Metabolism of D-myo-inositol 1,3,4,5-tetrakisphosphate by rat liver, including the synthesis of a novel isomer of myo-inositol tetrakisphosphate.

1. We have studied the metabolism of Ins(1,3,4,5)P4 (inositol 1,3,4,5-tetrakisphosphate) by rat liver homogenates incubated in a medium resembling intracellular ionic strength and pH. 2. Ins(1,3,4,5)P4 was dephosphorylated to a single inositol trisphosphate product, Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate), the identity of which was confirmed by periodate degradation, followed by reduction and dephosphorylation to yield altritol. 3. The major InsP2 (inositol bisphosphate) product was inositol 3,4-bisphosphate [Shears, Storey, Morris, Cubitt, Parry, Michell & Kirk (1987) Biochem. J. 242, 393-402]. Small quantities of a second InsP2 product was also detected in some experiments, but its isomeric configuration was not identified. 4. The Ins(1,3,4,5)P4 5-phosphatase activity was primarily associated with plasma membranes. 5. ATP (5 mM) decreased the membrane-associated Ins(1,4,5)P3 5-phosphatase and Ins(1,3,4,5)P4 5-phosphatase activities by 40-50%. This inhibition was imitated by AMP, adenosine 5'-[beta gamma-imido]triphosphate, adenosine 5'-[gamma-thio]triphosphate or PPi, but not by adenosine or Pi. A decrease in [ATP] from 7 to 3 mM halved the inhibition of Ins(1,3,4,5)P4 5-phosphatase activity, but the extent of inhibition was not further decreased unless [ATP] less than 0.1 mM. 6. Ins(1,3,4,5)P4 5-phosphatase was insensitive to 50 mM-Li+, but was inhibited by 5 mM-2,3-bisphosphoglycerate. 7. The Ins(1,3,4,5)P4 5-phosphatase activity was unchanged by cyclic AMP, GTP, guanosine 5'-[beta gamma-imido]triphosphate or guanosine 5'-[gamma-thio]triphosphate, or by increasing [Ca2+] from 0.1 to 1 microM. 8. Ins(1,3,4)P3 was phosphorylated in an ATP-dependent manner to an isomer of InsP4 that was partially separable on h.p.l.c. from Ins(1,3,4,5)P4. The novel InsP4 appears to be Ins(1,3,4,6)P4. Its metabolic fate and function are not known.     

Replacement of Ca by Ni in a Perovskite Titanate to Yield a Novel Perovskite Exsolution Architecture for Oxygen‐Evolution Reactions

For efficient catalysis and electrocatalysis well‐designed, high‐surface‐area support architectures covered with highly dispersed metal nanoparticles with good catalyst‐support interactions are required. In situ grown Ni nanoparticles on perovskites have been recently reported to enhance catalytic activities in high‐temperature systems such as solid oxide cells (SOCs). However, the micrometer‐scale primary particles prepared by conventional solid‐state reactions have limited surface area and tend to retain much of the active catalytic element within the bulk, limiting efficacy of such exsolution processes in low‐temperature systems. Here, a new, highly efficient, solvothermal route is demonstrated to exsolution from smaller scale primary particles. Furthermore, unlike previous reports of B‐site exsolution, it seems that the metal nanoparticles are exsolved from the A‐site of these perovskites. The catalysts show large active site areas and strong metal‐support interaction (SMSI), leading to ≈26% higher geometric activity (25 times higher mass activity with 1.4 V of E_on‐set) and stability for oxygen‐evolution reaction (OER) with only 0.72 µg base metal contents compared to typical 20 wt% Ni/C and even commercial 20 wt% Ir/C. The findings obtained here demonstrate the potential design and development of heterogeneous catalysts in various low‐temperature electrochemical systems including alkaline fuel cells and metal–air batteries.     

A species of Cryptonemia new to Europe

The morphology, reproduction and seasonal variation of a large foliose species of Cryptonemia collected in the drift in 1971–73 at Cork Harbour, Republic of Ireland, is described and compared with the known species of the genus. Although obviously closely related to a group of foliose Pacific species: C. borealis Kylin, C. obovata J. Ag. and C. angustata (Setch. et Gardn.) Dawson, the Irish Cryptonemia is not completely identifiable with any one of these and is therefore described as C. hibernica sp. nov., pending a revision of the genus.     

Developmental analysis of elongation factor-1 alpha expression in transgenic tobacco.

The developmental regulation of the translational elongation factor EF-1 alpha has been analyzed in tobacco. A gene fusion was constructed consisting of the 5' and 3' regions of the tomato genomic clone LeEF-A from the EF-1 alpha gene family and the beta-glucuronidase coding region. Analysis of the transgenic plants containing this chimeric gene demonstrated that the tomato LeEF-A flanking sequences were sufficient to confer expression patterns similar to those of the endogenous tobacco EF-1 alpha gene. The patterns of beta-glucuronidase activity in this system indicated that during plant growth and development EF-1 alpha is regulated with increased expression corresponding to regions of high protein synthesis, including meristems, rapidly growing tissues, and developing gametophytes. In addition, EF-1 alpha expression responds rapidly to changes in growth patterns induced by hormone treatment. Our results are in agreement with studies in animals indicating that EF-1 alpha expression may be rate limiting for protein synthesis and demonstrate that the analysis of EF-1 alpha is of value for studying interrelationships between protein synthesis and developmental control.     

A myo-inositol D-3 hydroxykinase activity in Dictyostelium.

A soluble ATP-dependent enzyme which phosphorylates myo-inositol has been characterized in Dictyostelium. The myo-inositol kinase activity was partially purified from amoebae by chromatography on DEAE-Sepharose and phenyl-Sepharose columns. The product of both the partially purified activity and of a crude cytosolic fraction was myo-inositol 3-phosphate. The partially purified preparations of myo-inositol kinase (a) possessed a Km for myo-inositol of 120 microM (in the presence of 5 mM-ATP) and for ATP of 125 microM (in the presence of 1 microM-myo-inositol), (b) did not recognize allo-, epi-, muco-, neo-, scyllo-, 1 D-chiro or 1 L-chiro-inositol as substrates, (c) were competitively inhibited by three naturally occurring analogues of myo-inositol: 1 L-chiro-inositol (Ki 49.5 +/- 0.7 microM: the structural equivalent of myo-inositol, except that the D-3 hydroxy moiety is axial), D-3-deoxy-myo-inositol [Ki 103 +/- 1 microM: (-)-viburnitol], and sequoyitol (Ki 271 +/- 7 microM; unlike 1 L-chiro-inositol and D-3-deoxy-myo-inositol, this was a substrate for the kinase), and finally (d) were apparently non-competitively inhibited by myo-inositol 3-phosphate. The product of myo-inositol kinase could be detected in intact amoebae and was a substrate for the first in a series of inositol polyphosphate kinases present in Dictyostelium which ultimately yield myo-inositol hexakisphosphate. The activity of myo-inositol D-3-hydroxykinase in Dictyostelium lysates showed evidence of developmental regulation.     

Relationships between Methylobacteria and Glyphosate with Native and Invasive Plant Species: Implications for Restoration

After removing invasive plants, whether by herbicides or other means, typical restoration design focuses on rebuilding native plant communities while disregarding soil microbial communities. However, microbial–plant interactions are known to influence the relative success of native versus invasive plants. Therefore, the abundance and composition of soil microorganisms may affect restoration efforts. We assessed the effect of herbicide treatment on phytosymbiotic pink‐pigmented facultative methylotrophic (PPFM) bacteria and the potential consequences of native and invasive species establishment post‐herbicide treatment in the lab and in a coastal sage scrub (CSS)/grassland restoration site. Lab tests showed that 4% glyphosate reduced PPFM abundance. PPFM addition to seeds increased seedling length of a native plant (Artemisia californica) but not an invasive plant (Hirschfeldia incana). At the restoration site, methanol addition (a PPFM substrate) improved native bunchgrass (Nassella pulchra) germination and size by 35% over controls. In a separate multispecies field experiment, PPFM addition stimulated the germination of N. pulchra, but not that of three invasive species. Neither PPFM nor methanol addition strongly affected the growth of any plant species. Overall, these results are consistent with the hypothesis that PPFMs have a greater benefit to native than invasive species. Together, these experiments suggest that methanol or PPFM addition could be useful in improving CSS/grassland restorations. Future work should test PPFM effects on additional species and determine how these results vary under different environmental conditions.     

Raised Soluble P-Selectin Moderately Accelerates Atherosclerotic Plaque Progression

Soluble P-selectin (sP-selectin), a biomarker of inflammatory related pathologies including cardiovascular and peripheral vascular diseases, also has pro-atherosclerotic effects including the ability to increase leukocyte recruitment and modulate thrombotic responses in vivo. The current study explores its role in progressing atherosclerotic plaque disease. Apoe ^−/− mice placed on a high fat diet (HFD) were given daily injections of recombinant dimeric murine P-selectin (22.5 µg/kg/day) for 8 or 16 weeks. Saline or sE-selectin injections were used as negative controls. In order to assess the role of sP-selectin on atherothrombosis an experimental plaque remodelling murine model, with sm22α-hDTR Apoe^−/− mice on a HFD in conjunction with delivery of diphtheria toxin to induce targeted vascular smooth muscle apoptosis, was used. These mice were similarly given daily injections of sP-selectin for 8 or 16 weeks. While plaque mass and aortic lipid content did not change with sP-selectin treatment in Apoe^−/− or SM22α-hDTR Apoe^−/− mice on HFD, increased plasma MCP-1 and a higher plaque CD45 content in Apoe ^−/− HFD mice was observed. As well, a significant shift towards a more unstable plaque phenotype in the SM22α-hDTR Apoe^−/− HFD mice, with increased macrophage accumulation and lower collagen content, leading to a lower plaque stability index, was observed. These results demonstrate that chronically raised sP-selectin favours progression of an unstable atherosclerotic plaque phenotype.     

Agonist-stimulated inositol phosphate metabolism in avian erythrocytes.

1. A screen for agonists capable of stimulating the formation of inositol phosphates in erythrocytes from 5-day-old chickens revealed the presence of a population of phosphoinositidase C-linked purinergic receptors. 2. If chicken erythrocytes prelabelled with [3H]Ins were exposed to a maximal effective dose of adenosine 5'-[beta-thio]diphosphate for 30 s, the agonist-stimulated increment in total [3H]inositol phosphates was confined to [3H]Ins(1,4,5)P3, Ins(1,3,4,5)P4 and InsP2. After 40 min stimulation, the radiolabelling of nearly all of the [3H]inositol phosphates that have been detected in these extracts [Stephens, Hawkins & Downes (1989) Biochem. J. 262, 727-737] had risen. However, some of these increases [especially those in Ins(3,4,5,6)P4 and Ins(1,3,4,5,6)P5] were accountable for almost entirely by increases in specific radioactivity rather than in mass. 3. The effect of purinergic stimulation on the rate of incorporation of [32P]Pi in the medium into the gamma-phosphate group of ATP and InsP4 and InsP5 was also measured. After 40 min stimulation, the incorporation of 32P into Ins(1,3,4,6)P4, Ins(1,3,4,5)P4, Ins(3,4,5,6)P4 and Ins(1,3,4,5,6)P5 was significantly elevated, whereas the mass of the last two and the specific radioactivity of the gamma-phosphate of ATP were unchanged compared with control erythrocyte suspensions. 4. In control suspensions of avian erythrocytes, the specific radioactivity of the individual phosphate moieties of Ins(1,3,4,6)P4 increased through the series 1, 6, 4 and 3 [Stephens & Downes (1990) Biochem. J. 265, 435-452]. This pattern of 32P incorporation is not the anticipated outcome of 6-hydroxy phosphorylation of Ins(1,3,4)P3 [the assumed route of synthesis of Ins(1,3,4,6)P4]. Although adenosine [beta-thio]diphosphate significantly stimulated the accumulation of [3H]Ins(1,3,4)P3, and despite the fact that avian erythrocyte lysates were shown to possess a chromatographically distinct, soluble, ATP-dependent, Ins(1,3,4)P3 6-hydroxykinase activity, purinergic stimulation of intact cells did not significantly alter the pattern of incorporation of [32P]Pi into the individual phosphate moieties of Ins(1,3,4,6)P4. These results suggest that the route of synthesis of this inositol phosphate species is not changed during the presence of an agonist.     

Selenocysteine's mechanism of incorporation and evolution revealed in cDNAs of three glutathione peroxidases

The nonsense codon, UGA, has for the first time recently been shown to encode selenocysteine in two proteins, mouse glutathione peroxidase (GSH-Px) (EC 1.11.1.9) and bacterial formate dehydrogenase. A co-translational rather than post-translational selenium-incorporation mechanism has been implicated. Furthermore, high expression levels of GSH-Px have suggested that suppression of termination is efficient and specific. We have isolated and characterized pituitary, kidney and placenta cDNAs for bovine, human and mouse GSH-Px respectively. It is demonstrated that this novel suppression event occurs in diverse tissues, in at least three mammalian species and at the translational step. Surprisingly, GSH-Px is shown to be extramitochondrially encoded, indicating a cytosolic suppression event rather than one utilizing the mitochondria's well-documented extended codon-reading ability. Sequence analysis reveals that a simple proximal contextual pattern responsible for readthrough does not exist. Analysis of predicted secondary structures of mRNAs, however, has revealed a conformation which may be unique to selenocysteine proteins and may prove useful as a tool for artificial incorporation of selenocysteines. A human intron for GSH-Px from an unspliced mRNA has been isolated whose position indicates an ancient, divergent evolutionary relationship with thioredoxin-S2, rather than an independent convergent one.     

Inositol 1,3,4,5-tetrakisphosphate is essential for sustained activation of the Ca2+-dependent K+ current in single internally perfused mouse lacrimal acinar cells

Summary We have examined the effects of various inositol polyphosphates, alone and in combination, on the Ca²⁺-activated K⁺ current in internally perfused, single mouse lacrimal acinar cells. We used the patch-clamp technique for whole-cell current recording with a set-up allowing exchange of the pipette solution during individual experiments so that control and test periods could be directly compared in individual cells. Inositol 1,4,5-trisphosphate (Ins 1,4,5 P₃) (10–100 m) evoked a transient increase in the Ca²⁺-sensitive K⁺ current that was independent of the presence of Ca²⁺ in the external solution. The transient nature of the Ins 1,4,5 P₃ effect was not due to rapid metabolic breakdown, as similar responses were obtained in the presence of 5mm 2,3-diphosphoglyceric acid, that blocks the hydrolysis of Ins 1,4,5 P₃, as well as with the stable analoguedl-inositol 1,4,5-trisphosphorothioate (Ins 1,4,5 P(S)₃) (100 m). Ins 1,3,4 P₃ (50 m) had no effect, whereas 50 m Ins 2,4,5 P₃ evoked responses similar to those obtained by 10 m Ins 1,4,5 P₃. A sustained increase in Ca²⁺-dependent K⁺ current was only observed when inositol 1,3,4,5-tetrakisphosphate (Ins 1,3,4,5 P₄) (10 m) was added to the Ins 1,4,5 P₃ (10 m)-containing solution and this effect could be terminated by removal of external Ca²⁺. The effect of Ins 1,3,4,5 P₄ was specifically dependent on the presence of Ins 1,4,5 P₃ as it was not found when 10 m concentrations of Ins 1,3,4 P₃ or Ins 2,4,5 P₃ were used. Ins 2,4,5 P₃ (but not Ins 1,3,4 P₃) at the higher concentration of 50 m did, however, support the Ins 1,3,4,5 P₄-evoked sustained current activation. Ins 1,3,4 P₃ could not evoke sustained responses in combination with Ins 1,4,5 P₃ excluding the possibility that the action of Ins 1,3,4,5 P₄ could be mediated by its breakdown product Ins 1,3,4 P₃. Ins 1,3,4,5 P₄ also evoked a sustained response when added to an Ins 1,4,5 P(S)₃-containing solution. Ins 1,3,4,5,6 P₅ (50 m) did not evoke any effect when administered on top of Ins 1,4,5 P₃. In the absence of external Ca²⁺, addition of Ins 1,3,4,5 P₄ to an Ins 1,4,5 P₃-containing internal solution evoked a second transient K⁺ current activation. Readmitting external Ca²⁺ in the continued presence internally of Ins 1,4,5 P₃ and Ins 1,3,4,5 P₄ made the response reappear. We conclude that both Ins 1,4,5 P₃ and Ins 1,3,4,5 P₄ play crucial and specific roles in controlling intracellular Ca²⁺ homeostasis.