADS)

    Engel, A.; Borchard, C.; Loginova, A.; Meyer, J.; Hauss, H.; Kiko, R.

    2015-10-01

    Gel particles such as the polysaccharidic transparent exopolymer particles (TEP) and the proteinaceous Coomassie stainable particles (CSP) play an important role in marine biogeochemical and ecological processes like particle aggregation and export, or microbial nutrition and growth. So far, effects of nutrient availability or of changes in nutrient ratios on gel particle production and fate are not well understood. The tropical ocean includes large oxygen minimum zones, where nitrogen losses due to anaerobic microbial activity result in a lower supply of nitrate relative to phosphate to the euphotic zone. Here, we report of two series of mesocosm experiments that were conducted with natural plankton communities collected from the eastern tropical North Atlantic (ETNA) close to Cape Verde in October 2012. The experiments were performed to investigate how different phosphate (experiment 1, Varied P: 0.15-1.58 μmol L-1) or nitrate (experiment 2, Varied N: 1.9-21.9 μmol L-1) concentrations affect the abundance and size distribution of TEP and CSP. In the days until the bloom peak was reached, a positive correlation between gel particle abundance and Chl a concentration was determined, linking the release of dissolved gel precursors and the subsequent formation of gel particles to autotrophic production. After the bloom peak, gel particle abundance remained stable or even increased, implying a continued partitioning of dissolved into particulate organic matter after biomass production itself ceased. During both experiments, differences between TEP and CSP dynamics were observed; TEP were generally more abundant than CSP. Changes in size distribution indicated aggregation of TEP after the bloom, while newly formed CSP decomposed. Abundance of gel particles clearly increased with nitrate concentration during the second experiment, suggesting that changes in [DIN] : [DIP] ratios can affect gel particle formation with potential consequences for carbon and nitrogen

  12. A cell factory of Bacillus subtilis engineered for the simple bioconversion of myo-inositol to scyllo-inositol, a potential therapeutic agent for Alzheimer's disease

    PubMed Central

    2011-01-01

    Background A stereoisomer of inositol, scyllo-inositol, is known as a promising therapeutic agent for Alzheimer's disease, since it prevents the accumulation of beta-amyloid deposits, a hallmark of the disease. However, this compound is relatively rare in nature, whereas another stereoisomer of inositol, myo-inositol, is abundantly available. Results Bacillus subtilis possesses a unique inositol metabolism involving both stereoisomers. We manipulated the inositol metabolism in B. subtilis to permit the possible bioconversion from myo-inositol to scyllo-inositol. Within 48 h of cultivation, the engineered strain was able to convert almost half of 10 g/L myo-inositol to scyllo-inositol that accumulated in the culture medium. Conclusions The engineered B. subtilis serves as a prototype of cell factory enabling a novel and inexpensive supply of scyllo-inositol. PMID:21896210

  13. 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.

  14. 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.

  15. 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.

  16. Prevention of Prostate Cancer by Inositol Hexaphosphate

    DTIC Science & Technology

    2004-02-01

    growth factor signaling pathways thereby inhibit growth. A large number of studies have pointed out that inositol hexaphosphate ( IP6 ) could have...beneficial effect on variety of cancers. The specific aims of this proposal are to determine (1) the in vivo effects of IP6 on the growth of PCa (2) the...efficacy of IP6 in inhibiting growth factor-induced DNA synthesis of the PCa cells in vitro, and (3) the molecular mechanisms by which IP6 inhibit growth

  17. Constitutive Optimized Production of Streptokinase in Saccharomyces cerevisiae Utilizing Glyceraldehyde 3-Phosphate Dehydrogenase Promoter of Pichia pastoris

    PubMed Central

    Vellanki, Ravi N.; Doddapaneni, Kiran K.; Anubrolu, Naveen; Mangamoori, Lakshmi N.

    2013-01-01

    A novel expression vector constructed from genes of Pichia pastoris was applied for heterologous gene expression in Saccharomyces cerevisiae. Recombinant streptokinase (SK) was synthesized by cloning the region encoding mature SK under the control of glyceraldehyde 3-phosphate dehydrogenase (GAP) promoter of Pichia pastoris in Saccharomyces cerevisiae. SK was intracellularly expressed constitutively, as evidenced by lyticase-nitroanilide and caseinolytic assays. The functional activity was confirmed by plasminogen activation assay and in vitro clot lysis assay. Stability and absence of toxicity to the host with the recombinant expression vector as evidenced by southern analysis and growth profile indicate the application of this expression system for large-scale production of SK. Two-stage statistical approach, Plackett-Burman (PB) design and response surface methodology (RSM) was used for SK production medium optimization. In the first stage, carbon and organic nitrogen sources were qualitatively screened by PB design and in the second stage there was quantitative optimization of four process variables, yeast extract, dextrose, pH, and temperature, by RSM. PB design resulted in dextrose and peptone as best carbon and nitrogen sources for SK production. RSM method, proved as an efficient technique for optimizing process conditions which resulted in 110% increase in SK production, 2352 IU/mL, than for unoptimized conditions. PMID:24171161

  18. A pulse radiolysis investigation of the reactions of tributyl phosphate with the radical products of aqueous nitric acid irradiation.

    PubMed

    Mincher, Bruce J; Mezyk, Stephen P; Martin, Leigh R

    2008-07-17

    Tributyl phosphate (TBP) is the most common organic compound used in liquid-liquid separations for the recovery of uranium, neptunium, and plutonium from acidic nuclear fuel dissolutions. The goal of these processes is to extract the actinides while leaving fission products in the acidic, aqueous phase. However, the radiolytic degradation of TBP has been shown to reduce separation factors of the actinides from fission products and to impede the back-extraction of the actinides during stripping. As most previous investigations of the radiation chemistry of TBP have focused on steady state radiolysis and stable product identification, with dibutylphosphoric acid (HDBP) invariably being the major product, here we have determined room temperature rate constants for the reactions of TBP and HDBP with the hydroxyl radical [(5.00 +/- 0.05) x 10(9), (4.40 +/- 0.13) x 10(9) M(-1) s(-1)], hydrogen atom [(1.8 +/-0.2) x 10(8), (1.1 +/- 0.1) x 10(8) M(-1) s(-1)], nitrate radical [(4.3 +/- 0.7) x 10(6), (2.9 +/- 0.2) x 10(6) M(-1) s(-1)], and nitrite radical (<2 x 10 (5), <2 x 10(5) M(-1) s(-1)), respectively. These data are used to discuss the mechanism of TBP radical-induced degradation.

  19. Characterization of myo-inositol hexakisphosphate deposits from larval Echinococcus granulosus.

    PubMed

    Casaravilla, Cecilia; Brearley, Charles; Soulé, Silvia; Fontana, Carolina; Veiga, Nicolás; Bessio, María I; Ferreira, Fernando; Kremer, Carlos; Díaz, Alvaro

    2006-07-01

    The abundant metabolite myo-inositol hexakisphosphate (InsP6) can form vesicular deposits with cations, a widespread phenomenon in plants also found in the cestode parasite, Echinococcus granulosus. In this organism, the deposits are exocytosed, accumulating in a host-exposed sheath of extracellular matrix termed the laminated layer. The formation and mobilization of InsP6 deposits, which involve precipitation and solubilization reactions, respectively, cannot yet be rationalized in quantitative chemical terms, as the solids involved have not been formally described. We report such a description for the InsP6 deposits from E. granulosus, purified as the solid residue left by mild alkaline digestion of the principal mucin component of the laminated layer. The deposits are largely composed of the compound Ca5H2L.16H2O (L representing fully deprotonated InsP6), and additionally contain Mg2+ (6-9% molar ratio with respect to Ca2+), but not K+. Calculations employing recently available chemical constants show that the precipitation of Ca5H2L.16H2O is predicted by thermodynamics in secretory vesicle-like conditions. The deposits appear to be similar to microcrystalline solids when analysed under the electron microscope; we estimate that each crystal comprises around 200 InsP6 molecules. We calculate that the deposits increase, by three orders of magnitude, the surface area available for adsorption of host proteins, a salient ability of the laminated layer. The major inositol phosphate in the deposits, other than InsP6, is myo-inositol (1,2,4,5,6) pentakisphosphate, or its enantiomer, inositol (2,3,4,5,6) pentakisphosphate. The compound appears to be a subproduct of the intracellular pathways leading to the synthesis and vesicular accumulation of InsP6, rather than arising from extracellular hydrolysis of InsP6.

  20. 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

  1. 21 CFR 137.175 - Phosphated flour.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 2 2014-04-01 2014-04-01 false Phosphated flour. 137.175 Section 137.175 Food and... CONSUMPTION CEREAL FLOURS AND RELATED PRODUCTS Requirements for Specific Standardized Cereal Flours and Related Products § 137.175 Phosphated flour. Phosphated flour, phosphated white flour, and...

  2. 21 CFR 137.175 - Phosphated flour.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 2 2013-04-01 2013-04-01 false Phosphated flour. 137.175 Section 137.175 Food and... CONSUMPTION CEREAL FLOURS AND RELATED PRODUCTS Requirements for Specific Standardized Cereal Flours and Related Products § 137.175 Phosphated flour. Phosphated flour, phosphated white flour, and...

  3. 21 CFR 137.175 - Phosphated flour.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 2 2012-04-01 2012-04-01 false Phosphated flour. 137.175 Section 137.175 Food and... CONSUMPTION CEREAL FLOURS AND RELATED PRODUCTS Requirements for Specific Standardized Cereal Flours and Related Products § 137.175 Phosphated flour. Phosphated flour, phosphated white flour, and...

  4. Engineering a pyridoxal 5’-phosphate supply for cadaverine production by using Escherichia coli whole-cell biocatalysis

    PubMed Central

    Ma, Weichao; Cao, Weijia; Zhang, Bowen; Chen, Kequan; Liu, Quanzhen; Li, Yan; Ouyang, Pingkai

    2015-01-01

    Although the routes of de novo pyridoxal 5′-phosphate (PLP) biosynthesis have been well described, studies of the engineering of an intracellular PLP supply are limited, and the effects of cellular PLP levels on PLP-dependent enzyme-based whole-cell biocatalyst activity have not been described. To investigate the effects of PLP cofactor availability on whole-cell biocatalysis, the ribose 5-phosphate (R5P)-dependent pathway genes pdxS and pdxT of Bacillus subtilis were introduced into the lysine decarboxylase (CadA)-overexpressing Escherichia coli strain BL-CadA. This strain was then used as a whole-cell biocatalyst for cadaverine production from L-lysine. Co-expression strategies were evaluated, and the culture medium was optimised to improve the biocatalyst performance. As a result, the intracellular PLP concentration reached 1144 nmol/gDCW, and a specific cadaverine productivity of 25 g/gDCW/h was achieved; these values were 2.4-fold and 2.9-fold higher than those of unmodified BL-CadA, respectively. Additionally, the resulting strain AST3 showed a cadaverine titre (p = 0.143, α = 0.05) similar to that of the BL-CadA strain with the addition of 0.1 mM PLP. These approaches for improving intracellular PLP levels to enhance whole-cell lysine bioconversion activity show great promise for the engineering of a PLP cofactor to optimise whole-cell biocatalysis. PMID:26490441

  5. Effects of varied nitrate and phosphate supply on polysaccharidic and proteinaceous gel particles production during tropical phytoplankton bloom experiments

    NASA Astrophysics Data System (ADS)

    Engel, A.; Borchard, C.; Loginova, A.; Meyer, J.; Hauss, H.; Kiko, R.

    2015-04-01

    It has been suggested that oxygen minimum zones (OMZ) will expand in the tropical oceans as a result of global change with potential consequences for marine element cycling, such as an increase in anaerobic nitrogen loss, resulting in a lower supply of nitrate relative to phosphate to the euphotic zone. So far, the effects of changes in nutrient ratios on organic matter recycling and export fluxes are not well understood. Here, were investigated how different phosphate (Varied P: 0.15-1.58 μmol L-1) or nitrate (Varied N: 1.9-21.9 μmol L-1) concentrations affect the abundance and size distribution of polysaccharidic transparent exopolymer particles (TEP), which are suggested to enhance particle aggregation and export fluxes, and on proteinaceous coomassie stainable particles (CSP), a supposedly good substrate for heterotrophic bacteria. Two series of mesocosm bloom experiments were conducted with natural plankton communities collected from the Eastern Tropical North Atlantic (ETNA) close to Cape Verde in October 2012. Until bloom peak, a positive correlation between gel particle abundance and Chl a concentration was determined, linking the release of dissolved gel precursors and the subsequent formation of gel particles to autotrophic production. After bloom peak, gel particle abundance remained stable or even increased, implying a continued partitioning of dissolved into particulate organic matter after biomass production itself ceased. During both experiments, differences between TEP and CSP dynamics were observed; TEP were generally more abundant than CSP. Changes in size distribution indicated aggregation of TEP during the bloom, while newly formed CSP decomposed. Abundance of gel particles clearly increased with nitrate concentration during the second experiment, suggesting that changes in [DIN]:[DIP] ratios can affect gel particle formation with potential consequences for carbon and nitrogen cycling as well as food web dynamics in tropical ecosystems.

  6. Engineering a pyridoxal 5'-phosphate supply for cadaverine production by using Escherichia coli whole-cell biocatalysis.

    PubMed

    Ma, Weichao; Cao, Weijia; Zhang, Bowen; Chen, Kequan; Liu, Quanzhen; Li, Yan; Ouyang, Pingkai

    2015-10-22

    Although the routes of de novo pyridoxal 5'-phosphate (PLP) biosynthesis have been well described, studies of the engineering of an intracellular PLP supply are limited, and the effects of cellular PLP levels on PLP-dependent enzyme-based whole-cell biocatalyst activity have not been described. To investigate the effects of PLP cofactor availability on whole-cell biocatalysis, the ribose 5-phosphate (R5P)-dependent pathway genes pdxS and pdxT of Bacillus subtilis were introduced into the lysine decarboxylase (CadA)-overexpressing Escherichia coli strain BL-CadA. This strain was then used as a whole-cell biocatalyst for cadaverine production from L-lysine. Co-expression strategies were evaluated, and the culture medium was optimised to improve the biocatalyst performance. As a result, the intracellular PLP concentration reached 1144 nmol/gDCW, and a specific cadaverine productivity of 25 g/gDCW/h was achieved; these values were 2.4-fold and 2.9-fold higher than those of unmodified BL-CadA, respectively. Additionally, the resulting strain AST3 showed a cadaverine titre (p = 0.143, α = 0.05) similar to that of the BL-CadA strain with the addition of 0.1 mM PLP. These approaches for improving intracellular PLP levels to enhance whole-cell lysine bioconversion activity show great promise for the engineering of a PLP cofactor to optimise whole-cell biocatalysis.

  7. Development of vanadium-phosphate catalysts for methanol production by selective oxidation of methane

    SciTech Connect

    McCormick, R.L.; Jha, M.C.

    1993-03-04

    Amax R D will perform laboratory scale development of a promising, practical catalyst for the selective oxidation of methane to methanol. The primary component of this catalyst is vanadium-phosphate (VPO) which has shown good activity and selectivity in the partial oxidation of n-butane and propane but has not been studied in detail for methane oxidation. The goal of the project is to develop a catalyst which allows methane oxidation to methanol to be conducted at high conversion and selectivity. A low CH[sub 4]/O[sub 2] ratio will be employed with air as the source of oxygen. Temperatures below 600[degrees]C and pressures up to 20 atm are to be investigated. The use of steam in the feed gas will also be investigated. The catalyst development strategy will be to utilize promoters and supports to improve the activity and selectivity of the unmodified VPO catalyst. The catalyst testing reactor system was used to perform blank (empty) reactor runs over a wide range of temperatures, pressure, and flow rates. No methane conversion was observed at temperatures of 500[degrees]C or lower in any of the tests. At higher temperatures, significant methane conversion to carbon dioxide was observed. At 550[degrees]C, 300 psig, and the highest flow rate studied, reactor ignition was observed. Based on the results of these blank runs, we conclude that catalyst testing should be performed at temperatures not to exceed 500[degrees]C.

  8. Effects of Inositol(s) in Women with PCOS: A Systematic Review of Randomized Controlled Trials

    PubMed Central

    Nestler, John E.; Kamenov, Zdravko A.; Prapas, Nikos; Facchinetti, Fabio

    2016-01-01

    Polycystic ovary syndrome (PCOS) is a common endocrine disorder, with complex etiology and pathophysiology, which remains poorly understood. It affects about 5–10% of women of reproductive age who typically suffer from obesity, hyperandrogenism, ovarian dysfunction, and menstrual irregularity. Indeed, PCOS is the most common cause of anovulatory infertility in industrialized nations, and it is associated with insulin resistance, type 2 diabetes mellitus, and increased cardiovascular risk. Although insulin resistance is not included as a criterion for diagnosis, it is a critical pathological condition of PCOS. The purpose of this systematic review is the analysis of recent randomized clinical trials of inositol(s) in PCOS, in particular myo- and D-chiro-inositol, in order to better elucidate their physiological involvement in PCOS and potential therapeutic use, alone and in conjunction with assisted reproductive technologies, in the clinical treatment of women with PCOS. PMID:27843451

  9. The D-chiro-inositol paradox in the ovary.

    PubMed

    Carlomagno, Gianfranco; Unfer, Vittorio; Roseff, Scott

    2011-06-30

    The D-chiro-inositol-to-myo-inositol ratio is regulated by an insulin-dependent epimerase. Enzyme activity varies among tissues, likely owing to the specific needs of the two different molecules. We hypothesize that in the ovaries of polycystic ovary syndrome patients, epimerase activity is enhanced, leading to a local myo-inositol deficiency which in turn is responsible for the poor oocyte quality.

  10. 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.

  11. 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.

  12. L-Ribose isomerase and mannose-6-phosphate isomerase: properties and applications for L-ribose production.

    PubMed

    Xu, Zheng; Sha, Yuanyuan; Liu, Chao; Li, Sha; Liang, Jinfeng; Zhou, Jiahai; Xu, Hong

    2016-11-01

    L-Ribose is a synthetic L-form monosaccharide. It is a building block of many novel nucleotide analog anti-viral drugs. Bio-production of L-ribose relies on a two-step reaction: (i) conversion of L-arabinose to L-ribulose by the catalytic action of L-arabinose isomerase (L-AI) and (ii) conversion of L-ribulose to L-ribose by the catalytic action of L-ribose isomerase (L-RI, EC 5.3.1.B3) or mannose-6-phosphate isomerase (MPI, EC 5.3.1.8, alternately named as phosphomannose isomerase). Between the two enzymes, L-RI is a rare enzyme that was discovered in 1996 by Professor Izumori's group, whereas MPI is an essential enzyme in metabolic pathways in humans and microorganisms. Recent studies have focused on their potentials for industrial production of L-ribose. This review summarizes the applications of L-RI and MPI for L-ribose production.

  13. Pyrolysis of rice straw with ammonium dihydrogen phosphate: Properties and gaseous potassium release characteristics during combustion of the products.

    PubMed

    Li, Hui; Han, Kuihua; Wang, Qian; Lu, Chunmei

    2015-12-01

    The effect of ammonium dihydrogen phosphate (NH4H2PO4) on rice straw (RS) carbonization was evaluated at temperatures of 350-650°C. The carbonized products of RS with NH4H2PO4 show higher solid and energy yields, but lower higher heating values than the carbonized RS at every carbonization temperature. The optimum carbonization operation of RS with NH4H2PO4 which has a higher energy yield at a lower solid volume may be determined between 350 and 450°C, and RS with NH4H2PO4 carbonized at 450°C presents better pore properties than carbonized RS. The carbonized products of RS with NH4H2PO4 all have lower gaseous potassium release ratios than those of RS carbonized at the same temperature at combustion temperatures of 700-1000°C by retaining potassium in non-volatile phosphorus compounds with high melting points. It is an effective method for inhibiting the gaseous potassium release during combustion of the carbonized products. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Heterogeneous photocatalysis of tris(2-chloroethyl) phosphate by UV/TiO2: Degradation products and impacts on bacterial proteome.

    PubMed

    Ye, Jinshao; Liu, Juan; Li, Chongshu; Zhou, Pulin; Wu, Shuang; Ou, Huase

    2017-11-01

    The widespread, persistent and toxic organophosphorus esters (OPEs) have become one category of emerging environmental contaminants. Thus, it is in urgent need to develop a cost-effective and safe treatment technology for OPEs control. The current study is a comprehensive attempt to use UV/TiO2 heterogeneous photocatalysis for the degradation of a water dissolved OPEs, tris(2-chloroethyl) phosphate (TCEP). A pseudo-first order degradation reaction with a kobs of 0.3167 min(-1) was observed, while hydroxyl radical may be the dominating reactive oxidative species. As the reaction proceeded, TCEP was transformed to a series of hydroxylated and dechlorinated products. The degradation efficiency was significantly affected by pH value, natural organic matters and anions, implying that the complete mineralization of TCEP would be difficult to achieve in actual water treatment process. Based on the proteomics analysis regarding the metabolism reactions, pathways and networks, the significant activation of transmembrane transport and energy generation in Escherichia coli exposed to preliminary degrading products suggested that they can be transported and utilized through cellular metabolism. Furthermore, the descending trend of stress resistance exhibited that the toxicity of products was obviously weakened as the treatment proceeded. In conclusion, hydroxylation and dechlorination of TCEP with incomplete mineralization were likewise effective for its detoxification, indicating that UV/TiO2 will be an alternative treatment method for OPEs control. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Effect of caries in mentally handicapped children of addition of fluoride and bicarbonate-phosphate to dietary sugar products.

    PubMed

    Luoma, H; Nyman, A; Toivonen, A; Söderholm, S; Nuuja, T; Kantero, R L; Hassinen, M L; Jokela, M; Nummikoski, P; Ranta, H; Thesleff, I

    1979-06-01

    Mentally handicapped children, aged 5--15 years and living in institutions, received fluoride supplement in several sugar products of their diet; in candies, marmalades, jams, fruit juices and in sweet desserts corresponding to 10 mg F as NaF per kg of the sugar (sucrose or glucose) of each product. To two of the four daily candies was also added a NaHCO3 + KH2PO4 mixture (mole ratio 9.8/l, resp.) to substitute for 2.5% of the sugar of the candy. The control children received the respective products without the additives. After stepwise exclusions of subjects for various reasons, e.g. for the absence of permanent teeth, low initial caries activity, strong medication, Down's syndrome, etc., the mean DMFS-increment in the remaining 43 control subjects was 4.5 and in the 41 test subjects 2.6 lesions/100 surfaces at risk, i.e. 42% reduction. Caries arrestment had occurred in these test subjects after the first year, while in the respective controls it was continuously increasing. Among numerous oral and body parameters studied, only surface enamel fluoride in primary teeth was increased by the fluoride supplements and urinary phosphate and calcium excretion decreased.

  16. Improvement in lipids extraction processes for biodiesel production from wet microalgal pellets grown on diammonium phosphate and sodium bicarbonate combinations.

    PubMed

    Shah, Syed Hasnain; Raja, Iftikhar Ahmed; Mahmood, Qaisar; Pervez, Arshid

    2016-08-01

    Biomass productivity and growth kinetics for microalgae grown on sodium bicarbonate and diammonium phosphate were investigated. Different carbon and nitrogen ratios have shown different growth rates and biomass productivity and C:N ratio 50:10 as mgL(-1) has shown the best production than all. For effective lipids extraction from biomass thermolysis and sonolysis were carried out from wet biomass. Sonolysis at 2.3W intensity for 5min has released 8.58mg at neutral pH. More quantity of lipids was extracted when extraction was made at pH 4 and 10 which resulted 9mg and 9.28mg lipids respectively. Thermal treatment at 100°C for 10min has released 12.82mg lipid at neutral pH. In the same thermolysis at pH 4 and 10 more quantity of lipids was extracted which were 15.16mg and 14.81mg respectively. Finally transesterified lipids were analyzed through GC-MS for FAME composition analysis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Lily pollen alkaline phytase is a histidine phosphatase similar to mammalian multiple inositol polyphosphate phosphatase (MINPP).

    PubMed

    Mehta, Bakul Dhagat; Jog, Sonali P; Johnson, Steven C; Murthy, Pushpalatha P N

    2006-09-01

    Phytic acid is the most abundant inositol phosphate in cells; it constitutes 1-5% of the dry weight of cereal grains and legumes. Phytases are the primary enzymes responsible for the hydrolysis of phytic acid and thus play important roles in inositol phosphate metabolism. A novel alkaline phytase in lily pollen (LlALP) was recently purified in our laboratory. In this paper, we describe the cloning and characterization of LlALP cDNA from lily pollen. Two isoforms of alkaline phytase cDNAs, LlAlp1 and LlAlp2, which are 1467 and 1533 bp long and encode proteins of 487 and 511 amino acids, respectively, were identified. The deduced amino acid sequences contains the signature heptapeptide of histidine phosphatases, -RHGXRXP-, but shares < 25% identity to fungal histidine acid phytases. Phylogenetic analysis reveals that LlALP is most closely related to multiple inositol polyphosphate phosphatase (MINPP) from humans (25%) and rats (23%). mRNA corresponding to LlAlp1 and LlAlp2 were expressed in leaves, stem, petals and pollen grains. The expression profiles of LlAlp isoforms in anthers indicated that mRNA corresponding to both isoforms were present at all stages of flower development. The expression of LlAlp2 cDNA in Escherichia coli revealed the accumulation of the active enzyme in inclusion bodies and confirmed that the cDNA encodes an alkaline phytase. In summary, plant alkaline phytase is a member of the histidine phosphatase family that includes MINPP and exhibits properties distinct from bacterial and fungal phytases.

  18. Calcium mobilization in permeabilized fibroblasts: effects of inositol trisphosphate, orthovanadate, mitogens phorbol ester, and guanosine triphosphate

    SciTech Connect

    Muldoon, L.L.; Jamieson, G.A. Jr.; Villereal, M.L.

    1987-01-01

    Utilizing a digitonin-permeabilized cell system, the authors have studied the release of calcium from a non-mitochondrial intracellular compartment in cultured human fibroblasts (HSWP cells). Addition of 1 mM MgATP to a monolayer of permeabilized cells in a cytosolic media buffered to 150 nM Ca with EGTA rapidly stimulates /sup 45/Ca uptake, and the subsequent addition of the putative intracellular messenger inositol trisphosphate (InsP/sub 3/) induces rapid release of 85% of the /sup 45/Ca taken up in response to ATP. Mitogenic peptides (bradykinin, vasopressin, epidermal growth factor (EGF), and insulin) and orthovanadate, which are effective in mobilizing intracellular Ca in intact cells, have little or no effect when added alone to permeabilized cells. However, in the presence of GTP these agents stimulate accumulation of inositol phosphates and release Ca from the InsP/sub 3/-sensitive pool. These data suggest that a GTP binding protein is involved in receptor mediated activation of phospholipase C, which leads to release of inositol phosphates. The GTP-dependent release of InsP/sub 3/ and the mobilization of /sup 45/Ca from the intracellular compartment are inhibited by pretreatment of cells, prior to permeabilization, with the protrein kinase C activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA). These results suggest that protein kinase C is involved in down-regulation or inhibition of phospholipase C, or the GTP binding protein responsible for relaying the mitogenic signal from the cell surface receptor to the phospholipase C activity.

  19. Crystal structures of Bacillus alkaline phytase in complex with divalent metal ions and inositol hexasulfate.

    PubMed

    Zeng, Yi-Fang; Ko, Tzu-Ping; Lai, Hui-Lin; Cheng, Ya-Shan; Wu, Tzu-Hui; Ma, Yanhe; Chen, Chun-Chi; Yang, Chii-Shen; Cheng, Kuo-Joan; Huang, Chun-Hsiang; Guo, Rey-Ting; Liu, Je-Ruei

    2011-06-03

    Alkaline phytases from Bacillus species, which hydrolyze phytate to less phosphorylated myo-inositols and inorganic phosphate, have great potential as additives to animal feed. The thermostability and neutral optimum pH of Bacillus phytase are attributed largely to the presence of calcium ions. Nonetheless, no report has demonstrated directly how the metal ions coordinate phytase and its substrate to facilitate the catalytic reaction. In this study, the interactions between a phytate analog (myo-inositol hexasulfate) and divalent metal ions in Bacillus subtilis phytase were revealed by the crystal structure at 1.25 Å resolution. We found all, except the first, sulfates on the substrate analog have direct or indirect interactions with amino acid residues in the enzyme active site. The structures also unraveled two active site-associated metal ions that were not explored in earlier studies. Significantly, one metal ion could be crucial to substrate binding. In addition, binding of the fourth sulfate of the substrate analog to the active site appears to be stronger than that of the others. These results indicate that alkaline phytase starts by cleaving the fourth phosphate, instead of the third or the sixth that were proposed earlier. Our high-resolution, structural representation of Bacillus phytase in complex with a substrate analog and divalent metal ions provides new insight into the catalytic mechanism of alkaline phytases in general.

  20. Old-Field Thinned Loblolly Pine Plantation Fertilization With Diammonium Phosphate Plus Urea and Poultry Litter -- 4 Year Growth and Product Class Distribution Results

    Treesearch

    E. David Dickens; Beth W. Richardson; Bryan C. McElvany

    2004-01-01

    A study area was installed in the Coastal Plain of South Carolina to determine the effects of diammonium phosphate (DAP) plus urea and poultry litter fertilization on growth, yield, diameter distributions, and product class distribu-tions in an old-field (Norfolk soil) thinned loblolly pine (Pinus taeda L.) plantation. Treatments included: (1)...

  1. The Effect of Phytase on the Oxygen Isotope Composition of Phosphate

    NASA Astrophysics Data System (ADS)

    von Sperber, C.; Tamburini, F.; Bernasconi, S. M.; Frossard, E.

    2013-12-01

    for acid phosphatases (6). In contrast, the results from assays with phytase from Aspergillus niger indicate that the exchange of oxygen occurs at more than one third of the total 24 oxygen which are associated to the phosphates in IP6. In addition, we observe a change in the oxygen isotope composition of Pi when using myo-inositol and potassium-dihydrogen-phosphate as sole substrates in the enzymatic assays with phytase from Aspergillus niger. These observations suggest that the reformation of IP6 from the two products of the reaction (myo-inositol and Pi) is taking place at a rate, which is within the time scale of the experiment. In this case, the isotopic fractionation caused by phytase from Aspergillus niger will be determined by the equilibrium of the reaction. Further experiments are in process to verify these findings.

  2. Effect of Phosphate on the Corrosion of Carbon Steel and on the Composition of Corrosion Products in Two-Stage Continuous Cultures of Desulfovibrio desulfuricans†

    PubMed Central

    Weimer, Paul J.; Van Kavelaar, Margaret J.; Michel, Charles B.; Ng, Thomas K.

    1988-01-01

    A field isolate of Desulfovibrio desulfuricans was grown in defined medium in a two-stage continuous culture apparatus with different concentrations of phosphate in the feed medium. The first state (V1) was operated as a conventional chemostat (D = 0.045 h−1) that was limited in energy source (lactate) or phosphate. The second stage (V2) received effluent from V1 but no additional nutrients, and contained a healthy population of transiently starved or resting cells. An increase in the concentration of phosphate in the medium fed to V1 resulted in increased corrosion rates of carbon steel in both V1 and V2. Despite the more rapid corrosion observed in growing cultures relative to that in resting cultures, corrosion products that were isolated under strictly anaerobic conditions from the two culture modes had similar bulk compositions which varied with the phosphate content of the medium. Crystalline mackinawite (Fe9S8), vivianite [Fe3(PO4)2 · 8H2O], and goethite [FeO(OH)] were detected in amounts which varied with the culture conditions. Chemical analyses indicated that the S in the corrosion product was almost exclusively in the form of sulfides, while the P was present both as phosphate and as unidentified components, possibly reduced P species. Some differential localization of S and P was observed in intact corrosion products. Cells from lactate-limited, but not from phosphate-limited, cultures contained intracellular granules that were enriched in P and Fe. The results are discussed in terms of several proposed mechanisms of microbiologically influenced corrosion. Images PMID:16347552

  3. Effect of Phosphate on the Corrosion of Carbon Steel and on the Composition of Corrosion Products in Two-Stage Continuous Cultures of Desulfovibrio desulfuricans.

    PubMed

    Weimer, P J; Van Kavelaar, M J; Michel, C B; Ng, T K

    1988-02-01

    A field isolate of Desulfovibrio desulfuricans was grown in defined medium in a two-stage continuous culture apparatus with different concentrations of phosphate in the feed medium. The first state (V1) was operated as a conventional chemostat (D = 0.045 h) that was limited in energy source (lactate) or phosphate. The second stage (V2) received effluent from V1 but no additional nutrients, and contained a healthy population of transiently starved or resting cells. An increase in the concentration of phosphate in the medium fed to V1 resulted in increased corrosion rates of carbon steel in both V1 and V2. Despite the more rapid corrosion observed in growing cultures relative to that in resting cultures, corrosion products that were isolated under strictly anaerobic conditions from the two culture modes had similar bulk compositions which varied with the phosphate content of the medium. Crystalline mackinawite (Fe(9)S(8)), vivianite [Fe(3)(PO(4))(2) . 8H(2)O], and goethite [FeO(OH)] were detected in amounts which varied with the culture conditions. Chemical analyses indicated that the S in the corrosion product was almost exclusively in the form of sulfides, while the P was present both as phosphate and as unidentified components, possibly reduced P species. Some differential localization of S and P was observed in intact corrosion products. Cells from lactate-limited, but not from phosphate-limited, cultures contained intracellular granules that were enriched in P and Fe. The results are discussed in terms of several proposed mechanisms of microbiologically influenced corrosion.

  4. 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

  5. Fagopyritol B1, O-alpha-D-galactopyranosyl-(1-->2)-D-chiro-inositol, a galactosyl cyclitol in maturing buckwheat seeds associated with desiccation tolerance.

    PubMed

    Horbowicz, M; Brenac, P; Obendorf, R L

    1998-05-01

    O-alpha-D-Galactopyranosyl-(1-->2)-D-chiro-inositol, herein named fagopyritol B1, was identified as a major soluble carbohydrate (40% of total) in buckwheat (Fagopyrum esculentum Moench, Polygonaceae) embryos. Analysis of hydrolysis products of purified compounds and of the crude extract led to the conclusion that buckwheat embryos have five alpha-galactosyl D-chiro-inositols: fagopyritol A1 and fagopyritol B1 (mono-galactosyl D-chiro-inositol isomers), fagopyritol A2 and fagopyritol B2 (di-galactosyl D-chiro-inositol isomers), and fagopyritol B3 (tri-galactosyl D-chiro-inositol). Other soluble carbohydrates analyzed by high-resolution gas chromatography included sucrose (42% of total), D-chiro-inositol, myo-inositol, galactinol, raffinose and stachyose (1% of total), but no reducing sugars. All fagopyritols were readily hydrolyzed by alpha-galactosidase (EC 3.2.1.22) from green coffee bean, demonstrating alpha-galactosyl linkage. Retention time of fagopyritol B1 was identical to the retention time of O-alpha-D-galactopyranosyl-(1-->2)-D-chiro-inositol from soybean (Glycine max (L.) Merrill, Leguminosae), suggesting that the alpha-galactosyl linkage is to the 2-position of D-chiro-inositol. Accumulation of fagopyritol B1 was associated with acquisition of desiccation tolerance during seed development and maturation in planta, and loss of fagopyritol B1 correlated with loss of desiccation tolerance during germination. Embryos of seeds grown at 18 degrees C, a condition that favors enhanced seed vigor and storability, had a sucrose-to-fagopyritol B1 ratio of 0.8 compared to a ratio of 2.46 for seeds grown at 25 degrees C. We propose that fagopyritol B1 facilitates desiccation tolerance and storability of buckwheat seeds.

  6. A study of organic acid production in contrasts between two phosphate solubilizing fungi: Penicillium oxalicum and Aspergillus niger

    NASA Astrophysics Data System (ADS)

    Li, Zhen; Bai, Tongshuo; Dai, Letian; Wang, Fuwei; Tao, Jinjin; Meng, Shiting; Hu, Yunxiao; Wang, Shimei; Hu, Shuijin

    2016-04-01

    Phosphate solubilizing fungi (PSF) have huge potentials in enhancing release of phosphorus from fertilizer. Two PSF (NJDL-03 and NJDL-12) were isolated and identified as Penicillium oxalicum and Aspergillus niger respectively in this study. The quantification and identification of organic acids were performed by HPLC. Total concentrations of organic acids secreted by NJDL-03 and NJDL-12 are ~4000 and ~10,000 mg/L with pH values of 3.6 and 2.4 respectively after five-days culture. Oxalic acid dominates acidity in the medium due to its high concentration and high acidity constant. The two fungi were also cultured for five days with the initial pH values of the medium varied from 6.5 to 1.5. The biomass reached the maximum when the initial pH values are 4.5 for NJDL-03 and 2.5 for NJDL-12. The organic acids for NJDL-12 reach the maximum at the initial pH = 5.5. However, the acids by NJDL-03 continue to decrease and proliferation of the fungus terminates at pH = 2.5. The citric acid production increases significantly for NJDL-12 at acidic environment, whereas formic and oxalic acids decrease sharply for both two fungi. This study shows that NJDL-12 has higher ability in acid production and has stronger adaptability to acidic environment than NJDL-03.

  7. A study of organic acid production in contrasts between two phosphate solubilizing fungi: Penicillium oxalicum and Aspergillus niger

    PubMed Central

    Li, Zhen; Bai, Tongshuo; Dai, Letian; Wang, Fuwei; Tao, Jinjin; Meng, Shiting; Hu, Yunxiao; Wang, Shimei; Hu, Shuijin

    2016-01-01

    Phosphate solubilizing fungi (PSF) have huge potentials in enhancing release of phosphorus from fertilizer. Two PSF (NJDL-03 and NJDL-12) were isolated and identified as Penicillium oxalicum and Aspergillus niger respectively in this study. The quantification and identification of organic acids were performed by HPLC. Total concentrations of organic acids secreted by NJDL-03 and NJDL-12 are ~4000 and ~10,000 mg/L with pH values of 3.6 and 2.4 respectively after five-days culture. Oxalic acid dominates acidity in the medium due to its high concentration and high acidity constant. The two fungi were also cultured for five days with the initial pH values of the medium varied from 6.5 to 1.5. The biomass reached the maximum when the initial pH values are 4.5 for NJDL-03 and 2.5 for NJDL-12. The organic acids for NJDL-12 reach the maximum at the initial pH = 5.5. However, the acids by NJDL-03 continue to decrease and proliferation of the fungus terminates at pH = 2.5. The citric acid production increases significantly for NJDL-12 at acidic environment, whereas formic and oxalic acids decrease sharply for both two fungi. This study shows that NJDL-12 has higher ability in acid production and has stronger adaptability to acidic environment than NJDL-03. PMID:27126606

  8. POTENTIAL EFFECTS OF PHOSPHATE PRODUCTS ON LEAD SOLUBILITY IN PLUMBING SYSTEMS

    EPA Science Inventory

    Lead concentrations in drinking wa